US008535891B2

(12) United States Patent (10) Patent No.: US 8,535,891 B2 Kentsis et al. (45) Date of Patent: Sep. 17, 2013

(54) METHOD OF PREDICTING ACUTE 2009/0175844 A1* 7/2009 Nakamura et al...... 435/6 APPENDICITIS 2010.0184049 A1* 7, 2010 Goodison et al...... 435/6 2010/0190662 A1* 7/2010 Sutphen et al...... 506,18 (75) Inventors: Alex Kentsis, Brookline, MA (US); FOREIGN PATENT DOCUMENTS Hanno Steen, Cambridge, MA (US); WO 2006/125973 11, 2006 Richard Bachur, North Reading, MA (US) OTHER PUBLICATIONS (73) Assignee: Children's Medical Center Mahmoud et al., (Tikrit Medical Journal 2007: 13(1): 81-83).* Corporation, Boston, MA (US) panel et al., (Journal of Leukocyte Biology, 2002; 72(3), 478 (*) Notice: Subject to any disclaimer, the term of this Murphy et al., (Nature 2008; vol. 1 No. 4, published online May

patent is extended or adjusted under 35 2.guci ettal. al., Biochem.51Ocnem. J. (2005) 387, 343-353)).* U.S.C. 154(b) by 0 days. Life Science application, Life Technologies 2008 catalog. Aspenbio Pharma, Inc., 'Appendicitis product pipeline.” http://www. (21) Appl. No.: 13/142,598 aspenbiopharma.com/rdproduct/appendicitis; accessed Apr. 7, 2009. (22) PCT Filed: Dec. 30, 2009 Kentsis et al., Annals of Emergency Medicine, 1-9.e4 (2009). “Dis covery and validation of urine markers of acute pediatric appendicitis (86). PCT No.: PCT/US2O09/0698OO using high-accuracy mass spectrometry.” S371 (c)(1), Grosel-Grenc et al., Scandinavian Journal of Clinical and Labora (2), (4) Date: Oct. 14, 2011 torylipopolysaccharide-binding Investigation, 67(2): 197-206protein in(2007). acute appendicitis"Interleukin-6 in chiland dren. (87) PCT Pub. No.: WO2010/0784.11 Lycopoulou et al., Clinical Chemistry and Laboratory Medicine, PCT Pub. Date: Jul. 8, 2010 43(1):49-53 (2005). "Serum amyloid A protein levels as a possible aid in the diagnosis of acute appendicitis in children.” (65) Prior Publication Data Sack et al., BMC Surgery, Biomed Central. 6(1): 15-22 (2006). “Diagnostic value of blood inflammatory markers for detection of US 2012/OO28268 A1 Feb. 2, 2012 acute appendicitis in children.” Related U.S. Application Data * cited by examiner (60) Provisional application No. 61/141.283, filed on Dec. 30, 2008, provisional application No. 61/185,676, Primary Examiner — Jacob Cheu filed on Jun. 10, 2009. Assistant Examiner — Carmencita MBelei 74) Attorney,ey, AgAgent, or Firm — David S. Resnick; Tari W. (51) E's (2006.01) Mills: Nixon Peabody LLP GOIN33/536 (2006.01) GOIN 33/537 (2006.01) (57) ABSTRACT GOIN33/54 (2006.01) Embodiments of the invention provide method and devices GOIN33/543 (2006.01) for predicting the likelihood of acute appendicitis without (52) U.S. Cl. invasive exploratory medical procedures. Several protein USPC ...... 435/7.1:436/501 biomarkers: leucine-rich C-2-glycoprotein (LRG); S100-A8 (58) Field of Classification Search (calgranulin); C-1-acid glycoprotein 1 (ORM); plasminogen None (PLG); mannan-binding lectin serine protease 2 (MASP2); See application file for complete search history. zinc-C-2-glycoprotein (AZGP1); Apollipoprotein D (Apol)); and C-1-antichymotrypsin (SERPINA3); are increased in the (56) References Cited urine of patients with appendicitis. The method and devices comprise detecting the levels of these biomarkers and com U.S. PATENT DOCUMENTS paring with reference levels found in healthy individuals. 2009/0018026 A1 1/2009 Kim et al...... 506.7 2009.0035875 A1 2/2009 Jemmerson 2009/0104605 A1 4/2009 Siuzdak et al...... 435/6 18 Claims, 22 Drawing Sheets U.S. Patent Sep. 17, 2013 Sheet 1 of 22 US 8,535,891 B2

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US 8,535,891 B2 1. 2 METHOD OF PREDICTING ACUTE In some embodiments, an appendicitis biomarker is leu APPENDICITIS cine C-2 glycoprotein (LRG). In some embodiments, an appendicitis biomarker is mannan-binding lectin serine pro tease 2 (MASP2). In some embodiments, an appendicitis CROSS REFERENCE TO RELATED biomarker is C.-1.-acid glycoprotein 1 (ORM). In some APPLICATIONS embodiments, an appendicitis biomarker is selected from the groups selected from leucine-rich C-2-glycoprotein (LRG); This application is a 35 U.S. C. S371 National Phase Entry S100-A8 (calgranulin); O.-1.-acid glycoprotein 1 (ORM); Application of International Application No. PCT/US2009/ plasminogen (PLG); mannan-binding lectin serine protease 2 069800 filed Dec. 30, 2009, which designates the United (MASP2); zinc-C-2-glycoprotein (AZGP1); apolipoprotein States, and which claims benefit under 35 U.S. C. S 119(e) of 10 D (Apol)); and C-1-antichymotrypsin (SERPINA3). In some U. S. Provisional Application No. 61/141.283, filed Dec. 30, embodiments, an appendicitis biomarker is selected from at 2008, and U. S. Provisional Application No. 61/185,676, filed least 1, or at least about 2, or at least about 3, or at least about Jun. 10, 2009, the contents of which are incorporated herein 4, or at least about 5, or more than 5 of any and all combina by reference in their entireties. tions of appendicitis biomarkers disclosed in Table 1. 15 One aspect of the present invention relates to a device for SEQUENCE LISTING detecting at least one protein biomarker in a urine sample from a subject to identify if the subject is likely to have acute appendicitis, the device comprising: (a) at least one protein The instant application contains a Sequence Listing which binding agent which specifically binds to at least one biom has been submitted in ASCII format via EFS-Web and is arker protein selected from the group of leucine C-2 glyco hereby incorporated by reference in its entirety. This ASCII protein (LRG), mannan-binding lectin serine protease 2 copy, created on Jun. 28, 2011, is named (MASP2), C.-1.-acid glycoprotein 1 (ORM); and (b) at least 20110629 SequenceListing TextFile 701039 one solid Support for the at least one protein binding-agent in 064443 US.txt and is 244,035 bytes in size. (a), wherein the protein-binding agent is deposited on the Solid Support. In some embodiments, a protein-binding agent BACKGROUND 25 deposited on the solid support specifically binds the leucine C-2 glycoprotein (LRG) of SEQ ID NO: 1. In another Appendicitis is a condition characterized by inflammation embodiment, a protein-binding agent deposited on the Solid of the appendix. All cases require removal of the inflamed Support specifically binds to the polypeptide of C-1-acid gly appendix, either by laparotomy or laparoscopy. Untreated, coprotein 1 (ORM) of SEQ ID NO: 3. In another embodi mortality is high, mainly because of peritonitis and shock. 30 ment, a protein-binding agent deposited on the Solid Support Appendicitis is among many human diseases, for which the specifically binds to the polypeptide of mannan-binding lec diagnosis is complicated by the heterogeneity of its clinical tin serine protease 2 (MASP2) of SEQID NO: 5. presentation. Patients with many other disorders can present In some embodiment, the device is useful for detecting with symptoms similar to those of appendicitis. Examples multiple appendicitis biomarkers, for example where the include the following: pelvic inflammatory disease (PID) or device further comprises at least one additional different pro tubo-Ovarian abscess, Endometriosis, ovarian cyst or torsion, 35 tein-binding agent deposited on the Solid Support, wherein the ureterolithiasis and renal colic, degenerating uterine lei additional protein-binding agent specifically binds to a biom omyomata, diverticulitis, Crohn's disease, colonic carci arker protein selected from the group consisting of leucine noma, rectus sheath hematoma, cholecystitis, bacterial rich C-2-glycoprotein (LRG); S100-A8 (calgranulin); O.-1- enteritis, mesentericadenitis, and omental torsion. It remains acid glycoprotein 1 (ORM); plasminogen (PLG); mannan the most common Surgical emergency of children, with initial 40 binding lectin serine protease 2 (MASP2); zinc-C-2- diagnosis accuracy additionally challenged because of non glycoprotein (AZGP1); Apollipoprotein D (Apol)); and C-1- specific but similar symptoms of many other childhood con antichymotrypsin (SERPINA3). ditions. Delays in accurate diagnosis lead to increased mor In some embodiment, the device is useful for detecting tality, morbidity, and costs associated with the complications multiple appendicitis biomarkers, for example where the of appendicitis. device further comprises at least one additional different pro The use of high resolution computed tomography (CT) to 45 tein-binding agent deposited on the Solid Support, wherein the identify appendiceal inflammation was hoped to improve additional protein-binding agent specifically binds to a biom both the diagnosis and treatment of acute appendicitis. arker protein selected from the group consisting of adipocyte Though variable, these improvements have been modest at specific adhesion molecule: AMBP: amyloid-like protein 2: best, with rates of unnecessary appendectomies and ruptures angiotensin converting enzyme 2: BAZ1B: carbonic anhy of 3-30% and 30-45%, respectively. In addition, availability 50 drase 1: CD14; chromograminA, FBLN7, FXR2; hemoglobin of and experience with CT limit the usefulness of this C.; hemoglobin B; interleukin-1 receptor antagonist protein; approach. Furthermore, recently its use has been re-evaluated inter-C-trypsin inhibitor, lipopolysaccharide binding protein; due to concerns of cancer risk. lymphatic vessel endothelial hyaluronan acid receptor 1; Development of non invasive diagnostics are therefore MLKL; nicastrin; novel protein (Accession No: needed and desirable. 55 IPIO0550644); PDZK1 interacting protein 1: PRIC285; pros taglandin-H2 D-isomerase: Rcl: S100-A9; serum amyloid A SUMMARY OF THE INVENTION protein; SLC13A3: SLC2A1; SLC2A2: SLC4A1; SLC9A3: SORBS1, SPRX2; supervillin: TGFbeta2R; TTYH3; The present invention generally relates to devices, kits and VAOD1; vascular adhesion molecule 1: versican; VIP36: C-1- methods to determine acute appendicitis in a Subject, Such as acid glycoprotein 2: B-1,3-galactosyltransferase, also dis a human Subject. In particular, the inventors have discovered closed in Table 1. a set of appendicitis biomarkers which are present in a urine In some embodiments, the solid support of the device is in sample obtained from a Subject with acute appendicitis. As the format of a dipstick, a microfluidic chip or a cartridge. In Such, one aspect of the present invention provides devices, Some embodiments, the dipstick is a lateral flaw immunoas kits and methods to detect the presence of Such appendicitis say test strip. In some embodiments, a single test Strip tests for biomarkers in a urine sample from a subject, Such as a human 65 one appendicitis biomarker, such as LRG or ORM or S100 subject. In some embodiments, the device is in the format of A8. In other embodiments, a single test strip test for several a dipstick test, in particular, a lateral flow immunoassay. appendicitis biomarkers, for example, a single test strip test US 8,535,891 B2 3 4 for all three appendicitis biomarkers: LRG, ORM and S100 In some embodiments, the reference level in the method is A8; or a single test strip test for two appendicitis biomarkers: a level of the particular appendicitis biomarker measured in a LRG and ORM: LRG and S100-A8; or ORM and S100-A8. urine sample of a healthy human not having acute appendi In some embodiments, a protein-binding agent is an anti citis. In some embodiments, the reference level is an average body, antibody fragment, aptamer, Small molecule or variant 5 level of the appendicitis biomarker in a plurality of urine or fragment thereof. In some embodiments, a Subject is a samples from a population of healthy humans not having mammalian Subject Such as a human Subject. In some acute appendicitis. In some embodiments, the reference level embodiments, a Subject with at least one symptom of appen is a normalized level of the appendicitis biomarker in a urine dicitis, as disclosed herein. sample of a healthy human not having acute appendicitis, In some embodiments, a protein-binding agent deposited 10 wherein the normalization is performed against the level of on the device specifically binds to the specific appendicitis albumin in the urine sample of a healthy human not having biomarker protein when the level of the appendicitis biomar acute appendicitis. ker protein is at least 2-fold above a reference level for that In some embodiments, the method comprises measuring appendicitis biomarker protein. Typically, a reference level the level of at least one the appendicitis biomarker in a urine for a particular appendicitis biomarker is an average level of 15 sample is collected in mid-stream. In some embodiments, the the appendicitis biomarker protein in a plurality of urine method comprises measuring the level of at least one the samples from a population of healthy humans not having appendicitis biomarker by depositing the urine sample from acute appendicitis. the Subject on a device. Such as a test strip or dipstick device, Another aspect of the present invention relates to the use of as disclosed herein. a device as disclosed herein to identify if a subject has acute appendicitis, wherein if at least one biomarker specifically BRIEF DESCRIPTION OF THE DRAWINGS binds to at least one protein-binding agent, the Subject is likely to have acute appendicitis. FIG. 1 is a representative SDS-PAGE separation of 17,000 Another aspect of the present invention provides a kit, g, 210,000 g, and TCA fractions of three urine specimens (1, where the kit comprises (a) a device as disclosed herein, and 2, 3) demonstrating Small differences in total protein abun (b) a first agent, wherein the first agent produces a detectable 25 dance among differenturine specimens, and preferential frac signal in the presence of a protein-binding agent which tionation of albumin (O) and uromodulin ( ) in the 17,000 g deposited on the device is specifically bound to a biomarker fraction, enabling improved detection of the remaining uri protein. In some embodiments, a kit optionally further com nary proteins. The majority of albumin and uromodulin prises a second agent, wherein the second agent produces a appears to sediment at 17,000 g, demonstrating that they exist different detectable signal in the presence of a second protein 30 in high molecular weight complexes, consistent with uro binding agent deposited on the device which is specifically modulin's ability to polymerize in urine. bound to a second biomarker protein. FIGS. 2A-2B show representative mass spectra. FIG. 2A is Another aspect of the present invention relates to a method the relative ion intensity as a function of m/z values of pre to identify the likelihood of a subject to have acute appendi cursor ions (MS), with the doubly charged peptide LDI citis comprising: (a) measuring the level of at least one appen TAEILAVR from plunc labeled by arrow, and FIG. 2B is the dicitis biomarker protein selected from the group listed in 35 fragmentation spectrum with fragmentions labeled as y- and Table 1 in a urine sample from the human subject; (b) com b-series fragment ions (MS/MS). paring the level of the at least one biomarker protein measured FIGS. 3A-3B shows the apparent mass accuracy error of in step (a) to a reference level for the measured appendicitis the LTQ-Orbitrap. FIG. 3A is a cumulative probability graph biomarker, where if the level of a measured appendicitis of the mass accuracy error, and FIG. 3B is the histogram of the biomarker is at least 2-fold increased than the reference level 40 LTQ-Orbitrap mass accuracy error, as assessed by compari for the particular appendicitis biomarker measured, it identi Son of observed masses of the trypsin autolysis peptide fies that the subject is likely to have acute appendicitis. In VATVSLPR, as compared to its expected monoisotopic mass, Some embodiments, the method can be used to guide a clini indicating that most peptides have apparent mass errors of cian to direct an appropriate therapy to a Subject which is less than 2 ppm. identified to have acute appendicitis. FIG. 4 is a Venn diagram showing the comparisons of the In some embodiments, the method further comprises deter 45 observed aggregate urine proteome with those published by mining the level of albumin in the urine sample from the Adachietal 13, and Pisitkun et al 10, demonstrating high human Subject. In some embodiments, the Subject is a human concordance with the previous studies of human urine, as Subject and the human Subject has exhibited at least one well as discovery of not previously observed proteins. symptom of acute appendicitis. FIG. 5 is a histogram showing the variability in the com In some embodiments, the method comprises measuring 50 position of individual urine proteomes, as assessed by the an appendicitis biomarker level by any method known by one coefficients of variation of their proteins spectral counts, of ordinary skill in the art, such as for example with the use of demonstrating a broad distribution, including proteins that an immunoassay oran automated immunoassay, or a dipstick are relatively invariant (A: Albumin, cubilin, megalin), and test, as disclosed herein. In some embodiments, the method those that appear to vary among individual proteomes (B: C. comprises measuring the level of the appendicitis biomarker 55 1-anti-trypsin, fibrinogen, C. 2-macroglobulin). leucine C-2 glycoprotein (LRG). In some embodiments, the FIG. 6 is a scatter plot showing the relative enrichment of method comprises measuring the level of the appendicitis appendicitis protein biomarkers as a function of appendicitis biomarker C-1-acid glycoprotein 1 (ORM). In some embodi tissue overexpression of the corresponding genes, demon ments, the method comprises measuring the level of the strating that more than 50% of markers with tissue overex appendicitis biomarker mannan-binding lectin serine pro pression exhibit urine enrichment (D), but that only 3 of these tease 2 (MASP2). 60 () were identified as markers by urine proteome profiling. In some embodiments, the method comprises measuring FIG. 7 is a flow diagram showing an experimental Scheme, the level of at least one the appendicitis biomarker selected outlining methods used for protein capture and fractionation, from a group consisting of leucine C-2 glycoprotein (LRG), of the identification and discovery of appendicitis biomarkers calgranulin A (S100-A8), C.-1.-acid glycoprotein 1 (ORM), using urine proteomics, and the validation of appendicitis plasminogen (PLG), mannan-binding lectin serine protease 2 65 diagnostic biomarkers. (MASP2), zinc-C-2-glycoprotein (AZGP1), C-1-antichy FIG. 8 is a boxplot showing the relative urine protein motrypsin (SERPINA3) and apolipoprotein D (Apol)). abundance (logarithm normalized ion current units) of the US 8,535,891 B2 5 6 validated diagnostic markers for the non-appendicitis (open) FIG. 17A-F are schematic diagrams showing the different and appendicitis (hatched) patient groups. Normalized value results that can obtained using the LFIA test strip shown in of 1 corresponds to the apparent abundance of internal refer FIG. 16. ence standard. Boxes contain the 25-75% interquartile range, FIG. 18 shows a schematic diagram of an alternative ver with the dividing bars representing means, whiskers repre sion on how the levels of four biomarker proteins can be senting 10-90% range, and crosses representing 1-99% range. determined simultaneously using four separate LFIA test Square symbols represent medians. Abundance of LRG in strips, one test strip for a different biomarker protein. A diag patients with pyelonephritis (solid dot, O) and those who nostic kit can comprise multiple LFIA test strips, one strip for underwent appendectomies with findings of histologically a different biomarker protein. normal appendices (open dot, O). 10 FIG. 19 shows a schematic diagram of an alternative ver FIGS. 9A-9B show validation of selected appendicitis sion how the levels of three biomarker proteins are deter biomarkers. FIG.9A shows receiver operating characteristics mined simultaneously on the same LFIA test strip. A diag of appendicitis protein biomarkers from urine validated by nostic kit can comprise a single composite LFIA test strip for target mass spectrometry, demonstrating the relative diagnos determining the levels of several biomarker proteins. tic performance of leucine-rich C-2-glycoprotein (LRG), cal 15 granulin A (S100-A8), C-1-acid glycoprotein 1 (ORM), and DETAILED DESCRIPTION OF THE INVENTION apolipoprotein D (Apol)). FIG.9B shows the enrichment of LRG in a random sample of urine of patients with histologi Embodiments of the present invention are based on the cally proven appendicitis (+) as compared to those without discovery of eight biomarkers whose increase in urinary con (-) by using Western immunoblotting. LRG signal was centration correlate accurately with acute appendicitis. These observed in 5/5 patients with appendicitis and no signal was eight biomarkers are leucine-rich C. 2-glycoprotein (LRG), observed in 5/6 patients without appendicitis. calgranulin A (S100-A8), C-1-acid glycoprotein 1 (orosomu FIGS. 10A-10B show clinical validation of selected appen coid) (ORM), plasminogen (PLG), mannan-binding lectin dicitis biomarkers. FIG. 10A is a boxplot showing the relative serine protease 2 (MASP2), zinc-C-2-glycoprotein (AZGP1), appendicitis protein biomarker abundance (normalized ion C-1-antichymotrypsin (SERPINA3) and apolipoprotein D current units) of leucine-rich C-2-glycoprotein (LRG) (top 25 (Apod). These appendicitis biomarker proteins have been panel) and calgranulin A (S100-A8) (bottom panel) as a func confirmed by Western immunoblotting (Example 2, FIGS. 9 tion of appendicitis severity, as assessed using histologic and 10) and further validated by target mass spectrometry classification. Note that the group with histologically normal (Example 2). appendices includes both patients who underwent appendec Accordingly, in some embodiments, these biomarkers can 30 be used as indicators of acute appendicitis. By simply mea tomies and patients without clinical diagnosis of appendicitis. Suring the levels of these biomarkers in a urine sample from FIG. 10B shows representative micrographs of appendec an individual having some symptoms of acute appendicitis or tomy specimens and immunohistochemistry staining against that is suspected of having acute appendicitis, a physician can LRG, demonstrating increased LRG signal in appendectomy quickly make a diagnosis and administer appropriate medical specimens with more severe grade of appendicitis. treatment in a timely manner. When the levels of these biom FIG. 11A (top view) and 11B (side view) shows the sche 35 arkers in an individual is greater than the reference level or matic diagrams of an exemplary lateral flow immunoassay reference value of the respective biomarkers, at least one (LFIA) dipstick test strip for determining that the level of an order of magnitude greater than that found in healthy indi appendicitis biomarker protein in urine is greater than (or vidual not having acute appendicitis, it is indicative that the increased as compared to) a predetermined reference level. individual is indeed having acute appendicitis. FIG. 12A-D are schematic diagrams of the top views of 40 In one embodiment, a Subjectorindividual is a mammalian exemplary LFIA dipstick test strips shown in FIG. 11, show Subject, Such as a human. ing the different results that can obtained using the simpletest Non-limiting symptoms of acute appendicitis include pain strip shown in FIG. 11. starting centrally (periumbilical) before localizing to the right FIG. 13 shows a schematic diagram of how the levels of iliac fossa (the lower right side of the abdomen); loss of three biomarker proteins can be determined simultaneously appetite and fever, nausea or vomiting; the feeling of drowsi using three independent LFIA test strips, one test strip for a 45 ness; the feeling of general bad health; pain beginning and different biomarker protein. A diagnostic kit can comprise staying in the right iliac fossa, diarrhea and a more prolonged, several LFIA test strips, one strip for a different biomarker Smoldering course; increased frequency of urination; marked protein. retching; tenesmus or "downward urge' (the feeling that a FIG. 14 shows a schematic diagram of how the levels of bowel movement will relieve discomfort); positive Rovsings three biomarker proteins are determined simultaneously on 50 sign, Psoas sign, and/or Obturator sign. the same LFIA test strip. A diagnostic kit can comprise a In one embodiment, the invention provides a kit for pre single composite or multiplex LFIA test strip for determining dicting acute appendicitis in a human comprising an indicator the levels of several biomarker proteins simultaneously. The or device that is responsive to a level of at least one biomarker single composite test trip has three distinct protein binding in a sample of urine from a human upon contact with the agent specific respectively for three appendicitis biomarker 55 sample of urine, wherein the appendicitis biomarker protein proteins. in a sample of urine is selected from a group consisting of FIG. 15A-D are schematic diagrams of an alternative leucine C-2 glycoprotein (LRG), calgranulin A (S100-A8), embodiment of an exemplary LFIA dipstick test strip shown C-1-acid glycoprotein 1 (ORM), plasminogen (PLG), man in FIG. 11 for determining whether the level of a biomarker nan-binding lectin serine protease 2 (MASP2), Zinc-C-2-gly protein in a fluid sample is above or below a reference/control coprotein (AZGP1), C.-1-antichymotrypsin (SERPINA3) and value for that biomarker and the interpretation of the results 60 apolipoprotein D (Apod), and wherein the indicator provides obtained. Two differentanti-biomarker antibodies are used on a positive test result when the appendicitis biomarker level the test strip. exceeds a reference value. FIG. 16A (top view) and 16B (side view) shows a sche In some embodiments, the present invention provides a kit matic diagram of an alternative embodiment of a LFIA test or device for predicting acute appendicitis in a subject, (e.g. a strip for determining the level of a biomarker protein in a fluid 65 human Subject) that are responsive to at least one marker sample and comparing the determined level with a reference selected from the list of appendicitis biomarkers listed in value. S, T, C definition are as in FIG. 11. Table 1. In one embodiment, the kit or device for predicting US 8,535,891 B2 7 8 acute appendicitis in a subject is responsive to leucine C-2 In one embodiment, the determination of the appendicitis glycoprotein (LRG). In one embodiment, the kit or device for biomarker level is completed with the use of an immunoassay. predicting acute appendicitis in a Subject, is responsive to In some embodiments, the immunoassay is a lateral flow leucine C-2 glycoprotein (LRG) and at least one marker immunoassay test, also known as the immunochromato selected from C.-1.-acid glycoprotein 1 (ORM), and/or man 5 graphic assay, or strip test. In some embodiments, the lateral nan-binding lectin serine protease 2 (MASP2). In some flow immunoassay is a double antibody sandwich assay, a embodiments, the kit or device for predicting acute appendi competitive assay, a quantitative assay or variations thereof. citis in a Subject, is responsive to leucine C-2 glycoprotein In one embodiment, the appendicitis biomarker protein is (LRG) and at least one marker selected from the group con leucine C-2 glycoprotein (LRG). In one embodiment, the sisting of calgranulin A (S100-A8), C-1-acid glycoprotein 1 10 appendicitis biomarker protein is selected from a group con (ORM), plasminogen (PLG), mannan-binding lectin serine sisting of leucine C-2 glycoprotein (LRG), calgranulin A protease 2 (MASP2), zinc-C-2-glycoprotein (AZGP1), C.-1- (S100-A8), C.-1-acid glycoprotein 1 (ORM), plasminogen antichymotrypsin (SERPINA3) and apolipoprotein D (PLG), mannan-binding lectin serine protease 2 (MASP2), (Apod). As used herein, the term “responsive' refers to the Zinc-C-2-glycoprotein (AZGP1), C-1-antichymotrypsin (SERPINA3) and apolipoprotein D (Apol)). In another ability to detect the level of an appendicitis biomarkers of 15 interest in a urine sample. embodiment, the appendicitis biomarker is selected from the In another embodiment, the kit or device for predicting group of biomarkers selected from any of those listed in Table acute appendicitis in a Subject, is responsive to leucine C-2 1. glycoprotein (LRG) and at least 1, or a least 2 or at least 3, or In other embodiments, for the method and kit or devices, at least 4 or at least 5, or at least 6, or at least 7, or at least 8, various combinations of appendicitis biomarkers can be or at least 9, or at least 10 other marker(s), in all and any selected. For examples: LRG and S100-8A: LRG and ORM; combination, selected from the group consisting of the list of ORM and S100-A8, LRG and PLG: LRG and MASP2: LRG biomarkers listed in Table 1. In another embodiment, the kit and AZGP1; LRG and SERPINA3: LRG and ApolD; LRG, or device for predicting acute appendicitis in a Subject, is MASP2 and ORM: ORM and MASP2, LRG, S100-A8 and responsive to leucine C-2 glycoprotein (LRG) and at least one ORM: LRG, ORM and PLG: LRG, ORM and ApolD; LRG, marker selected from the group consisting of adipocyte spe 25 S100-A8, and PLG: LRG, S100-A8, and ApolD; LRG, S100 cific adhesion molecule: AMBP: amyloid-like protein 2: A8, ORM and SERPINA3; LRG, S100-8A and SERPINA3; angiotensin converting enzyme 2: BAZ1B: carbonic anhy LRG, SERPINA3 and AZGP1; LRG, SERPINA3 and Apo D drase 1: CD14; chromograminA, FBLN7, FXR2; hemoglobin and so forth. C.; hemoglobin 13; interleukin-1 receptor antagonist protein; In one embodiment, the method of predicting acute appen inter-C-trypsin inhibitor, lipopolysaccharide binding protein; 30 dicitis in a human comprises the step of determining the level lymphatic vessel endothelial hyaluronan acid receptor 1; of leucine-rich C-2-glycoprotein (LRG) in a sample of urine MLKL, nicastrin; novel protein (Accession No: from the human. IPIO0550644); PDZK1 interacting protein 1: PRIC285; pros In one embodiment, the method of predicting acute appen taglandin-H2 D-isomerase: Rcl: S100-A9; serum amyloid A dicitis in a human comprises the step of determining the levels protein; SLC13A3: SLC2A1; SLC2A2: SLC4A1; SLC9A3: 35 of LRG and S100-A8 (calgranulin) in a sample of urine from SORBS1; SPRX2; supervillin: TGFbeta2R; TTYH3; the human. VAOD1; vascular adhesion molecule 1: versican; VIP36: C-1- acid glycoprotein 2; and B-1,3-galactosyltransferase. In one embodiment, the method of predicting acute appen In one embodiment, the indicator is in the form of a test dicitis in a human comprises the step of determining the levels strip Such as a dipstick. In one embodiment, the test strip is a of LRG and C-1-acid glycoprotein 1 (ORM) in a sample of lateral flow immunoassay (LFIA). In one embodiment, the 40 urine from the human. test strip is a double sandwich LFIA. In another embodiment, In one embodiment, the method of predicting acute appen test strip is a competitive LFIA. dicitis in a human comprises the step of determining the levels In one embodiment, the reference value is an average level of LRG and plasminogen (PLG) in a sample of urine from the of the appendicitis biomarker in urine samples from a popu human. lation of healthy humans not having acute appendicitis. In Some embodiments, healthy humans not having acute appen 45 In one embodiment, the method of predicting acute appen dicitis do not exhibit any symptom associated with acute dicitis in a human comprises the step of determining the levels appendicitis as disclosed herein. of LRG and mannan-binding lectin serine protease 2 In one embodiment, the responsiveness of the indicator of (MASP2) in a sample of urine from the human. the kit is by way of an immunoassay. In one embodiment, the In one embodiment, the method of predicting acute appen immunoassay is a lateral flow immunoassay test, also known 50 dicitis in a human comprises the step of determining the levels as the immunochromatographic assay, or strip test. of LRG and zinc-C-2-glycoprotein (AZGP1) in a sample of In one embodiment, the invention provides a method of urine from the human. predicting acute appendicitis in a human comprising the steps In one embodiment, the method of predicting acute appen of: (a) determining the level of at least one biomarker protein dicitis in a human comprises the step of determining the levels in a sample of urine from the human; and comparing the level 55 of LRG and apolipoprotein D (Apod) in a sample of urine of step (a) to a reference value to determine whether the from the human. human is Suffering from acute appendicitis. In one embodiment, the method of predicting acute appen In one embodiment, the invention further comprises deter mining the level of albumin in the sample of urine from the dicitis in a human comprises the step of determining the levels human. of ORM and S100-A8 in a sample of urine from the human. In one embodiment, the sample of urine is collected by the 60 In one embodiment, the method of predicting acute appen human. dicitis in a human comprises the step of determining the levels In one embodiment, the human exhibits at least one symp of LRG, ORM and S100-A8 in a sample of urine from the tom of acute appendicitis described herein. human. In one embodiment, the human had an inconclusive CT to In one embodiment, the method of predicting acute appen determine inflammation of the appendix. 65 dicitis in a human comprises the step of determining the levels In one embodiment, the human did not have a CT to deter of LRG and C-1-antichymotrypsin (SERPINA3) in a sample mine inflammation of the appendix. of urine from the human. US 8,535,891 B2 9 10 TABLE 1. In one embodiment, the reference level or reference value is a normalized level of the appendicitis biomarker in a urine List of appendicitis biomarkers for use in the kits, devices sample of a healthy human not having acute appendicitis, and methods as disclosed herein for predicting acute appendicitis in the subject, for example a human subject. wherein the normalization is performed against the level of 5 albumin in the urine sample of a healthy human not having Protein biomarker Accession no SEQID acute appendicitis, or not having been diagnosed with acute appendicitis. The normalized reference value for leucine C-2 Leucine-rich C-2-glycoprotein (LRG) PIOOO22417 1 S100-A8 (calgranulin) PIOOOO7047 2 glycoprotein (LRG), calgranulin A (S100-A8), C-1-acid gly C-1-acid glycoprotein 1 (ORM) PIOOO22429 3 coprotein 1 (ORM), plasminogen (PLG), mannan-binding Plasminogen PIOOO1958O 4 10 lectin serine protease 2 (MASP2), Zinc-C-2-glycoprotein Mannan-binding lectin serine protease 2 (MASP2) IPIO0306378 5 (AZGP1), C.-1-antichymotrypsin (SERPINA3) and apolipo Zinc-C-2-glycoprotein (AZGP1) PIOO166729 6 Apollipoprotein D (ApoD) PIOOOO6.662 7 protein D (Apolo) is 0.001. When the normalized value for C-1-antichymotrypsin (SERPINA3) PIOO550991 8 any of the described biomarker from a human is at least one Adipocyte specific adhesion molecule PIOOO24929 9 order of magnitude greater that the normalized reference AMBP PIOOO22426 10 15 value, i.e. 0.01 and greater, this is indicative that the human Amyloid-like protein 2 PIOOO31 O30 11 has acute appendicitis. Angiotensin converting enzyme 2 PIOO4651.87 12 BAZ1B PIOO216695 13 In one embodiment, the urine sample is collected in mid Carbonic anhydrase 1 PIOO215983 14 Stream. CD14 PIOOO29260 15 chromogramin A PIOO383975 16 In one embodiment, the urine sample is obtained by depos FBLN7 PIOO167710 17 iting the urine on to a test strip. In one embodiment, the test FXR2 PIOOO162SO 18 strip is a lateral flow immunoassay test, also known as the Hemoglobin C. PIOO41071.4 19 Hemoglobin B PIOO654755 2O immunochromatographic assay. In some embodiments, the Interleukin-1 receptor antagonist protein PIOOOOOO45 21 lateral flow immunoassay is a double antibody sandwich Inter-C-trypsin inhibitor PIOO218.192 22 assay, a competitive assay, a quantitative assay or variations Lipopolysaccharide binding protein PIOOO32311 23 25 thereof (See FIGS. 11-19). Lymphatic vessel endothelial hyaruronan acid PIOO29O856 24 receptor 1 Appendicitis Biomarker Proteins MLKL PIOO180781 25 Nicastrin PIOOO21983 26 As discussed herein, in some embodiments, the present Novel protein PIOOSSO644 27 invention provides kits or devices for predicting acute appen PDZK1 interacting protein 1 PIOOO11858 28 30 PRIC285 PIOO24930S 29 dicitis in a subject, for example, a human Subject that is Prostaglandin-H2 D-isomerase PIOOO13179 30 responsive to at least one appendicitis biomarker selected Rc PIOOOO7926 31 S100-A9 PIOOO27462 32 from the list of appendicitis biomarkers listed in Table 1. In Serum amyloid A protein PIOO552.578 33 one embodiment, the kit or device for predicting acute appen SLC13A3 PIOO103426 34 35 SLC2A1 PIOO22O194 35 dicitis in a subject is responsive to leucine C-2 glycoprotein SLC2A2 PIOOOO3905 36 (LRG). In one embodiment, the kit or device for predicting SLC4A1 PIOOO22361 37 SLC9A3 PIOOO11184 38 acute appendicitis in a Subject, is responsive to leucine C-2 SORBS1 PIOOOO2491 39 glycoprotein (LRG) and at least one marker selected from SPRX2 PIOOOO4446 40 40 Supervillin PIOO412650 41 C-1-acid glycoprotein 1 (ORM), and/or mannan-binding lec TGFbeta2R PIOO3834.79 42 tin serine protease 2 (MASP2). TTYH3 PIOO749.429 43 VAOD1 PIOOO34159 44 LRG: leucine-rich alpha-2-glycoprotein 1 (LRG) is also Vascular adhesion molecule 1 PIOOO18136 45 known in the art as LRG; HMFT1766; LRG1. The leucine Versican PIOOOO98O2 46 45 rich repeat (LRR) family of proteins, including LRG1, has VIP36 PIOOOO99SO 47 been shown to be involved in protein-protein interaction, C-1-acid glycoprotein 2 PIOOO2O091 48 signal transduction, and cell adhesion and development. 3-1,3-galactosyltransferase PIOOO32O34 49 LRG1 is expressed during granulocyte differentiation. The SEQD NO refers to the amino acid sequence encoding the protein biomarker, and are In some embodiments, LRG can be detected in the meth incorporated herein by reference, ods, kits and devices using commercially available assay kits, 50 e.g., from Immuno-Biological Laboratories, Inc., Human In one embodiment, the reference level or reference value LRG Assay Kit, catalog number 27769. LRG can also be is a level of a appendicitis biomarker in a urine sample of a detected using the kits as disclosed in U.S. patent application healthy human not having acute appendicitis, or not having Ser. No. 1 1/627,164 filed Jan. 25, 2007, and provisional been diagnosed with acute appendicitis. A healthy human is patent application 60/761,808 filed Jan. 25, 2006, which are any person who exhibits no symptom which commonly 55 incorporated herein in their entirety by reference. known to be associated with acute appendicitis as described Commercial polyclonal and monoclonal antibodies herein. In another embodiment, the reference value is an against LRG are also useful as protein-binding agents to LRG average level of the appendicitis biomarker in a plurality of and are available from a variety of companies, e.g., but not urine samples from a population of healthy humans not hav limited to Assay Designs, SIGMA-ALDRICH and Novus ing acute appendicitis or not having been diagnosed with 60 Biologicals. acute appendicitis. A population of healthy Subjects that have Antibodies or protein binding agents which recognize and not been diagnosed with acute appendicitis is at least five specifically bind the LRG1 protein of SEQ ID NO: 1, the healthy humans, at least 10 healthy humans, preferably 20 or sequence of which is reproduced below, can be readily pro more healthy humans. The average urine level of an appen duced by one of ordinary skill in the art and are useful for the dicitis biomarker can be obtained by taking the sum of the 65 methods, kits and devices as disclosed herein. SEQID NO: 1 level of an appendicitis biomarker from a number of humans is the polypeptide sequence for LRG (Leucine-rich alpha-2- divided by the number of humans. glycoprotein) and has the amino acid sequence as follows: US 8,535,891 B2 11 12

MSSWSRORPKSPGGIOPHVSRTLFLLLLLAASAWGVTLSPKDCOVFRSDHGSSISCOPPAEIPG

YLPADTWHLAVEFFNLTHLPANLLQGASKLQELHLSSNGLESLSPEFLRPVPOLRVLDLTRNAL

TGLPPGLFOASATLDTLVLKENOLEWLEWSWLHGLKALGHLDLSGNRLRKLPPGLLANFTLLRT

LDLGENOLETLPPDLLRGPLOLERLHLEGNKLOWLGKDLLLPOPDLRYLFLNGNKLARVAAGAF

QGLROLDMLDLSNNSLASVPEGLWASLGOPNWDMRDGFDISGNPWICDONLSDLYRWLOAOKDK

MFSONDTRCAGPEAVKGOTLLAVAKSO

S1 OOA8: In some embodiments, commercial polyclonal and mono S100A8 is also known in the art as synonyms 60B8AG: clonal antibodies against S100A8 are also useful as protein CAGA; CFAG; CGLA; CP-10; L1Ag: MA387: MIF: Migra binding agents to S100A8 and are available from a variety of companies, e.g., but not limited to commercial polyclonal and tion inhibitory factor-related protein 8 (MRP8); NIF; monoclonal antibodies against S100A8 are available from a OTTHUMP00000015329; OTTHUMP00000015330; P8: variety of companies, e.g. Assay Designs, SIGMA-ALD S100 calcium-binding protein A8; S100 calcium-binding RICH, R & D Systems, Novus Biologicals and Santa Cruz protein A8 (calgranulin A); S100A8; calgranulin A; cystic Biotechnology. fibrosis antigen. Antibodies or protein binding agents which recognize and Without wishing to be bound by theory, S100 calcium 20 specifically bind the S100 A8 protein of SEQID NO: 2, the binding protein A8 (S100 A8), also known as migration sequence of which is reproduced below, can be readily pro inhibitory factor-related protein (MRP-8) belongs to the duced by one of ordinary skill in the art and are useful for the S-100 family of calcium binding proteins associated with methods, kits and devices as disclosed herein. myeloid cell differentiation. They are highly expressed in SEQ ID NO: 2 is the polypeptide sequence for S100 A8 and resting neutrophils, keratinocytes (particularly in psoriasis), 25 has the amino acid sequence as follows: in infiltrating tissue macrophages and on epithelial cells in active inflammatory disease. The heterogeneity of macroph age Subpopulations in chronic or acute inflammation is MLTELEKALNSIIDWYHKYSLIKGNFHAVYRDDLKKLLETECPOYIRKK reflected by different expression of MRP8 and migration inhibitory factor-related proteins-14 (MRP14). Phagocytes so GADVWFKELDINTDGAVNFOEFLILVIKMGVAAHKKSHEESHKE expressing MRP8 and MRP14 belong to the early infiltrating ORM: cells, while MRP8 alone is found in chronic inflammatory alpha-1-acid-glycoprotein 1 (ORM) is also known in the tissues. The partially antagonistic functions of MRP8, art as orosomucoid 1, AGP1; AGP-A; ORM1. This gene MRP14 and of the Ca"-dependent MRP8/14 heterocomplex encodes a key acute phase plasma protein. Because of its makes them versatile mediators. increase due to acute inflammation, this protein is classified Human S100A8 (MRP8) has a molecular weight of 11.0 as an acute-phase reactant. The specific function of this pro kD, while human MRP14 exists in a 13.3 kD and a truncated tein has not yet been determined; however, it may be involved 12.9 kD form. Ca2" induces the formation of heterocom in aspects of immunosuppression. plexes of the form (MRP8)(MRP14) (abbreviated MRP8/14), In some embodiments, ORM can be detected in the meth (MRP8)2(MRP14), and (MRP8/14)2. There are two EF-hand ods, kits and devices using commercial assays, such as, but motifs each on MRP8 and MRP14. MRP14 shows a higher 40 without limitation, Human Orosomucoid ELISA Quantita affinity for calcium than MRP8, and the affinity of the C tion Kit from GenWay Biotech, Inc. catalog No. 40-288 terminal EF2 is higher than that of the N-terminal EF1. The 22927F. C-terminal domain also mainly determines the specificity of In some embodiments, commercial polyclonal and mono dimerization. The helix in EF2 undergoes a large conforma clonal antibodies against ORM are also useful as protein tional change upon calcium binding and may play a role as a binding agents to ORM and are available from a variety of trigger for Ca" induced conformational change. 45 companies, e.g., but not limited to Assay Designs, SIGMA In some embodiments, S100A8 can be detected in the ALDRICH, Novus Biologicals, Lifespan Biosciences, R&D methods, kits and devices using commercial assays, such as, Systems, and Santa Cruz, Biotechnology but without limitation, S100A8 assay kits from R & D Sys Antibodies or protein binding agents which recognize and tems’s Human MIF QUANTIKINE(R) ELISA KitCatalog specifically bind the ORM protein of SEQ ID NO: 3, the number: DMF00; and BMA Biomedicals, MRP8 Enzyme 50 sequence of which is reproduced below, can be readily pro Immunoassay Product Code: S-1007. S100A8 can also be duced by one of ordinary skill in the art and are useful for the detected using the kits as disclosed in U.S. Pat. No. 7,501.256 methods, kits and devices as disclosed herein. and WO/2006/012588 which is incorporated herein in its SEQID NO:3 is the polypeptide sequence for ORM and has entirety by reference. the amino acid sequence as follows:

MALSWVLTVLSLLPLLEAOIPLCANLVPVPITNATLDOITGKWFYIASAFRNEEYNKSVOEIQA

TFFYFTPNKTEDTIFLREYOTRODOCIYNTTYLNVORENGTISRYWGGOEHFAHLLILRDTKTY

MLAFDVNDEKNWGLSWYADKPETTKEOLGEFYEALDCLRIPKSDVVYTDWKKDKCEPLEKOHEK

ERKOEEGES US 8,535,891 B2 13 14 Plasminogen (PLG): MASP2: Plasminogen, is also known in the art as PLG or DKFZp779M0222 and is a circulating Zymogen that is con mannan-binding lectin serine peptidase 2 (MASP2) is also Verted to the active enzyme plasmin by cleavage of the pep known in the art as aliases sMAP: MAP19; MASP-2: MASP2 tide bond between arg560 and valS61, which is mediated by and is a Ra-reactive factor (RARF) which is a complement urokinase (PLAU; MIM 191840) and tissue plasminogen dependent bactericidal factor that binds to the Ra and R2 activator (PLAT; MIM 173370). The main function of plas minis to dissolve fibrin (see, e.g., FGA, MIM 134820) clots. polysaccharides expressed by certain enterobacteria. Alter Plasmin, like trypsin, belongs to the family of serine protein nate splicing of this gene results in two transcript variants a SCS. encoding two RARF components that are involved in the In some embodiments, PLG can be detected in the meth 10 ods, kits and devices using commercial assays, such as, but mannan-binding lectin pathway of complement activation. without limitation, commercial assay kits from Human Plas The longer isoform is cleaved into two chains which form a minogen ELISA Kit from Alpco Diagnostics 41-PLAHU heterodimer linked by a disulfide bond. The encoded proteins E01; Human Plasminogen ELISA Kit from AMERICAN are members of the trypsin family of peptidases. DIAGNOSTICA, 640; Plasminogen Colorimetric Assay Kit 15 from AMERICAN DIAGNOSTICA, 851; Human Plasmino In some embodiments, MASP2 can be detected in the gen total antigen ELISA Assay Kit from Innovative Research, methods, kits and devices using commercial assays, such as, 1HPLGKT TOT. PLG can also be detected using the kits as but without limitation, commercial assay kits such as Human disclosed in International Patent Application WO/1991/ MASP-2 ELISA Kit from Cell Sciences, HK326. MASP2 005257 and European Patent Application EP 1990914430 can also be detected using the kits as disclosed in Interna which is incorporated herein in its entirety by reference. tional Patent Application WO/2007/028795 which is incor In some embodiments, commercial polyclonal and mono porated herein in its entirety by reference. clonal antibodies against PLG are also useful as protein In some embodiments, commercial polyclonal and mono binding agents to PLG and are available from a variety of clonal antibodies against MASP2 are also useful as protein companies, e.g., but not limited to Rockland, Abcam, Assay binding agents to MASP2 and are available from a variety of Designs, EMD Biosciences, SIGMA-ALDRICH, Novus 25 Biologicals, Lifespan BioSciences, R&D Systems, and Santa companies, e.g., but not limited to Cell Sciences, USBIO, and Cruz, Biotechnology. Santa Cruz, Biotechnology. Antibodies or protein binding agents which recognize and Antibodies or protein binding agents which recognize and specifically bind the PLG protein of SEQ ID NO: 4, the specifically bind the MASP2 protein of SEQ ID NO: 5, the sequence of which is reproduced below, can be readily pro 30 sequence of which is reproduced below, can be readily pro duced by one of ordinary skill in the art and are useful for the duced by one of ordinary skill in the art and are useful for the methods, kits and devices as disclosed herein. methods, kits and devices as disclosed herein. SEQID NO. 4 is the polypeptide sequence for PLG and has SEQ ID NO: 5 is the polypeptide sequence for MASP5 and the amino acid sequence as follows: has the amino acid sequence as follows:

MEHKEWWLLLLLFLKSGOGEPLDDYVNTOGASLFSVTKKOLGAGSIEECAAKCEEDEEFTCRAF

OYHSKEQQCVIMAENRKSSIIIRMRDVWLFEKKWYLSECKTGNGKNYRGTMSKTKNGITCOKWS

STSPHRPRFSPATHPSEGLEENYCRNPDNDPOGPWCYTTDPEKRYDYCDILECEEECMHCSGEN

YDGKISKTMSGLECOAWDSOSPHAHGYIPSKFPNKNLKKNYCRNPDRELRPWCFTTDPNKRWEL

CDIPRCTTPPPSSGPTYOCLKGTGENYRGNVAVTVSGHTCOHWSAOTPHTHNRTPENFPCKNLD

ENYCRNPDGKRAPWCHTTNSOVRWEYCKIPSCDSSPVSTEOLAPTAPPELTPVVODCYHGDGOS

YRGTSSTTTTGKKCOSWSSMTPHRHOKTPENYPNAGLTMNYCRNPDADKGPWCFTTDPSVRWEY

CNLKKCSGTEASWWAPPPVVLLPDVETPSEEDCMFGNGKGYRGKRATTWTGTPCODWAAOEPHR

HSIFTPETNPRAGLEKNYCRNPDGDWGGPWCYTTNPRKLYDYCDVPOCAAPSFDCGKPOVEPKK

CPGRVVGGCVAHPHSWPWOWSLRTRFGMHFCGGTLISPEWVLTAAHCLEKSPRPSSYKVILGAH

OEVNLEPHVOEIEVSRLFLEPTRKDIALLKLSSPAVITDKVIPACLPSPNYWVADRTECFITGW

GETOGTFGAGLLKEAOLPVIENKVCNRYEFLNGRVOSTELCAGHLAGGTDSCOGDSGGPLVCFE US 8,535,891 B2 15 16

MRLLTLLGLLCGSWATPLGPKWPEPWFGRLASPGFPGEYANDOERRWTLTAPPGYRLRLYFTHF

DLELSHLCEYDFWKLSSGAKVLATLCGOESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFT

GFEAFYAAEDIDECOWAPGEAPTCDHHCHNHLGGFYCSCRAGYWLHRNKRTCSEOSL

AZGP1: (ZA2G, ZAG) ELISA Kit, HRP Detection, from BioVendor alpha-2-glycoprotein 1 (AZGP1) is also known in the art as Laboratory Medicine, Inc., RD191093100R, The assay is aliases zinc-alpha-2-glycoprotein (ZAG); ZA2G, AZGP1, intended for the determination of human Zinc-alpha-2-gly Azgp1, ZNGP1 and lipid-Mobilizing Factor (LMF). AZGP1 10 coprotein in serum, plasma, cerebrospinal fluid, urine and cell is a soluble 41 kDa glycoprotein belonging to the immuno lysate; Human/Mouse/Rat ZAG EIA Kit from Raybiotech, globuline protein family and consisting of a single polypep Inc or Biovendor lab medicine Inc., EIA-ZAG-1. tide chain. Human ZAG shares 59% sequence identity with In some embodiments, commercial polyclonal and mono the murine homolog. AZGP1 is closely related to antigens of clonal antibodies against AZGP1 are also useful as protein the class 1 major histocompatibility complex (MHC I) and 15 binding agents to AZGP1 and are available from a variety of shares 30-40% sequence identity with the heavy chain of companies, e.g., but not limited to Abcam (Zinc Alpha 2 MHC I. Most MHC-I members heterodimerize with beta-2- Glycoprotein antibody, catalog hab47116) and Novus Bio microglobuline (b2m) and bind peptides derived from intra logicals (AZGP1 Antibody, catalog #H00000563-B01). Cell cellular proteins to present them to cytotoxic T cells. In con Sciences, USBIO, and Santa Cruz, Biotechnology. trast, AZGP1 is a soluble protein rather than being anchored to plasma membranes that acts independently on b2m and Antibodies or protein binding agents which recognize and binds the hydrophobic ligand which may relate to its function specifically bind the AZGP1 protein of SEQ ID NO: 6, the in lipid metabolism. sequence of which is reproduced below, can be readily pro AZGP1 is widespread in body fluids and is also found in duced by one of ordinary skill in the art and are useful for the various human tissues such as adipose tissue, prostate, breast, 25 methods, kits and devices as disclosed herein. skin, salivary gland, trachea, broncheus, lung, gastrointesti SEQID NO: 6 is the polypeptide sequence for AZGP1 and nal tract, pancreas, liver and kidney. AZGP1 acts as a lipid has the amino acid sequence as follows:

MVRMVPWLLSLLLLLGPAVPOENODGRYSLTYIYTGLSKHVEDVPAFOALGSLNDLOFFRYNSK

DRKSOPMGLWROVEGMEDWKODSOLOKAREDIFMETLKDIVEYYNDSNGSHVLOGRFGCEIENN

RSSGAFWKYYYDGKDYIEFNKEIPAWWPFDPAAQITKOKWEAEPVYWORAKAYLEEECPATLRK

YLKYSKNILDRODPPSVVVTSHOAPGEKKKLKCLAYDFYPGKIDVHWTRAGEVOEPELRGDVLH

NGNGTYOSWVVVAVPPODTAPYSCHVOHSSLAOPLVWPWEAS mobilizing factor to induce lipolysis in adipocytes and plays APOD: Apollipoprotein D (Apol) or APOD) is a polypep an important role in lipid utilization and loss of adipose tissue, tide which is a high density lipoprotein that has no marked especially during cachexia, which occurs in patient Suffering 40 similarity to other apolipoprotein sequences. It has a high from cancer, AIDS and other chronic illnesses. The role of degree of homology to plasma retinol-binding protein and AZGP1 in cancer cachexia is also connected with its ability to other members of the alpha 2 microglobulin protein Super directly influence expression of uncoupling proteins (UCPs) family of carrier proteins, also known as lipocalins. This which are implicated in the regulation of energy balance. In glycoprotein is closely associated with the enzyme lecithin: human adipocytes, AZGP1 expression is regulated particu cholesterol acyltransferase—an enzyme involved in lipopro larly through TNF-alpha and the PPARgamma nuclear recep 45 tein metabolism. tor. AZGP1 expression is also upregulated by glucocorti In some embodiments, Apol) can be detected in the meth coides and attenuated by eicosapentaenoic acid (EPA) and ods, kits and devices using as disclosed in International Patent beta-3-adrenoreceptor antagonists. Application WO/1996/019500 or U.S. Pat. No. 5,804,368 or AZGP1 is overexpressed in certain human malignant 5,804,368 or European Patent EP0301667 which are incor tumors such as prostate, breast, lung or bladder cancer and 50 porated herein in their entirety by reference. can relate to tumor differentiation. Additionally, AZGP1 Antibodies or protein binding agents which recognize and plays a role in obesity, diabetic kidney disorders, frontotem specifically bind the ApoD protein of SEQ ID NO: 7, the poral dementia and regulation of melanin production by mel sequence of which is reproduced below, can be readily pro anocytes. AZGP1 is proposed to have a therapeutic use in duced by one of ordinary skill in the art and are useful for the obesity and cachexia. It can be used as a marker for clinical 55 methods, kits and devices as disclosed herein. analysis of diabetic nephropathy and as a marker for certain SEQID NO: 7 is the polypeptide sequence for Apol) and has tumors. the amino acid sequence as follows:

MVMLLLLLSALAGLFGAAEGOAFHLGKCPNPPVOENFDVNKYLGRWYEIEKIPTTFENGRCIOA

NYSLMENGKIKVLNOELRADGTVNOIEGEATPVNLTEPAKLEVKFSWFMPSAPYWILATDYENY

ALWYSCTCIIOLFHVDFAWILARNPNLPPETVDSLKNILTSNNIDWKKMTVTDOWNCPKLS

In some embodiments, AZGP1 can be detected in the meth- 65 SERPINA3: ods, kits and devices using commercial assays, such as, but C-1-antichymotrypsin (SERPINA3) is also known in the without limitation, Human Zinc-Alpha-2-Glycoprotein art as aliases serpin peptidase inhibitor, Glade A (alpha-1 US 8,535,891 B2 17 18 antiproteinase, antitrypsin), member 3, ACT; AACT: GIG24; protein, and can be for example, a synthetic peptide, chemi GIG25 and MGC88254. The SERPINA3 polypeptide is a cal, Small molecule, or antibody or antibody fragment or plasma protease inhibitor and member of the serine protease variants thereof. In some embodiments, a protein-binding inhibitor class. Polymorphisms in this protein appear to be agent is a ligand or antibody or antibody fragment, and in tissue specific and influence protease targeting. Variations in Some embodiments, a protein-binding agent is preferably this protein's sequence have been implicated in Alzheimer's detectably labeled. disease, and deficiency of this protein has been associated In one embodiment of the invention, immunoassays using with liver disease. Mutations have been identified in patients antibodies are used to measure the levels of biomarker pro with Parkinson disease and chronic obstructive pulmonary teins in urine. As used herein, the term “antibody' is intended disease. to include immunoglobulin molecules and immunologically In some embodiments, SERPINA3 can be detected in the 10 active determinants of immunoglobulin molecules, e.g., mol methods, kits and devices using as disclosed in International ecules that contain an antigen binding site which specifically Patent Application WO/2005/0395.88 which is incorporated binds (immunoreacts with) to the appendicitis biomarker to herein in its entirety by reference. be measured. The term “antibody' is intended to include In some embodiments, commercial polyclonal and mono whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, clonal antibodies against SERPINA3 are also useful as pro 15 etc), and includes fragments thereof which are also specifi tein-binding agents to SERPINA3 and are available from a cally reactive with the appendicitis biomarker proteins to be variety of companies, e.g., but not limited to Proteintech measured, e.g. leucine C-2 glycoprotein (LRG), calgranulin Group, Lifespan BioSciences, and Santa Cruz, Biotechnology. A (S100-A8), C.-1.-acid glycoprotein 1 (ORM), plasminogen Antibodies or protein binding agents which recognize and (PLG), mannan-binding lectin serine protease 2 (MASP2), specifically bind the SERPINA3 protein of SEQID NO: 8, the Zinc-C-2-glycoprotein (AZGP1), C-1-antichymotrypsin sequence of which is reproduced below, can be readily pro (SERPINA3) or apolipoprotein D (Apol)). Antibodies can be duced by one of ordinary skill in the art and are useful for the fragmented using conventional techniques. Thus, the term methods, kits and devices as disclosed herein. “antibody' includes segments of proteolytically-cleaved or SEQ ID NO: 8 is the polypeptide sequence for SERPINA3 recombinantly-prepared portions of an antibody molecule and has the amino acid sequence as follows: that are capable of selectively reacting with a certain protein.

MKIHYSROTALESTSYIQLPEAELRMERMLPLLALGLLAAGFCPAVLCHPNSPLDEENLTOENO

DRGTHVDLGLASANVDFAFSLYKOLVLKAPDKNWIFSPLSISTALAFLSLGAHNTTLTEILKGL

KFNLTETSEAEIHOSFOHLLRTLNQSSDELOLSMGNAMFWKEOLSLLDRFTEDAKRLYGSEAFA

TDFODSAAAKKLINDYWKNGTRGKITDLIKDLDSOTMMVLVNYIFFKAKWEMPFDPODTHOSRF

YLSKKKWVMVPMMSLHHLTIPYFRDEELSCTV WELKYTGNASALFILPDODKMEEVEAMLLPET

LKRWRDSLEFREIGELYLPKFSISRDYNLNDILLOLGIEEAFTSKADLSGITGARNLAVSOVVH

KAVLDVFEEGTEASAATAVKITLLSALVETRTIVRFNRPFLMIIWPTDTONIFFMSKVTNPKOA

Measuring Levels of Appendicitis Biomarker Proteins Non limiting examples of Such proteolytic and/or recombi In embodiments of the invention, the level of appendicitis 40 nant fragments include Fab, F(ab')2, Fab'. Fv, dAbs and single biomarker proteins, such as those disclosed in Table 1, and in chain antibodies (scFv) containing a VL and VH domain particular, the following appendicitis biomarker: leucine C-2 joined by a peptide linker. The scFv's can be covalently or glycoprotein (LRG), calgranulin A (S100-A8), C-1-acid gly non-covalently linked to form antibodies having two or more coprotein 1 (ORM), plasminogen (PLG), mannan-binding binding sites. Thus, “antibody' includes polyclonal, mono lectin serine protease 2 (MASP2), zinc-C-2-glycoprotein clonal, or other purified preparations of antibodies and (AZGP1), C-1-antichymotrypsin (SERPINA3) or apolipo 45 recombinant antibodies. The term “antibody' is further protein D (Apod), is measured to obtain a determination of intended to include humanized antibodies, bispecificantibod whether a human patient has acute appendicitis. A urinary ies, and chimeric molecules having at least one antigenbind biomarker protein level can be measured using any assay ing determinant derived from an antibody molecule. In one known to those of ordinary skilled in the art, including, but not embodiment, the antibody is detectably labeled. limited to, Enzyme-LinkedImmunosorbent Assay (ELISA), 50 Antibodies to the appendicitis biomarker proteins can be immunoprecipitation assays, radioimmunoassay, mass spec generated using methods known to those skilled in the art. trometry, Western Blotting, and via dipsticks using conven Alternatively, commercially available antibodies can be used. tional technology. Antibodies to LRG, S100-A8, ORM1, PLG, MASP2, For purposes of comparison, the levels of an appendicitis AZGP1, Apod and SERPINA3 are commercially available. biomarker protein in a urine sample from the patient should 55 As used herein “detectably labeled, includes antibodies be measured in the same manner as the reference value is that are labeled by a measurable means and include, but are measured. For example, the levels of appendicitis biomarker not limited to, antibodies that are enzymatically, radioac proteins can be represented in arbitrary units dependent upon tively, fluorescently, and chemiluminescently labeled. Anti the assay used to measure the levels of appendicitis biomarker bodies can also be labeled with a detectable tag, such as proteins, e.g., the intensity of the signal from the detectable c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. label can correspond to the amount of appendicitis biomarker 60 In the diagnostic methods of the invention that use an proteins present (e.g. as determined by eye, densitometry, an antibody for the detection of biomarker proteins levels, the ELISA plate reader, a luminometer, or a scintillation level of biomarker proteins present in the urine samples cor counter). relates to the intensity of the signal emitted from the detect The levels of an appendicitis biomarker protein present in ably labeled antibody. a urine sample can be determined using any protein-binding 65 In one embodiment, the antibody is detectably labeled by agent. In some embodiments, a protein-binding agent is a linking the antibody to an enzyme. The enzyme, in turn, when ligand that specifically binds to an appendicitis biomarker exposed to its substrate, will react with the substrate in such US 8,535,891 B2 19 20 a manner as to produce a chemical moiety which can be biomarker in the urine. Thus, this defined amount of antibody detected, for example, by spectrophotometric, fluorometric, is used to indicate whether the amount of antigen in the or by visual means. Enzymes which can be used to detectably biological sample is at least at, below or above the reference label the antibodies of the present invention include, but are level of biomarker (See FIGS. 11-12). not limited to, malate dehydrogenase, staphylococcal Detecting the quantity of antigen in the biological sample nuclease, delta-V-steroid isomerase, yeast alcohol dehydro can be achieved by a variety of methods. One of the most genase, alpha-glycerophosphate dehydrogenase, triose phos common is to label either the antigen or the antibody. The phate isomerase, horseradish peroxidase, alkaline phos label can consist of an enzyme (see enzyme immunoassay phatase, asparaginase, glucose oxidase, beta-galactosidase, (EIA)), colloidal gold (lateral flow assays), radioisotopes ribonuclease, urease, catalase, glucose-VI-phosphate dehy such as I-' Radioimmunoassay (RIA), magnetic labels drogenase, glucoamylase and acetylcholinesterase. Chemilu 10 (magnetic immunoassay—MIA) or fluorescence. Other tech minescence is another method that can be used to detect an niques include agglutination, nephelometry, turbidimetry and antibody. Western Blot. Detection can also be accomplished using any of a variety In one embodiment, the immunoassay is a competitive of other immunoassays. For example, by radioactively label immunoassay. In another embodiment, the immunoassay is a ing an antibody, it is possible to detect the antibody through 15 noncompetitive immunoassay. the use of radioimmune assays. The radioactive isotope can Immunoassays can be divided into those that involve be detected by Such means as the use of a gamma counter or labeled reagents and those which involve non-labeled a Scintillation counter or by audoradiography. Isotopes which reagents. Those which involve labeled reagents are divided a articularly useful for the purpose of the present invention into homogenous and heterogeneous (which require an extra are H, I, S, 'C, and preferably 'I. step to remove unbound antibody or antigen from the site, It is also possible to label an antibody with a fluorescent usually using a solid phase reagent) immunoassays. Hetero compound. When the fluorescently labeled antibody is geneous immunoassays can be competitive or non-competi exposed to light of the proper wave length, its presence can tive. then be detected due to fluorescence. Among the most com In a competitive immunoassay, the antigen in the unknown monly used fluorescent labeling compounds are CYE dyes, sample competes with labeled antigen to bind with antibod fluorescein isothiocyanate, rhodamine, phycoerytherin, phy 25 ies. The amount of labeled antigenbound to the antibody site cocyanin, allophycocyanin, o-phthaldehyde and fluorescam is then measured. In this method, the response will be 1C. inversely proportional to the concentration of antigen in the An antibody can also be detectably labeled using fluores unknown. This is because the greater the response, the less cence emitting metals such as "Eu, or others of the lan antigen in the unknown was available to compete with the thanide series. These metals can be attached to the antibody 30 labeled antigen. using such metal chelating groups as diethylenetriaminepen In noncompetitive immunoassays, also referred to as the taacetic acid (DTPA) or ethylenediaminetetraacetic acid 'sandwich assay, antigen in the unknown, e.g. urine sample, (EDTA). is bound to a first antibody site, then second antibody that is An antibody also can be detectably labeled by coupling it to labeled is bound to the antigen, forming a sandwich. The a chemiluminescent compound. The presence of the chemi amount of labeled antibody on the site is then measured. luminescent-antibody is then determined by detecting the 35 Unlike the competitive method, the results of the noncom presence of luminescence that arises during the course of a petitive method will be directly proportional to the concen chemical reaction. Examples of particularly useful chemilu tration of the antigen. This is because labeled antibody will minescent labeling compounds are luminol, luciferin, isolu not bind if the antigen is not present in the unknown sample, minol, theromatic acridinium ester, imidazole, acridinium e.g. urine sample. salt and oxalate ester. 40 In one embodiment, the levels of biomarker proteins in In one embodiment, the levels of biomarker proteins in urine are detected by ELISA assay. There are different forms urine are detected by an immunoassay. Immunoassays ofELISA which are well known to those skilled in the art, e.g. include but are not limited to enzyme immunoassay (EIA), standard ELISA, competitive ELISA, and sandwich ELISA. also called enzyme-linked immunosorbant assay (ELISA), The standard techniques for ELISA are described in “Meth radioimmunoassay (RIA), diffusion immunoassay (DIA), ods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, fluoroimmunoassay (FIA), chemiluminescent immunoassay 45 eds. John Wiley & Sons, 1980; Campbellet al., “Methods and (CLIA), counting immunoassay (CIA), lateral flow tests or Immunology”. W. A. Benjamin, Inc., 1964; and Oellerich, M. immunoassay (LFIA), also known as lateral flow immuno 1984, J. Clin. Chem. Clin. Biochem., 22:895-904. chromatographic assays, and magnetic immunoassay (MIA). Enzyme-linked immunosorbent assay, also called ELISA, An immunoassay is a biochemical test that measures the enzyme immunoAssay or EIA, is a biochemical technique concentration of a Substance in a biological sample, typically 50 used mainly in immunology to detect the presence of an serum or urine, using the reaction of an antibody orantibodies antibody oran antigen in a sample. The ELISA has been used to its antigen. The assay takes advantage of the specific bind as a diagnostic tool in medicine and plant pathology, as well ing of an antibody to its antigen. Monoclonal antibodies are as a quality control check in various industries. For the meth often used as they only usually bind to one site of a particular ods described herein, in the ELISA a known amount of anti molecule, and therefore provide a more specific and accurate 55 biomarker antibody is affixed to a solid surface, and then the test, which is less easily confused by the presence of other sample, e.g. urine, containing the biomarker of interest is molecules. The antibodies picked must have a high affinity for washed over the Surface so that the antigen biomarker can the antigen (if there is antigen available, a very high propor bind to the immobilized antibodies (a first antibody). The tion of it must bind to the antibody). Surface is washed to remove any unbound biomarker and also For numerical results, the response of the biological any non-biomarker proteins present in the urine sample. A sample being measured must be compared to standards of a 60 detection antibody (a second antibody) is applied to the Sur known concentration. This is usually done through the plot face. The detection antibody is specific to antibodies from the ting of a standard curve on a graph, the position of the curve Subject. For example, if the Subject is a human, the detection at response of the unknown is then examined, and so the antibody should be an anti-human IgG antibody. If the subject quantity of the unknown found. Alternatively, a defined is a dog, the detection antibody then should an anti-dog IgG amount of antibody is used in the assay where the defined 65 antibody. This detection antibody can be linked to an enzyme, amount of antibody binds completely to a fixed amount of and in the final step a substance is added that the enzyme can antigen. This fixed amount of antigen is the reference level of convert to Some detectable signal. For example, in the case of US 8,535,891 B2 21 22 fluorescence ELISA, when light is shone upon the sample, be measured under conditions permitting formation of a com any antigen/antibody complexes will fluoresce so that the plex between the antibody and the appendicitis biomarker amount of antibodies in the sample can be measured. proteins (e.g. LRG, S100-A8, ORM1, PLG, MASP2, The following is a general standard protocol for setting up AZGP1, ApoD and SERPINA3). The amount of complex and performing an indirect enzyme-linked immunosorbent formed is then measured as a measure of the level of the assay. Using 96-well microtiter plates (Falcon Pro-Bindassay appendicitis biomarker protein, and the amount of complex plate 3915; Becton Dickinson, Paramus, N.J.), test wells are formed is compared to the amount of complex formed coated with anti-biomarker antibody by incubation with 100 between the first antibody and a predetermined reference ul of purified anti-LRG antibody (3 ug/ml in PBS) per well amount of the appendicitis biomarker protein. This predeter overnight at room temperature, with PBS substituted for the mined reference level amount of the appendicitis biomarker antibody in control wells. After the plates have been washed 10 protein is the amount found in the urine of healthy humans. A three times with PBS-Tween, 250 ul of 2% BSA in PBS is level above the reference level amount of an appendicitis added to each well, and the plates are incubated for 1 h at biomarker protein indicates that the human has acute appen room temperature. The plates are washed three times with dicitis. PBS-Tween and incubated for 1 h at room temperature with In one embodiment, the firstantibody is detectably labeled. test urine sample and control urine sample from healthy indi 15 Detectably labeling the first antibody is appropriate for use, viduals diluted 1:100 in PBS-Tween-BSA; each urine sample for example, in standard ELISA assays where biomarker is tested in triplicate in anti-LRG antibody-coated wells as protein is absorbed to an ELISA plate, or in Western Blot well as in PBS control wells. The plate is then assayed (with analysis, or certain LFIA dipstick analyses. appropriate controls) for the presence and/or the level of LRG In one embodiment, the first antibody is immobilized on a by incubation for 1 hat room temperature with 100 ul of goat solid support, for example, when using a “Sandwich ELISA anti-LRG IgG conjugated with horseradish peroxidase (Bio ora dipstickanalysis, then the amount of complex formed can Rad, Richmond, Calif.) per well diluted 1:2,000 in PBS measured by detecting binding of a second antibody that Tween-BSA. After three washes in PBS-Tween, the substrate specifically binds to the appendicitis biomarker protein (e.g. Solution (o-phenylenediamine dihydrochloride: Sigma) is LRG, S100-A8, ORM1, PLG, MASP2, AZGP1, ApolD and added to each well. The plates are then incubated for 30 min SERPINA3) under conditions permitting formation of a com at room temperature in darkness, and the reaction is termi 25 plex between the second antibody and the appendicitis biom nated by the addition of 2N sulfuric acid. The optical density arker protein, wherein the second antibody does not substan values at 490 nm (OD490) are measured in a micro plate tially cross-react with the first antibody, and wherein the ELISA reader. For each urine sample, mean OD490 readings second antibody is detectably labeled. are calculated for the test wells and for the antigen control Any Solid Support can be used, including but not limited to, wells, the latter being subtracted from the former to obtain the 30 nitrocellulose, Solid organic polymers, such as polystyrene, net ELISA value. or laminated dipsticks such as described in U.S. Pat. Nos. Performing an ELISA involves at least one antibody with 5,550,375 and 5,656,448, which is specifically incorporated specificity for a particular biomarker. A known amount of herein by reference in their entirety. anti-biomarker antibody is immobilized on a solid Support In one embodiment, the levels of two appendicitis biomar (usually a polystyrene microtiter plate) either non-specifi ker proteins defining a first and a second appendicitis biom cally (via adsorption to the Surface) or specifically (via cap 35 arker protein, are measured using at least two antibodies ture by another antibody specific to the anti-biomarker anti specific to each appendicitis biomarker protein to be mea body, in a “sandwich ELISA). After the antigen is sured. Each antibody specifically reacts either the first appen immobilized, the detection antibody is added, forming a com dicitis biomarker protein or the second appendicitis biomar plex with the antigen. The detection antibody can be ker protein to be measured while not substantially cross covalently linked to an enzyme, or can itself be detected by a 40 reacting with the other appendicitis biomarker proteins to be secondary antibody which is linked to an enzyme through measured. bio-conjugation. Between each step the plate is typically In one embodiment, the levels of three biomarker proteins washed with a mild detergent solution to remove any proteins defining a first biomarker protein, a second biomarker pro or antibodies that are not specifically bound. After the final tein, and a third biomarker protein, are measured using at least wash step the plate is developed by adding an enzymatic three antibodies specific to each biomarker protein to be Substrate to produce a visible signal, which indicates the 45 measured, wherein each antibody specifically reacts either quantity of antigen in the sample. Older ELISAs utilize chro the first biomarker protein, the second biomarker protein, or mogenic Substrates, though newer assays employ fluorogenic the third biomarker protein to be measured while not substan Substrates with much higher sensitivity. tially cross-reacting with the other biomarker proteins to be In another embodiment, a competitive ELISA is used. Puri measured. fied anti-biomarker antibody is coated on the solid phase of 50 In one embodiment, the levels of four biomarker proteins multi-wells. Urine sample, a defined amount of purified defining a first, a second, a third and a fourth biomarker biomarker and horseradish peroxidase labeled with anti protein, are measured using at least four antibodies specific to biomarker antibody (secondary detection conjugated anti each biomarker protein to be measured, wherein each anti body) are added to coated wells to form competitive combi body specifically reacts either the first biomarker protein, the nation. After incubation, if the biomarker level in the urine 55 second biomarker protein, the third biomarker protein, or the sample is high, a complex of biomarker-anti-biomarker anti fourth biomarker protein to be measured while not substan body-anti-biomarker antibody labeled with HRP will form. tially cross-reacting with the other biomarker proteins to be Washing the wells will remove the complex. Incubation with measured. TMB (3,3',5,5'-tetramethylbenzidene) will result in color In one embodiment, the appendicitis biomarker proteins development substrate for the localization of horseradish per are selected from the group consisting of LRG, S100-A8, oxidase-conjugated antibodies in the wells. There will be no 60 ORM1, PLG, MASP2, AZGP1, ApolD and SERPINA3. color change or little color change. If the biomarker level in In one embodiment, the levels of biomarker proteins in the urine sample is low, there will be much color change. Such urine are detected by a lateral flow immunoassay test (LFIA), a competitive ELSA test is specific, sensitive, reproducible also known as the immunochromatographic assay, or strip and easy to operate. test. LFIAS are a simple device intended to detect the presence In one embodiment, the levels of appendicitis biomarker 65 (or absence) of a target antigen in a fluid sample. There are proteins are determined by contacting a urine sample with a currently many LFIA tests are used for medical diagnostics first antibody that specifically binds to a biomarker protein to either for home testing, point of care testing, or laboratory US 8,535,891 B2 23 24 use. LFIA tests are a form of immunoassay in which the test The use of "dip sticks' or LFIA test strips and other solid sample flows along a solid Substrate via capillary action. After supports have been described in the art in the context of an the sample is applied to the test it encounters a coloured immunoassay for a number of antigens. U.S. Pat. Nos. 4,943, reagent which mixes with the sample and transits the Sub 522; 6,485,982; 6,187,598; 5,770.460;5,622,871; 6,565,808, strate encountering lines or Zones which have been pretreated U.S. patent application Ser. No. 10/278,676; U.S. Ser. No. with an antibody or antigen. Depending upon the antigens 09/579,673 and U.S. Ser. No. 10/717,082, which are incor present in the sample the coloured reagent can become bound porated herein by reference in their entirety, are non-limiting at the test line or Zone. LFIAS are essentially immunoassays examples of such lateral flow test devices. Three U.S. patents adapted to operate along a single axis to Suit the test Strip (U.S. Pat. No. 4,444,880, issued to H. Tom; U.S. Pat. No. 4,305,924, issued to R. N. Piasio; and U.S. Pat. No. 4,135, format or a dipstick format. Strip tests are extremely versatile 10 884, issued to J. T. Shen) describe the use of "dip stick” and can be easily modified by one skilled in the art for detect technology to detect soluble antigens via immunochemical ing an enormous range of antigens from fluid samples Such as assays. The apparatuses and methods of these three patents urine, blood, water samples etc. Strip tests are also known as broadly describe a first component fixed to a solid surface on dip Stick test, the name bearing from the literal action of a "dip Stick” which is exposed to a solution containing a "dipping the test strip into a fluid sample to be tested. LFIA 15 soluble antigen that binds to the component fixed upon the strip test are easy to use, require minimum training and can "dip Stick, prior to detection of the component-antigen com easily be included as components of point-of-care test plex upon the stick. (POCT) diagnostics to be use on site in the field. A urine dipstick is a colorimetric chemical assay that can LFIA tests can be operated as either competitive or sand be used to determine the pH, specific gravity, protein, glu wich assays. Sandwich LFIAS are similar to sandwich cose, ketone, bilirubin, urobilinogen, blood, leukocyte, and ELISA. The sample first encounters coloured particles which nitrite levels of an individual’s urine. It consists of a reagent are labeled with antibodies raised to the target antigen. The Stick-pad, which is immersed in a fresh urine specimen and test line will also contain antibodies to the same target, then withdrawn. After predetermined times the colors of the although it may bind to a different epitope on the antigen. The reagent pad are compared to standardized reference charts. test line will show as a coloured band in positive samples. The urine dipstick offers an inexpensive and fast method to Example 5 illustrates a sandwich LFIA in the test strip format. 25 perform screening urinalyses, which help in identifying the Competitive LFIAS are similar to competitive ELISA. The presence of various diseases or health problems. A urine sample first encounters coloured particles which are labeled dipstick provides a simple and clear diagnostic guideline and with the target antigen or an analogue. The test line contains can be used in the methods and kits as described herein. antibodies to the target/its analogue. Unlabelled antigen in the Accordingly, one aspect of the presents invention relates to a sample will block the binding sites on the antibodies prevent 30 method for detecting acute appendicitis using a device. Such ing uptake of the coloured particles. The test line will show as as a dipstick, to test for the presence of appendicitis biomar a coloured band in negative samples. kers as described herein. Dipsticks useful in the present A typical test strip consists of the following components: invention can be used to test for at least one appendicitis (1) sample application area comprising an absorbent pad (i.e. biomarker, for example LRG or multiple biomarkers, such as the matrix or material) onto which the test sample is applied; any combination selected from the group of leucine-rich C-2- (2) conjugate or reagent pad—this contains antibodies spe 35 glycoprotein (LRG); S100-A8 (calgranulin); C-1-acid glyco cific to the target antigen conjugated to coloured particles protein 1 (ORM); plasminogen (PLG); mannan-binding lec (usually colloidal gold particles, or latex microspheres); test tin serine protease 2 (MASP2); zinc-C-2-glycoprotein results area comprising a reaction membrane—typically a (AZGP1); apolipoprotein D (Apod); C-1-antichymotrypsin hydrophobic nitrocellulose or cellulose acetate membrane (SERPINA3), or alternatively, multiple biomarkers selected onto which anti-antigen antibodies are immobilized in a line 40 from any combination listed in Table 1. Combination dip across the membrane as a capture Zone or test line (a control Sticks can be used to test for at least two appendicitis biom Zone may also be present, containing antibodies specific for arkers selected from the group of leucine-rich C-2-glycopro the conjugate antibodies); and (4) optional wick or waste tein (LRG); S100-A8 (calgranulin); C-1-acid glycoprotein 1 reservoir—a further absorbent pad designed to draw the (ORM); plasminogen (PLG); mannan-binding lectin serine sample across the reaction membrane by capillary action and protease 2 (MASP2); zinc-C-2-glycoprotein (AZGP1); apo collect it. The components of the strip are usually fixed to an 45 lipoprotein D (Apol)); C-1-antichymotrypsin (SERPINA3), inert backing material and may be presented in a simple or alternatively, multiple biomarkers selected from any com dipstick format or within a plastic casing with a sample port bination listed in Table 1. Examples of combinations of two and reaction window showing the capture and control Zones. appendicitis biomarkers are LRG and ORM: LRG and S100 While not strictly necessary, most tests will incorporate a A8; LRG and PLG: LRG and MASP2: LRG and AZGP1; second line which contains an antibody that picks up free 50 LRG and ApolD; LRG and SERPINA3: ORM and S100-A8: latex/gold in order to confirm the test has operated correctly. ORM and PLG; ORM and MASP2; ORM and ApolD; ORM FIGS. 11-19 show the various components and embodiments and SERPINA3; S100-A8 and PLG, S100-A8 and MASP2: of several test strips. S100-A8 and Apol); S100-A8 and SERPINA3; PLG and In some embodiments, the lateral flow immunoassay is a MASP2: PLG and ApolD; PLG and SEPRINA3; MASP2 and double antibody sandwich assay, a competitive assay, a quan 55 ApolD; MASP2 and SERPINA3; and Apo and SERPINA3. titative assay or variations thereof. FIGS. 15, 16, 17. Example Combination dipsticks can be used to test for at least three 5 and Example 6 exemplify double antibody sandwich LFIA appendicitis biomarkers, at least four appendicitis biomark in a test strip format. ers, at least five appendicitis biomarkers, or at least six appen There are a number of variations on lateral flow technol dicitis biomarkers selected from the group of leucine-rich ogy. It is also possible to apply multiple capture Zones to C-2-glycoprotein (LRG); S100-A8 (calgranulin); C-1-acid create a multiplex test. FIGS. 14 and 19 exemplify a multiplex 60 glycoprotein 1 (ORM); plasminogen (PLG); mannan-bind LFIA in a test strip format. In one embodiment, a diagnostic ing lectin serine protease 2 (MASP2); Zinc-C-2-glycoprotein kit can comprise multiple LFIA test strips, one strip for a (AZGP1); apolipoprotein D (Apod); C-1-antichymotrypsin different biomarker protein. In another embodiment, a diag (SERPINA3), or alternatively, multiple biomarkers selected nostic kit can comprise a single composite LFIA test strip for from any combination listed in Table 1. Combination dip determining the levels of several biomarker proteins. Such 65 Sticks can be used to test for at least seven appendicitis biom diagnostic kits and LFIA test strips can be used as POCT in arkers selected from the group of leucine-rich C-2-glycopro the field. tein (LRG); S100-A8 (calgranulin); C-1-acid glycoprotein 1 US 8,535,891 B2 25 26 (ORM); plasminogen (PLG); mannan-binding lectin serine For example, as a general guideline but not as a limitation, protease 2 (MASP2); zinc-C-2-glycoprotein (AZGP1); apo when using a MULTISTIX-2 by Bayer Aktiengesellschaft lipoprotein D (Apolo): C-1-antichymotrypsin (SERPINA3), (Fed. Rep. Germany) two minutes pass between the time that or alternatively, multiple biomarkers selected from any com the device is contacted with the sample and when it is read to bination listed in Table 1. An example of a combination of produce an experimental test result. The MULTISTIX-2 dip seven appendicitis biomarkers is LRG, ORM, S100-A8, stick is sold to test urine. The experimental test result is then PLG, MASP2, ApoD, and SERPINA3. Uses of dipsticks are compared to pre-determined test results that indicate either commonly known in the art, and are described in U.S. Pat. No. the presence or absence of acute appendicitis. 5,972,594 to Heine, which is incorporated herein in its In Some embodiments, the method to diagnose acute entirety by reference which is used to detect the presence of appendicitis in a Subject uses a quantitative device (such as, neutrophil defensins to diagnose reproductive tract inflam 10 for example, the MULTISTIX-2, MULTISTIX-10, URIS mation and preeclampsia. TIX-4, or any appendicitis biomarker-detecting device as Other dipsticks and related components are well known in disclosed herein) or the subject inventive device that has two the art, for example dipsticks to detect leukocytes and leuko indications, one for a positive result and one for a negative cyte enzymes in body fluids have been patented. For example, result. When using Such a quantitative device, it produces a U.S. Pat. No. 5,656,448 to Kang et al, which is incorporated 15 range of results. For example, the MULTISTIX-2 produces herein in its entirety discloses a dipstick encompassed for use quantitative results of 0, trace, +1, +2 and +3. Quantitative in the present invention. Additionally, U.S. Pat. No. 4,758, results also include "Between +1 and +2' and “Between +2 508 to Schnabel, et al. describes an agent and a method for and +3. A test result of 0, trace, and +1 corresponds to the detecting esterolytic and/or proteolytic enzymes in body flu absence of acute appendicitis). A test result of “Between +1 ids. U.S. Pat. No. 4,637,979 to Skjold, et al. describes a and +2”, “Moderate (+2)”, “Between +2 and +3, and “Large composition and test device for determining the presence of (+3)” corresponds to the presence of acute appendicitis). The leukocytes intest samples including body fluids such as urine. pre-determination is done using a study where the range of the U.S. Pat. No. 4,645,842 describes pyrrole compounds, and urine marker presence is determined based on the range in U.S. Pat. No. 4,704,460 (both to Corey) describes novel com urine from confirmed appendicitis Subjects as compared to pounds for detecting the presence of hydrolytic analytes the range of urine maker in the urine from healthy (i.e. con including leukocytes, esterase, and protease, in a test sample, 25 firmed non-appendicitis) subjects. including urine. U.S. Pat. No. 4.774,340 to Corey describes a In some embodiments, a device. Such as a dipstick immu method for preparing 3-hydroxypyrroles and esters thereof, nological device as disclosed herein can includes (1) a matrix which are used to test samples including urine. A composition (preferably filter paper) with diagnostic test reagents and (2) and test device for determining the presence of leukocytes, a mounting Substrate (preferably polystyrene film), which esterase, and protease in a body fluid including urine is 30 typically does not absorb the test (e.g. urine) sample, Such that described in U.S. Pat. No. 4,657,855 to Corey, et al. A method the user can hold onto the Substrate without contacting the for determining the concentration of white blood cells in sample. The device produces a visual change in the matrix urine or other biological fluid is described in U.S. Pat. No. upon contact with the urine sample. In some embodiments, 5,663,044 to Noffsinger, et al. A method for preparing an ester the matrix has two indicators—a first that indicates the pres used to detect leukocyte cells, esterase, and protease in body ence of acute appendicitis and a second that indicates the fluids such as urine is described in U.S. Pat. No. 4,716.236 to 35 absence of appendicitis. The first indicator produces a posi Ward, et al. All of these patents, which are incorporated herein tive test result and the second indicator produces a negative in their entirety by reference, identify an abnormally high result. The test result is positive when the test result is pre level of leukocytes in a patient’s urine and produce a signal to determined to correspond with a level of the appendicitis identify likelihood that the subject from which the urine was biomarker which is indicative of acute appendicitis. Con obtained has a pathological condition Such as kidney or uro 40 versely, a test result is negative when the test result is pre genital tract infection or other dysfunction. determined to be below the level of an appendicitis biomarker In some embodiments, the present invention provides a which indicates the absence of acute appendicitis. The device, LFIA device such as a dipstick to identify appendicitis biom Such as a dipstick device determines the presence of acute arkers in a urine test sample. In one embodiment is a method appendicitis with the positive test result, and the absence of for detecting acute appendicitis using a LFIA device. Such as acute appendicitis with the negative test result. a dipstick, having diagnostic test reagents to detect acute 45 In some embodiments, the diagnostic test reagents may be appendicitis. The diagnostic test reagents react with the test associated with the matrix by any physical or chemical sample, Such as urine test sample to produce a change upon means, including, for example impregnation, coating, link contact with the test sample, Such as urine. Another embodi ing, and covalent attachment. The matrix may take any con ment of the invention is a device. Such as a dipstick, that has Venient physical form, such as a card, pad, strip, or dipstick. (1) a positive indication for the presence of acute appendicitis 50 Such diagnostic test reagents include the compositions of the and (2) a negative indication for the absence of acute appen above-referenced patents, including an ester (preferably a dicitis. The difference between the positive indication and the chromogenic ester) and a diazonium salt such as those negative indication is pre-determined. described in U.S. Pat. No. 4,637,979. Another preferred In some embodiments, the present invention also provides reagent is a derivatized pyrrole amino acid ester, a diazonium a method for determining if a subject has a likelihood of acute 55 salt, a buffer, and non-reactive ingredients as described in appendicitis. In some embodiments, the method begins with U.S. Pat. Nos. 4,645,842; 4,637,979; 4,657,855; 4,704,460; obtaining a urine sample from a subject, Such as a symptom 4,758,508; and 4,774,340. The preferred amounts of these atic patient for appendicitis. Symptomatic patients for appen ingredients is based on dry weight at the time of impregnation dicitis are described herein. Once the sample is obtained, a and is as follows: about 0.4% w/w derivatized pyrrole amino device having diagnostic test reagents that detect the presence acid ester, about 0.2% w/w diazonium salt, about 40.9% w/w of at least one appendicitis biomarker, Such as leucine-rich 60 buffer, and about 58.5% w/w non-reactive ingredients. C-2-glycoprotein (LRG): 5100-A8 (calgranulin); C-1-acid In one embodiment, the test reagent, e.g. the anti-antigen glycoprotein 1 (ORM); plasminogen (PLG); mannan-bind antibody of the immunoassay is detectably labeled. In some ing lectin serine protease 2 (MASP2); Zinc-C-2-glycoprotein embodiments, the detectable label is selected from a group (AZGP1); apolipoprotein D (Apod); C-1-antichymotrypsin consisting of enzyme, fluorescent, biotin, gold, latex, hapten (SERPINA3) or any listed from Table 1 is contacted with the 65 and radioisotope labeling. A detectable-hapten includes but is urine sample. Depending on the type of device used, a certain not limited to biotin, fluorescein, digoxigenin, dinitrophenyl amount of time might have to pass before the device is read. (DNP). Other labels include but are not limited to colloidal US 8,535,891 B2 27 28 gold and latex beads. The latex beads can also be colored. Weeks, I. et al., Clin. Chem. 29:1480-1483 (1983)). Radio Methods of labeling antibodies, antibody-based moiety, or immunoassay (Kashyap, M. L. et al., J. Clin. Invest, 60:171 proteins are known in the art, for example, as described in 180 (1977)) is another technique in which antibody can be “Colloidal Gold. Principles. Methods and Applications'. used after labeling with a radioactive isotope such as 'I. Hayat MA (ed) (1989-91). Vols 1-3, Academic press, Lon Some of these immunoassays can be easily automated by the don; in “Techniques in Immunocytochemistry’, Bullock GR use of appropriate instruments such as the IMXTM (Abbott, and Petrusz P (eds) (1982-90) Vols 1, 2, 3, and 4, Academic Irving, Tex.) for a fluorescent immunoassay and Ciba Corn Press, London; in “Principles of Biological Microtechnique'. ing ACS180TM (Ciba Corning, Medfield, Mass.) for a chemi Baker J R (1970), Methuen, London: Lillie R D (1965), luminescent immunoassay. Histopathologic Technique and practical Histochemistry, 3rd A “LFIA test strip’ or "dip stick' can include one or more ed, McGraw Hill, New York; Berryman MA, et al (1992), J. 10 bibulous or non-bibulous materials or matrices. In reference Histochem Cytochem 40, 6, 845-857, all of which are incor to a “LFIA test strip’ or "dip stick', the terms “material and porated hereby reference in their entirety. "matrix” are used interchangeably. If a test Strip comprises In one embodiment, the detectable label is a dye. A “dye' more than one material, the one or more materials are pref refers to a Substance, compound or particle that can be erably in fluid communication. One material of a test strip detected, particularly by visual, fluorescent or instrumental 15 may be overlaid on another material of the test strip. Such as means. A dye can be, for example, but not limited to, a for example, filter paper overlaid on nitrocellulose mem pigment produced as a coloring agent or ink, Such as Brilliant brane. Alternatively or in addition, a test strip can include a Blue, 3132 Fast Red 2R and 4230 Malachite Blue Lake, all region comprising one or more materials followed by a region available from Hangzhou Hongyan Pigment Chemical Com comprising one or more different materials. In this case, the pany, China. The "dye can also be aparticulate label. Such as, regions are in fluid communication and may or may not but not limited to, blue latex beads or gold particles. The partially overlap one another. Suitable materials for test strips particulate labels may or may not be bound to a protein, include, but are not limited to, materials derived from cellu depending upon if it is desired for the particles to move in the lose, such as filter paper, chromatographic paper, nitrocellu test strip or not. If the particles are to be immobilized in the lose, and cellulose acetate, as well as materials made of glass test strip, the particles may be conjugated to a protein, e.g. the fibers, nylon, dacron, PVC, polyacrylamide, cross-linked anti-antigen antibody, which in turn is bound to the test Strip 25 dextran, agarose, polyacrylate, ceramic materials, and the by either physical or chemical means. like. The material or materials of the test strip may optionally In colloidal gold labeling technique, the unique redcolor of be treated to modify their capillary flow characteristics or the the accumulated gold label, when observed by lateral or trans characteristics of the applied sample. For example, the verse flow along a membrane on which an antigen is captured sample application region of the test strip may be treated with by an immobilized antibody, or by observation of the red 30 buffers to correct the pH or specific gravity of an applied urine color intensity in solution, provides an extremely sensitive sample, to ensure optimal test conditions. method for detecting sub nanogram quantities of proteins in The material or materials can be a single structure such as Solution. A colloidal gold conjugate consists of a suspension a sheet cut into strips or it can be several strips or particulate of gold particles coated with a selected protein or macromol material bound to a Support or Solid Surface Such as found, for ecule (such as an antibody or antibody-based moiety). The example, in thin-layer chromatography and may have an gold particles may be manufactured to any chosen size from 35 absorbent pad either as an integral part or in liquid contact. 1-250 nm. This gold probe detection system, when incubated The material can also be a sheet having lanes thereon, capable with a specific target, such as in a tissue section, will reveal of spotting to induce lane formation, wherein a separate assay the target through the visibility of the gold particles them can be conducted in each lane. The material can have a rect selves. For detection by eye, gold particles will also reveal angular, circular, oval, triagonal or other shape provided that immobilized antigen on a solid phase such as a blotting mem 40 there is at least one direction of traversal of a test solution by brane through the accumulated red color of the gold Sol. capillary migration. Other directions of traversal may occur Silver enhancement of this gold precipitate also gives further Such as in an oval or circular piece contacted in the center with sensitivity of detection. Suppliers of colloidal gold reagents the test solution. However, the main consideration is that for labeling are available from SPI-MARKTM. Polystyrene there beat least one direction of flow to a predetermined site. latex Bead size 200 nm colored latex bead coated with anti The support for the test strip, where a support is desired or body SIGMA ALDRICHR), Molecular Probes, Bangs Labo 45 necessary, will normally be water insoluble, frequently non ratory Inc., and AGILENTR Technologies. porous and rigid but may be elastic, usually hydrophobic, and Other detection systems can also be used, for example, a porous and usually will be of the same length and width as the biotin-streptavidin System. In this system, the antibodies strip but may be larger or Smaller. The Support material can be immunoreactive (i.e. specific for) with the biomarker of inter transparent, and, when a test device is assembled, a transpar est is biotinylated. Quantity of biotinylated antibody bound to 50 ent Support material can be on the side of the test strip that can the biomarker is determined using a streptavidin-peroxidase be viewed by the user, Such that the transparent Support mate conjugate and a chromagenic Substrate. Such streptavidin rial forms a protective layer over the test strip where it may be peroxidase detection kits are commercially available, e.g. exposed to the external environment. Such as by an aperture in from DAKO: Carpinteria, Calif. the front of a test device. A wide variety of materials, both Protein binding agents described herein Such as antibodies 55 natural and synthetic, and combinations thereof, may be and antibody-based moiety can alternatively be labeled with employed provided only that the support does not interfere any of a number of fluorescent compounds such as fluorescein with the capillary action of the material or materials, or non isothiocyanate, europium, lucifer yellow, rhodamine B specifically bind assay components, or interfere with the sig isothiocyanate (Wood, P. In: Principles and Practice of Immu nal producing system. Illustrative polymers include polyeth noassay, Stockton Press, New York, pages 365-392 (1991)) ylene, polypropylene, poly(4-methylbutene), polystyrene, for use in immunoassays. In conjunction with the known 60 polymethacrylate, poly(ethylene terephthalate), nylon, poly techniques for separation of antibody-antigen complexes, (vinylbutyrate), glass, ceramics, metals, and the like. Elastic these fluorophores can be used to quantify the biomarker of Supports may be made of polyurethane, neoprene, latex, sili interest. The same applies to chemiluminescent immunoas cone rubber and the like. say in which case antibody or biomarker of interest can be In some embodiments, a dipstick device has one indication labeled with isoluminol oracridinium esters (Krodel, E. et al., 65 of the presence of acute appendicitis and a second indication In: Bioluminescence and Chemiluminescence: Current Sta for the absence of acute appendicitis. The two indications tus. John Wiley and Sons Inc. New York, pp 107-110 (1991); preferably are a negative (-) symbol and a positive (+) sym US 8,535,891 B2 29 30 bol, but could be any two indications. In one embodiment, the embodiments, the instrument is capable of quantify a number device has the negative indication (e.g., the '-' portion of a of reagent bands as well as quantify the overall color intensity possible '+' symbol) containing reagents that reacts with all sensed on the band. samples. That is, the diagnostic test reagents react to some In some embodiments, the immunoassays operate on a constituent analyte. Such as urea which is present in all urine 5 purely qualitative basis. However it is possible to measure the samples. Alternatively, the diagnostic test reagents test an intensity of the test line to determine the quantity of antigen in aspect of the sample, such as pH, that every sample has. The the sample when using an immunoassay Such a LFIA. Imple positive indication (e.g., the portion of a “+' symbol) menting a magnetic immunoassay (MIA) in the lateral flow contains a reagent that the reacts only with a sample contain test form also allows for getting a quantified result. ing the presence of a test appendicitis biomarker which is 10 Instruments have been developed which both determine above a certain pre-defined level, such that it reacts in urine the chemical constituents of the urine and also assist in the samples which only contain the presence of the appendicitis microscopic analysis, for example the instrument disclosed in biomarker (i.e. of a LRG biomarker) above a certain level, i.e. U.S. Pat. No. 6,004,821 which is incorporated herein in its above a pre-defined level of the appendicitis biomarker. entirety by reference. Such an instrument is the Yellow IRIS, Another embodiment has the negative indicator (e.g., the “- 15 which automatically places the sample on the urine dipstick portion of a possible '+' symbol) which contains reagents and then reads the chemical results. FIG. 8 of U.S. Pat. No. that reacts with the sample which either has the absence of the 6,004.821 shows a schematic depiction of such an automated test appendicitis biomarker (i.e. absence of a LRG biomarker) calorimetric microscopical instrument assembly (which is or the level of the test appendicitis biomarker (i.e. the LRG denoted generally by the numeral 54), and which can be used biomarker) below a certain pre-defined or threshold level. to Scan a urine sample, and can, without significant human The positive indication (e.g., the “I” part of the “+' symbol) intervention, colorometrically analyze the wavelengths of the has a lower sensitivity to the presence of a test appendicitis colors imparted to the dipstick by the urine in the chamber 14, biomarker (i.e. LRG biomarker) and thus such the reagents either colorometrically and/or morphometrically. Accord react only with urine samples containing level of the urine ingly, such an instrument, which is specifically adapted to marker (i.e. LRG biomarker) above a pre-defined level. scan the reaction of the dipstick after contact with a urine In some embodiments, a test device, such as a dipstick 25 sample for the presence of the appendicitis biomarkers (such device has text on the device in two places. In one place the as at least one selected from Table 1) is encompassed for use text indicates a positive result (i.e. the likelihood the subject in the present invention. has acute appendicitis). In another, it indicates a negative In some embodiments, the dipstick uses reagents such as result (i.e. the likelihood the subject does not have acute copper-creatinine and iron-creatinine complexes have per appendicitis). Next to the indications are matrices having the 30 appropriate diagnostic test reagents. For example, next to the oxidase activity. Other dipstick reagents can use reagents negative indication is a matrix having diagnostic test reagents such as 3,3',5,5'-tetramethylbenzidine (TMB), and diisopro that react with all urine samples, regardless of the content of pyl benzene dihydroperoxide (DBDH) which are used with appendicitis biomarkers as disclosed herein. Next to the posi peroxidase. In some embodiments, a dipstick for use to detect tive indication is a matrix having diagnostic test reagents that the presence of appendicitis biomarkers is based upon the react only with samples that have the presence of the test 35 first-generation devices which relied on the same colorimet appendicitis biomarker, (e.g. LRG, or any or any combination ric reaction used for assessing the presence of glucose test of appendicitis biomarkers listed in Table 1) above a pre strips for urine. Besides glucose oxidase, a test kit for use defined level. In some embodiments, such a device Such as hereincan containabenzidine derivative, which is oxidized to one discussed in FIG. 12, does not require a chart. Such as a a blue polymer by the hydrogen peroxide formed in the oxi coloration chart, to interpret the results. In some embodi 40 dation reaction. Care must be taken if such a dipstick is ments of this aspect of the invention, this enables the detec generated to ensure the test strip is developed after a precise tion device. Such as a dipstick device (and the corresponding interval after contact with the urine test sample as well as method) to be used easily by one without special training and frequent calibration of the meter to read the test result. The provides a more rapid diagnostic (and method) for determin same principle is used in test strips that have been commer ing if a subject is likely to have acute appendicitis. In some cialized for the detection Diabetic ketoacidosis (DKA). These embodiments of this aspect of the invention, Such a device is 45 test Strips use a beta-hydroxybutyrate-dehydrogenase ideal for point-of-care testing application. enzyme instead of a glucose oxidizing enzyme and have been Production and manufacturer of dipsticks are well known used to detect and help treat Some of the complications that by ordinary skill in the art. Dipsticks are commercially avail can result from prolonged hyperglycaemia. Blood alcohol able from Bayer Corporation of Elkhart, Ind., as well as other sensors using the same approach but with alcohol dehydro commercial sources. The dipstick is dipped into a well mixed 50 genase enzymes have been developed. urine sample, and after a time period, for example between In another embodiment, the device. Such as a dipstick about thirty seconds (30s) to about two minutes (2 mins) or device uses an electrochemical method. Test strips contain a more, the various reagent bands are visually or optically capillary that Sucks up a reproducible amount of urine. The examined for color changes. The bands can be visually com presence of an appendicitis biomarker Such as any or a com pared to a preprinted color chart in order to determine the 55 bination of those listed in Table 1 in the urine reacts with an amount of each of the constituents or parameters being mea enzyme electrode containing protein-binding agents with the Sured. It is also possible to optically scan using a machine or test appendicitis biomarker. The coulometric method is a optical scanner the dipstick and thereby obtain instrument technique where the total amount of charge generated by the readings of color intensity or wave length through the use of specific binding of the appendicitis biomarker to the specific a particular instrument adapted for reading the reagents and protein-binding agent reaction is measured over a period of color of the dipstick. Examples of Such instruments or 60 time. This is analogous to throwing a ball and measuring the machines are manufactured by Ames. Examples of useful distance it has covered so as to determine how hard it was machines or instruments for optically scanning the dipstick thrown. The amperometric method is used by some meters bands are able to distinguish between positive and negative and measures the electrical current generated at a specific reaction or reagent bands, was well as differences in color point in time. This is analogous to throwing a ball and using distribution of the reagent bands in the presence (i.e. above a 65 the speed at which it is travelling at a point in time to estimate certain threshold level) or absence (or below a certain thresh how hard it was thrown. The coulometric method can allow old level) of the test appendicitis biomarker(s). In some for variable test times, whereas the test time on a meter using US 8,535,891 B2 31 32 the amperometric method is always fixed. Both methods give generated by ancillary materials cannot be ignored. In MIA an estimation of the concentration of the appendicitis biom based on non-linear magnetic properties of magnetic labels arker in the urine sample. the beads are exposed to an alternating magnetic field at two In one embodiment, the levels of appendicitis biomarker frequencies, fl and f2. In the presence of non-linear materials proteins in urine are detected by a magnetic immunoassay Such as Superparamagnetic labels, a signal can be recorded at (MIA). MIA is a type of diagnostic immunoassay using mag combinatorial frequencies, for example, at f=fl-2xf2. This netic beads as labels in lieu of conventional enzymes signal is exactly proportional to the amount of magnetic mate (ELISA), radioisotopes (RIA) or fluorescent moieties (fluo rial inside the reading coil. Ultrasensitive magnetic biosensor rescent immunoassays). This assay involves the specific bind for homogeneous immunoassay have been described by Y. R. ing of a protein binding agent to an appendicitis biomarker Chemla, et al., Proc Natl Acad Sci USA. 2000, 97:14268 protein, such as an antibody binding to its antigen, where a 10 14272. This is incorporate hereby reference in its entirety. magnetic label is conjugated to one element of the pair. The In one embodiment, the levels of biomarker proteins in presence of magnetic beads is then detected by a magnetic urine are detected by a diffusion immunoassay (DIA). In this reader (magnetometer) which measures the magnetic field assay, the transport of molecules perpendicular to flow in a change induced by the beads. The signal measured by the microchannel, e.g. in a microfluidic chip, is affected by bind magnetometer is proportional to the antigen or biomarker 15 ing between antigens and antibodies. By imaging the steady quantity in the initial sample. state position of labeled components in a flowing stream, the Magnetic beads are made of nanometric-sized iron oxide concentration of very dilute analytes, in this invention, the particles encapsulated or glued together with polymers. urine biomarkers, can be measured in a few microliters of These magnetic beads can range from 35 nm up to 4.5 um. sample in seconds. Microfluidics is the manipulation of The component magnetic nanoparticles range from 5 to 50 microliter volumes in channels with sub-millimeter dimen nm and exhibit a unique quality referred to as Superparamag sions. Microfluidic diffusion immunoassays for the detection netism in the presence of an externally applied magnetic field. of analytes or biomarkers in fluid samples have been Magnetic labels exhibit several features very well adapted for described in the art, for example, in U.S. Pat. Nos. 6,541,213; Such applications: they are not affected by reagent chemistry 6,949,377: 7.271,007; U.S. Patent Application No. or photo-bleaching and are therefore stable over time; the 20090194707; 20090181411; in Hatch et al., 2001, Nature magnetic background in a biomolecular sample is usually 25 Biotechnology 19(5): 461–465; K. Scott Phillips and Quan insignificant; sample turbidity or staining have no impact on Cheng, Anal. Chem., 2005, 77:327-334; J. Hsieh, et al., magnetic properties; and magnetic beads can be manipulated Nanotech 2007 Vol. 3, Technical Proceedings of the 2007 remotely by magnetism. NSTI Nanotechnology Conference and Trade Show, Chapter The use of MIA is well known in the art, for example, 4: Micro and Nano Fluidics, pp. 292-295; Frank Y. H. Linet Dittmer W U and colleagues (J Immunol Methods. 2008, 30 al., Clinical and Diagnostic Laboratory Immunology, 2005, 338:40-6) described a sensitive and rapid immunoassay for 12:418-425; and A. Bhattacharyya and C. M. Klapperich, detection and measurement parathyroid hormone using mag 2007, Biomedical Microdevices, 9:245-251. These are incor netic particle labels and magnetic actuation. The assay porated herein by reference in their entirety. U.S. Pat. No. involves a 1-step sandwich immunoassay with no fluid 6,541.213 describes the use of a credit-card sized microflu replacement steps. The detection limit is the 10 pM range and idic device to perform competitive immunoassays. The abil the assay took only 15 minutes; Kuma H and colleagues 35 ity to perform assays in this microscale dimension affords an (Rinsho Byori. 2007, 55:351-7) developed a sensitive immu extremely rapid, homogenous, and cost effective alternative noassay system using magnetic nanoparticles made from to current methods used commercially today. The credit-card FeO; and Kuramitz H. reviews the current state of concern sized microfluidic device can be integrated into the develop ing electrochemical immunoassays using magnetic micro ment of point-of-use systems that allow real-time answers to beads as a solid phase in Anal Bioanal Chem. 2009,394:61-9. 40 health questions while at the physicians office, home, work U.S. Pat. Nos. 5,252,493: 5,238,811: 5,236,824; 7,604,956; place, School, shopping mall and other public places. These U.S. Patent Application No. 20090216082; 20090181359: systems include portable and handheld instruments with inte and 20090263834 all describe various improvements and grated laboratory-tests-on-a-card (“lab cards”), as well as versions of MIA. These references are all incorporated herein stand alone, single use lab cards being developed to provide by reference in their entirety. rapid on-site results in infectious diseases testing, nucleic Magnetometers are instruments that can detect the pres 45 acid testing, blood type analysis, cancer testing, and respira ence and measure the total magnetic signal of a sample. An tory disease testing. effective MIA is one that is capable of separating naturally In one embodiment, the levels of biomarker proteins in occurring magnetic background (noise) from the weak mag urine are detected by an on-the-spot assay also known as netically labeled target (signal). Various approaches and point-of-care assay. Point-of-care testing (POCT) is defined devices have been employed to achieve a meaningful signal 50 as diagnostic testing at or near the site of patient care. Cur to-noise ratio (SNR) for bio-sensing applications: giant mag rently majority of the detection and diagnostic testing for neto-resistive sensors and spin valves, piezo-resistive canti analytes, toxin, pathogen toxins and antigens in Samples are levers, inductive sensors, Superconducting quantum largely restricted to centralized laboratories because of the interference devices, anisotropic magneto-resistive rings, and need for long assay times, complex and expensive equipment, miniature Hall sensors. MIA that exploits the non-linear mag 55 and highly trained technicians. POCT brings the test conve netic properties of magnetic labels can effectively use the niently and immediately to the patient. This increases the intrinsic ability of a magnetic field to pass through plastic, likelihood that the patient will receive the results in a timely water, nitrocellulose, and other materials, thus allowing for manner. POCT is accomplished through the use of transport true Volumetric measurements in various immunoassay for able, portable, and handheld instruments (e.g., blood glucose mats. Unlike conventional methods that measure the Suscep meter, nerve conduction study device) and test kits (e.g., CRP, tibility of Superparamagnetic materials, a MIA based on non 60 HBA1C, Homocystein, HIV salivary assay, etc.). POCTs are linear magnetization eliminates the impact of linear dia- or well known in the art, especially immunoassays. For paramagnetic materials such as sample matrix, consumable example, the LFIA test strip or dip Sticks can easily be inte plastics and/or nitrocellulose. Although the intrinsic magne grated into a POCT diagnostic kit. One skilled in the art would tism of these materials is very weak, with typical susceptibil be able to modify immunoassays for POCT using different ity values of -10-5 (dia) or +10-3 (para), when one is inves 65 format, e.g. ELISA in a microfluidic device format or a test tigating very small quantities of Superparamagnetic strip format. For example, U.S. Patent Application No. 2009/ materials, such as nanograms per test, the background signal 0181411 describes a microfluidic device-based point-of-care US 8,535,891 B2 33 34 immunoassay for biomarker molecules associated with “Clinical Use of Monoclonal Antibodies, in BIOTECH pathology in a vertebrate host, man or animal. The microflu NOLOGY AND PHARMACY 227-49, Pezzuto et al. (eds.) idic devices such as chips are formatted to either hand-held (Chapman & Hall 1993). cartridges (also termed "cards), or cartridges for automated For example, to generate a polyclonal antibody against or semi-automated, machine-aided testing. Microfluidic human LRG, S100-A8, ORM1, PLG, MASP2, AZGP1, device-based assays enable small-volume sampling, with point-of-care results from abroad variety of biological fluids Apold or SERPINA3. Methods of making recombinant pro and samples in real time. In addition, the assay cartridges can teins are well known in the art. For example, full-length be single use reagent packs, or be fully self-contained and cDNAs of LRG, S100-A8, ORM1, PLG, MASP2, AZGP1, operable entirely by hand. This reference is incorporated Apol) and SERPINA3 (Genbank Accession Nos. herein by reference in its entirety. 10 NM 052972.2, NM 002964.3, NM 000607.2, Embodiments of the invention further provide for diagnos NM 000301.2, NM 006610.2, NM 001185.2, tic kits and products of manufacture comprising the diagnos NM 001647.3, and NM 001085.4 respectively) can be tic kits. The kits can comprise a means for predicting acute cloned into the pGE30 vector containing an N-terminal hexa appendicitis in a human. histidine tag (QIAGEN, GmbH, Hilden, Germany), and then In one embodiment, the kit comprises an indicator respon 15 transformed into E. coli strain JM109 cells. Recombinant sive to the level of biomarker protein in a sample of urine, proteins is expressed and purified by affinity chromatography wherein the appendicitis biomarker protein is selected from using Ni-nitriloacetic acid agarose (QIAGEN) according to the group consisting of LRG, S100-A8, ORM1, PLG, the manufacturer's instructions. The final preparation yielded MASP2, AZGP1, ApolD and SERPINA3. In some embodi a single calculated molecular weight of 89707 kDa band on ments, the indicator is in the form of a LFIA test strip or a SDS-PAGE and is used for the immunization of rabbits. microfluidic device. In one embodiment, a diagnostic kit can Detection of anti-antibodies to the appendicitis biomarkers comprise multiple LFIA test strips, one strip for a different can be achieved by direct labeling of the antibodies them biomarker protein. In another embodiment, a diagnostic kit selves, with labels including a radioactive label such as H, can comprise a single composite LFIA test strip for determin C. S, 'I, or 'I, a fluorescent label (e.g. Cy3, Cy5, ing the levels of several biomarker proteins. In one embodi FITC), a hapten label such as biotin, heavy metal such as gold, ment, a diagnostic kit can comprise a single multichannel 25 or an enzyme such as horse radish peroxidase or alkaline microfluidic device for determining the levels of several phosphatase. Such methods are well known in the art. Alter biomarker proteins. In another embodiment, a diagnostic kit natively, unlabeled primary antibody is used in conjunction can comprise several microfluidic devices for determining the with labeled secondary antibody, comprising antisera, poly levels of several biomarker proteins, one microfluidic device clonal antisera or a monoclonal antibody specific for the for a different biomarker protein. 30 The kits can further comprise cups or tubes, or any other primary antibody. In another embodiment, the primary anti collection device for sample collection of urine. body or antisera is unlabeled, the secondary antisera or anti In one embodiment, the kit can optionally further comprise body is conjugated with biotin and enzyme-linked strepavi at least one diagram and/or instructions describing the inter din is used to produce visible staining for histochemical pretation of test results. analysis. Protein-Binding Agents, Antibodies or Antisera Against 35 In one embodiment, the levels of the appendicitis biomar Biomarker Proteins ker proteins described herein in a sample can be determined In one embodiment, the methods disclosed herein uses by mass spectrometry such as MALDI/TOF (time-of-flight), antibodies or anti-sera for detecting, quantifying, and/or SELDI/TOF, liquid chromatography-mass spectrometry labeling LRG, S100-A8, ORM1, PLG, MASP2, AZGP1, (LC-MS), gas chromatography-mass spectrometry (GC Apol) and SERPINA3 described herein. The antibodies can 40 MS), high performance liquid chromatography-mass spec be obtained from a commercial source. These commercial trometry (HPLC-MS), capillary electrophoresis-mass spec antibodies can also be conjugated with labels, e.g. Cy 3 or trometry, nuclear magnetic resonance spectrometry, or FITC. tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI Antibodies for use in the methods described hereincan also MS/MS, etc.). See for example, U.S. Patent Application Nos. be produced using standard methods to produce antibodies, 20030199001, 20030134304, 20030077616, which are for example, by monoclonal antibody production (Campbell, 45 herein incorporated by reference in their entirety. A. M. Monoclonal Antibodies Technology: Laboratory Mass spectrometry methods are well known in the art and Techniques in Biochemistry and Molecular Biology, Elsevier have been used to quantify and/or identify biomolecules, such Science Publishers, Amsterdam, the Netherlands (1984); St. as proteins (see, e.g., Li et al. (2000) Tibtech 18:151-160: Groth et al., J. Immunology, (1990) 35: 1-21; and Kozbor et Rowley et al. (2000) Methods 20: 383-397; and Kuster and al., Immunology Today (1983) 4:72). Antibodies can also be 50 Mann (1998) Curr. Opin. Structural Biol. 8:393-400). Fur readily obtained by using antigenic portions of the protein to ther, mass spectrometric techniques have been developed that Screen an antibody library, such as a phage display library by permitat least partial de novo sequencing of isolated proteins. methods well known in the art. For example, U.S. Pat. No. Chait et al., Science 262:89-92 (1993); Keough et al., Proc. 5,702,892 (U.S.A. Health & Human Services) and WO Natl. Acad. Sci. USA. 96:7131-6 (1999); reviewed in Berg 01/18058 (Novopharm Biotech Inc.) disclose bacteriophage 55 man, EXS 88:133-44 (2000). display libraries and selection methods for producing anti In certain embodiments, a gas phase ion spectrophotom body binding domain fragments. eter is used. In other embodiments, laser-desorption/ioniza Methods for the production of antibodies are disclosed in tion mass spectrometry is used to analyze the sample. Modern PCT publication WO 97/40072 or U.S. Application. No. laser desorption/ionization mass spectrometry (“LDI-MS) 2002/0182702, which are herein incorporated by reference. can be practiced in two main variations: matrix assisted laser The processes of immunization to elicit antibody production 60 desorption/ionization ("MALDI) mass spectrometry and in a mammal, the generation of hybridomas to produce mono surface-enhanced laser desorption/ionization (“SELDI). In clonal antibodies, and the purification of antibodies may be MALDI, the analyte is mixed with a solution containing a performed by described in “Current Protocols in Immunol matrix, and a drop of the liquid is placed on the surface of a ogy” (CPI) (John Wiley and Sons, Inc.) and Antibodies: A substrate. The matrix solution then co-crystallizes with the Laboratory Manual (Ed Harlow and David Lane editors, Cold 65 biological molecules. The substrate is inserted into the mass Spring Harbor Laboratory Press 1988) which are both incor spectrometer. Laser energy is directed to the substrate surface porated by reference herein in their entireties: - - - Brown, where it desorbs and ionizes the biological molecules without US 8,535,891 B2 35 36 significantly fragmenting them. See, e.g., U.S. Pat. No. 5,118, detecting the location of the protein-binding agent at the 937 (Hillenkamp et al.), and U.S. Pat. No. 5,045,694 appendix with an extracorporeal detection means capable of (Beavis & Chait). detecting the labeling means; and (c) quantifying the protein In SELDI, the substrate surface is modified so that it is an binding agent concentration in order to determine the pres active participant in the desorption process. In one variant, the ence and extent of inflammation in the appendix. In one Surface is derivatized with adsorbent and/or capture reagents embodiment, the intensity of the label is directly proportional that selectively bind the protein of interest. In another variant, to the concentration of the protein-binding agent that binds the Surface is derivatized with energy absorbing molecules specifically to an appendicitis biomarker protein. that are not desorbed when struck with the laser. In another In Some embodiment, the protein-binding agent concentra variant, the surface is derivatized with molecules that bind the tion measured by extracorporeal detection means in a patient protein of interest and that contain a photolytic bond that is 10 is compared to the protein-binding agent concentration in a broken upon application of the laser. In each of these meth healthy individual, wherein in the detectable label and the ods, the derivatizing agent generally is localized to a specific imaging method are the same for both the patient and the location on the Substrate Surface where the sample is applied. healthy individuals. In some embodiments, the patient has at See, e.g., U.S. Pat. No. 5,719,060 and WO 98/593.61. The two least one symptom associated with acute appendicitis as dis methods can be combined by, for example, using a SELDI 15 closed herein or as known to one skilled in the art such as a affinity Surface to capture an analyte and adding matrix-con physician. In some embodiments, the protein-binding agent taining liquid to the captured analyte to provide the energy concentration at the appendix of a patient is compared to the absorbing material. protein-binding agent concentration that is the average For additional information regarding mass spectrometers, obtained for a population, i.e. more than two individuals, see, e.g., Principles of Instrumental Analysis, 3rd edition. preferably ten or more, of healthy individuals, wherein in the Skoog. Saunders College Publishing, Philadelphia, 1985; and detectable label and the imaging method are the same for both Kirk-Othmer Encyclopedia of Chemical Technology, the patient and the healthy individual. 4.sup.thed. Vol. 15 (John Wiley & Sons, New York 1995), pp. In one embodiment, the protein-binding agent is intro 1071-1094. duced into the vascular system of the Subject, for example, Detection and quantification of the appendicitis biomarker intravenously. In one embodiment, the protein-binding agent proteins will typically depend on the detection of signal inten 25 is introduced into the abdomen cavity of the subject, prefer sity. This, in turn, can reflect the quantity and character of a ably within the vicinity of the appendix at the lower right polypeptide bound to the Substrate. For example, in certain abdomen. In one embodiment, the protein-binding agent is embodiments, the signal strength of peak values from spectra introduced into the peritoneal cavity, preferably within the of a first sample and a second sample can be compared (e.g., vicinity of the appendix at the lower right abdomen. visually, by computer analysis etc.), to determine the relative 30 In one embodiment, a fixed amount of time is allowed to amounts of particular biomolecules. Software programs such lapse before imaging is performed. as the appendicitis biomarker WIZARD program (Ciphergen In one embodiment, the protein-binding agent is an anti Biosystems, Inc., Fremont, Calif.) can be used to aid in ana body or fragment thereof. In one embodiment, the protein lyzing mass spectra. The mass spectrometers and their tech binding agent is a monoclonal antibody or active fragment niques are well known to those of skill in the art. thereof. In one embodiment, the protein-binding agent is a Diagnostic Imaging of Acute Appendicitis 35 polyclonal antibody or active fragment thereof. For example, In some embodiments, described herein is a method of the protein-binding agent is an anti-LRG antibody or frag diagnosing likelihood of acute appendicitis in a Subject by in ments thereof. In some embodiments, the protein-binding situ histochemical imaging of an appendix using at least a agent is an antibody that is specifically immunoreactive (i.e. protein binding agent that bind specifically to a biomarker binds specifically to) to a biomarker protein selected from the selected from the group consisting of leucine-rich C-2-gly 40 group consisting of leucine-rich C-2-glycoprotein (LRG); coprotein (LRG); S100-A8 (calgranulin); C-1-acid glycopro S100-A8 (calgranulin); O.-1.-acid glycoprotein 1 (ORM); tein 1 (ORM); plasminogen (PLG); mannan-binding lectin plasminogen (PLG); mannan-binding lectin serine protease 2 serine protease 2 (MASP2); zinc-C-2-glycoprotein (MASP2); zinc-C-2-glycoprotein (AZGP1); apolipoprotein (AZGP1); apolipoprotein D (Apod); and C-1-antichymot D (ApoD); C-1-antichymotrypsin (SERPINA3); AMBP: rypsin (SERPINA3). amyloid-like protein 2; angiotensin converting enzyme 2: In other embodiments, the method further comprises at 45 BAZ1B: carbonic anhydrase 1: CD14; chromogranin A; least one additional different protein-binding agent that bind FBLN7, FXR2; hemoglobin C.; hemoglobin 13; interleukin-1 specifically to a biomarker selected from the group consisting receptor antagonist protein; inter-O-trypsin inhibitor; AMBP: amyloid-like protein 2; angiotensin converting lipopolysaccharide binding protein; lymphatic vessel endot enzyme 2: BAZ1B: carbonic anhydrase 1: CD14; chromog helial hyaluronan acid receptor 1; MLKL: nicastrin; novel ranin A; FBLN7: FXR2; hemoglobin C.; hemoglobin B; inter 50 protein (Accession No: IPI 100550644); PDZK1 interacting leukin-1 receptor antagonist protein; inter-C-trypsin inhibi protein 1: PRIC285; prostaglandin-H2 D-isomerase; Rcl: tor, lipopolysaccharide binding protein; lymphatic vessel S100-A9; serum amyloid A protein; SLC13A3: SLC2A1; endothelial hyaluronan acid receptor 1; MLKL; nicastrin; SLC2A2: SLC4A1; SLC9A3; SORBS1, SPRX2; supervil novel protein (Accession No: IPI00550644); PDZK1 inter lin: TGFbeta2R; TTYH3; VAOD1; vascular adhesion mol acting protein 1: PRIC285; prostaglandin-H2 D-isomerase: 55 ecule 1: versican; VIP36; CL-1-acid glycoprotein 2: B-1,3- Rcl: S100-A9; serum amyloid A protein; SLC13A3: galactosyltransferase and a biomarker selected from Table 1. SLC2A1; SLC2A2: SLC4A1 SLC9A3; SORBS1, SPRX2: In one embodiment, the protein-binding agent is conju supervillin: TGFbeta2R:TTYH3; VAOD1; vascular adhesion gated to a label for extracorporeal detection of the protein molecule 1: versican; V1P36: C-1-acid glycoprotein 2; and binding agent located in the body of the Subject. 13-1,3-galactosyltransferase. In other embodiments, the In some embodiments, the detectable label on the protein method further comprises at least one additional different 60 binding agent is selected from the group comprising of radio protein-binding agent that bind specifically to a biomarker isotopes, paramagnetic labels, echogenic liposomes, biotin, selected from Table 1. and fluorescence. In one embodiment, the method for diagnosing likelihood In some embodiments, the extracorporeal detection of acute appendicitis in a Subject comprise (a) introducing a method is selected from the group comprising magnetic reso protein-binding agent into the Subject via a physiologically 65 nance imaging (MRI), computer axial tomography (CAT) compatible vehicle in an amount effective for detection, Scan, positron emission tomography (PET) scan, electron wherein the protein binding agent in detectably labeled; (b) beam, computed tomography (CT) scan, single photon emis US 8,535,891 B2 37 38 sion computed tomography (SPECT) imaging, gamma imag Conjugation of Protein Binding Agent, e.g. Antibody to ing, angiography, abdominal ultrasound, and abdominal Echogenic Liposomes for Ultrasound Imaging radioactive and fluorescent detection. Antibody-conjugated echogenic liposomes have been In one embodiment, radionuclide is used as the labeling developed for site-specific intravascular (30 MHz) and trans means and the step of detecting the location of the protein vascular (15 MHz) image enhancement. As examples, anti binding agent within the Subject further includes detecting fibrinogen and anti-intercellular adhesion molecule-1 (anti radiation therefrom with a radiation detector. In one embodi ICAM-1) antibodies have been conjugated to acoustically ment, a radionuclide is the detectable label conjugated to the reflective liposomes and images obtained in animal models of protein binding agent. thrombi and atherosclerotic lesions. These acoustic lipo somes consist of a 60:8:2:30 molar mixture of phosphatidyl In one embodiment, step of detecting radiation further 10 choline:phosphatidyl-ethanolamine:phosphatidylglycerol: includes employing a gamma camera to detect and make an cholesterol and are prepared by a dehydration/rehydration image of gamma radiation emitted by the labeling means of mixture. They are multilamellar with well separated lipid the protein binding reagent. bilayers and internal vesicles which confers echogenicity. Suitable radionuclides include Co-57, Cu-67, Ga-67, Their mean size is ~800 nm as measured by quasielastic light Ga-68, Ru-97, Tc-99m, In-111, In-113m, I-123, I-125, I-131, 15 scattering. These liposomes are stable in circulation, do not Hg-197, Au-198, and Pb-203. The radionuclides can be trap gas, pass through pulmonary capillaries and retain their linked by direct labeling (e.g., by acidic buffered reactions or properties at 37° C., even after conjugation with antibodies. oxidative procedures) or by ligand exchange orchelation. The Antibodies are modified by the addition of cysteines to the C radionuclides are preferably imaged with a radiation detec or N-terminus of the protein and conjugated to liposomes. A tion means capable of detecting gamma radiation, such as a 12 MHZ imaging catheter (Acuson) is used for imaging (reso gamma camera or the like. Methods of radiolabeling of pro lution <1 mm). The antibodies are thiolated with N-succin teins for imaging are well known to one skilled in the art, for imidyl-3-(2-pyridyldithio) propionate, reduced, and conju examples, D. Hnatowich, et al., 1983, Science 220:613-615; gated with the liposomes by creating a thioether linkage M. R. McDevitt, et al., 2000, Cancer Res. 60:6095-6100: DA between the antibody and phospholipid. The conjugated anti Scheinberg, et al., 1982, Science, 215:1511-1513; and W. J. bodies are stable and have a long shelfhalf-life. Imaging is by McBride, et al., 2009, J. Nucl. Med. 50, 991-998; and R. 25 ultrasound. Macklis, B. et al., 1988, Science 240:1024-1026; U.S. Pat. Gadolinium (Gd3)-Labeled Protein Binding Agent, e.g. Sclv Nos. 4,472,509:4454,106; 4,634,586; 4,994,560; 5,286,850; Antibodies (MAbs) U.S. Patent Application Nos. 2008/0241967 and An alternative imaging method that provides enhanced 20090297620. These are all incorporated herein by reference resolution (<0.5 mm), magnetic resonance imaging (MRI) is in their entirety. 30 using Gd3-labeling protein binding agent as a contrast agent. Typically, radiation imaging cameras employ a conversion MRI has the advantages of rapid acquisition, increased reso medium (wherein the high energy gamma ray is absorbed, lution, and absence of radioactivity However, because free displacing an electron which emits a photon upon its return to Gd3 as a contrast agent is toxic, it is used in clinical MRI the orbital state), photoelectric detectors arranged in a spatial imaging bound to diethylenetriaminepentaacetic acid detection chamber (to determine the position of the emitted (DTPA). Precedent exists for conjugating Gd3 to MAbs by photons), and circuitry to analyze the photons detected in the 35 reacting cyclic-diaminetriaminepentaacetic acid anhydride chamber and produce an image. (c-DTPA) with the MAb.Polylysine-DTPA-Gd3-coupled The invention can also be practiced with non-radioactive antibodies have been used for tumour imaging with up to 30 labeling means, such as magnetic contrast agents capable of Gd3 ions conjugated without significantly affecting antigen detection in magnetic resonance imaging (MRI) systems. In affinity. Previous studies using Gd3-labeled MAbs have Such systems, a strong magnetic field is used to align the 40 either directly bound Gd3 to available NH groups or chemi nuclear spin vectors of the atoms in a patient’s body. The field cally conjugated polylysine. The natural site for coupling is then disturbed and an image of the patient is read as the DTPA is limited in scFv (single chain antibody) molecules. nuclei return to their equilibrium alignments. In the present Therefore, genetic fusion of several clusters of polylysine invention, the protein binding agent can be linked to diamag groups (6-30 in length) to the N-terminal or C-terminal of netic contrast agents, such as gadolinium, cobalt, nickel, scFv MAb can be used and this fusion can be reacted with manganese or copper complexes, to form conjugate diagnos 45 c-DTPA. Although otheramino groups may potentially react, tic reagents that are imaged extracorporeally with an MRI the availability of polylysine in the tail of the molecule should system. Other imaging techniques include plethysmography, allow preferential site-directed labeling. The bioengineering thermography and ultrasonic scanning of the polylysine site was done by PCR using primers encod In one embodiment, the protein binding agent Such as an ing six lysine residues and restriction site for cloning at both antibody can be genetically or chemically engineered to con 50 5' and 3' ends. tain "Tc binding sites for nuclear Scintigraphy imaging. In Imaging with "Tc-Labeled Protein Binding Agent, e.g. vivo localized quantitative imaging is performed (SPECT Antibody imaging) can be carried out on the Subject. "Tc-labeling of oxidation specific antibodies has been In one embodiment, the protein binding agent can be previously described (Tsimikas et al., 1999, J Nucl Cardiol. labeled with gadolinium or echogenic liposomes for mag 55 1999; 6:41-53). "Tc-protein binding agent specific for the netic resonance and abdomen ultrasound imaging, respec biomarkers described herein can be intravenously injected tively. into the patient and is analyzed for the pharmacokinetics, Methods and regents such as detectably labeled antibodies organ distribution and appendix uptake. For in Vivo imaging, for in situ imaging are been described and are well known in 1-5 mCi are intravenously injected in the patient and imaging the art, for example, U.S. Pat. Nos. 3,899,675; 4,660,563: can be performed with a dual detector ADAC vertex model 4,877,599; 4,647,445; 5,605,831; 6,716.410; U.S. Patent 60 gamma camera set to a 20% window for "Tc (VXUR col Application Nos. 2009/0016965 and 20070059775. Addi limator) equipped with ADAC PegasysTM computer software. tional methods and regents for in situ imaging are described in In vivo images planar (anterior, posterior and 45' oblique J H Tseng, 2001, Abdominal Imaging, 26: 171-177; Liu, positions) and SPECT can be acquired on a 256x256x12 Qing-Yu, 2009, Abdominal Imaging, in press; DA Schein matrix for a minimum of 1x10 counts at 10 minutes post berg, et al., 1982, Science, 215:1511-1513; and W. J. 65 injection. Repeat imaging can be performed for 3-500,000 McBride, et al., 2009, J. Nucl. Med. 50,991-998. These are all counts at various time points based on the optimal target to incorporated herein by reference in their entirety. background ratio derived from in vivo uptake data. Previous US 8,535,891 B2 39 40 imaging studies using whole monoclonal antibody have medium and methods as disclosed herein that is indicative of shown that whole monoclonal antibody often give a low acute appendicitis is at a level of at least about two-fold (2x) signal to noise ratio due to the prolonged half-life of the above the control or reference appendicitis biomarker level ”Tc-MAb in the circulation. The use of Fab, scFv, or for that biomarker. For example, if the appendicitis biomarker Smaller fragments can abrogate this problem under certain is LRG, if the level of LRG in the test urine sample from the imaging conditions as the Fabs and ScHVS have a very short subject is at least about 2-fold above the reference LRG half lives (<30 minutes). When the signal to noise ratio is not biomarker level, it is indicative of a subject likely to have or be favorable, injections of MDA-LDL, Cu-OxLDL, or other at risk of acute appendicitis. In some embodiments a thresh appropriate antigen can be injected to clear the background old level is at least about 3-fold, or at least about 4-fold, or at signal. least about 5-fold, or at least about 6-fold, or at least about Imaging with Gd3-Labeled Protein Binding Agent, e.g. Anti 10 7-fold, or at least about 8-fold, or at least about 9-fold, or at body least about 10-fold or more than 10-fold above the reference Labeling of Gd3 to an antibody-DTPA complex has been level for that biomarker, and thus a the level of the appendi previously described (Lister-James, et al., 1996, J Nucl Med. citis biomarker in the test urine sample above the threshold 1996: 40:221-233; Wu et al., 1995, Arterioscler Thromb Vasc level it is indicative of a subject likely to have or be at risk of Biol. 1995; 15:529-533). Initial testing by in vivo uptake 15 acute appendicitis. assays can be carried out with 153Gd-antibody in mice and In Some embodiments, the system, computer-readable rabbits and the pharmacokinetics, biodistribution and aortic media and methods as disclosed herein is used to measure an plaque uptake of antibody is determined. In vivo imaging can appendicitis biomarker levelina biological sample, where the be performed in rabbits with a 1.5 T GE MRI scanner with a appendicitis biomarker level is the level of a polypeptide Small Surface coil. biomarker, for example any biomarker of Table 1 or of any Computer Systems and Computer Readable Media to Assay SEQID NOs 1-49. In some embodiments, the level of at least Appendicitis Biomarkers in Urine Samples. one biomarker protein is measured by immuno assay, for One aspect of the present invention relates to a system for example western blot analysis or ELISA, or a highthrough analyzing a urine biological sample from a subject, where the put protein detection method, for example but are not limited system comprises: (a) a determination module configured to to automated immunohistochemistry apparatus, for example, receive a urine biological sample and to determine an appen 25 robotically automated immunohistochemistry apparatus dicitis biomarker level information, wherein the appendicitis which in an automated system section the tissue or biological biomarker level information comprises determination of at sample specimen, prepare slides, perform immunohis least one appendicitis biomarker level, i.e. at the level or tochemistry procedure and detect intensity of immunostain amount of an appendicitis biomarker, Such as LRG, or any or ing, such as intensity of an antibody binding to a biomarker a combination of appendicitis biomarkers listed in Table 1: 30 protein in the urine sample and produce output data. (b) a connection from the determination module to transmit Examples of Such automated immunohistochemistry appara the appendicitis biomarker level information to an electronic tus are commercially available, for example such Autostain computer, wherein the computer comprises a storage device, ers 360, 480, 720 and Labvision PT module machines from a comparison module and a display module; (c) the storage LabVision Corporation, which are disclosed in U.S. Pat. Nos. device configured to store appendicitis biomarker level infor 7,435,383; 6,998,270; 6,746,851, 6,735,531; 6,349,264; and mation from the determination module; (d) the comparison 35 5.839; 091 which are incorporated herein in their entirety by module adapted to compare the appendicitis biomarker level reference. Other commercially available automated immuno information stored on the storage device with reference data, histochemistry instruments are also encompassed for use in and to provide a comparison result, wherein the comparison the present invention, for example, but not are limited result comprises: (i) a comparison of the appendicitis biom BONDTM Automated Immunohistochemistry & In Situ arker level in the urine biological sample with the reference 40 Hybridization System, Automate slide loader from GTI appendicitis biomarker level, and (ii) a determination of the vision. Automated analysis of immunohistochemistry can be appendicitis biomarker level in the biological sample above performed by commercially available systems such as, for or below a threshold level relative to the reference appendi example, IHC Scorer and Path EX, which can be combined citis biomarker level, whereina appendicitis biomarker level with the Applied spectral Images (ASI) CytoLab view, also above the threshold level for that biomarker is indicative of available from GTI vision or Applied Spectral Imaging (ASI) acute appendicitis (i.e. a positive test result); and wherein a 45 which can all be integrated into data sharing systems such as, appendicitis biomarker level below the threshold level is for example, Laboratory Information System (LIS), which indicative of absence of acute appendicitis (i.e. a negative test incorporates Picture Archive Communication System result); and (e) the display module for displaying a content (PACS), also available from Applied Spectral Imaging (ASI) based in part on the comparison result for the user, wherein (see world-wide-web: spectral-imaging.com). Other a deter the content is a signal indicative of the likelihood of a subject 50 mination module can be an automated immunohistochemis having acute appendicitis (i.e. a positive test result) or try systems such as NexES(R) automated immunohistochem unlikely to have acute appendicitis (i.e. a negative test result). istry (IHC) slide staining system or BenchMark R. LT Another aspect of the present invention relates to a com automated IHC instrument from Ventana Discovery SA, puter readable medium having computer readable instruc which can be combined with VIASTM image analysis system tions recorded thereon to define software modules including 55 also available Ventana Discovery. BioGenex Super Sensitive a comparison module and a display module for implementing MultiLink R. Detection Systems, in either manual or auto a method on a computer, the method comprising: (a) compar mated protocols can also be used as the detection module, ing with the comparison module the data stored on a storage preferably using the BioGenex Automated Staining Systems. device with reference data to provide a comparison result, Such systems can be combined with a BioGenex automated wherein the comparison result is the appendicitis biomarker staining systems, the ió000TM (and its predecessor, the Opti level information in the urine biological above a threshold 60 Max(R) Plus), which is geared for the Clinical Diagnostics lab, level relative to a reference appendicitis biomarker level for and the GenoMX 6000TM, for Drug Discovery labs. Both that biomarker tested which is indicative of acute appendici systems BioGenex systems perform All-in-One, All-at tis; and (b) displaying a content based in part on the compari Once” functions for cell and tissue testing. Such as Immuno son result for the user, wherein the content is a signal indica histochemistry (IHC) and In Situ Hybridization (ISH). tive of acute appendicitis. 65 As an example, a determination module used in the system, In some embodiments, the appendicitis biomarker thresh computer-readable media and methods as disclosed herein old level which is used in the system, computer-readable for determining appendicitis biomarker level measures the US 8,535,891 B2 41 42 level of at least one appendicitis biomarker polypeptide, for In Some embodiments, the system, computer-readable instance the determination module is configured to detect the media and methods as disclosed herein is used to measure at total level (i.e. amount) of at least one appendicitis biomarker least one appendicitis biomarker level in a urine biological polypeptide of Table 1 using any known systems for auto sample obtained from a Subject. mated protein expression analysis, including for example, but Another aspect of the present invention relates to a method not limited Mass Spectrometry systems including MALDI of treating a subject identified to have acute appendicitis TOF, or Matrix Assisted Laser Desorption Ionization Time comprising: (a) determining if the Subject has, or is likely to of Flight systems: SELDI-TOF-MS ProteinChip array profil have or is at risk of having acute appendicitis by measuring at ing systems, e.g. Machines with Ciphergen Protein Biology least one appendicitis biomarker level in a urine sample System IITM software; systems for analyzing gene expression 10 obtained from the Subject, and if high levels (e.g. at least data (see for example U.S. 2003/0194711); systems for array about 2-fold above a reference level for the measured biom based expression analysis, for example HTarray systems and arker) of the appendicitis biomarker protein exists in the urine cartridge array systems available from Affymetrix (Santa biological sample from the Subject, it indicates that the Sub Clara, Calif. 95051) AutoLoader, Complete GeneChip(R) ject is likely to have acute appendicitis, and (b) administering Instrument System, Fluidics Station 450, Hybridization Oven 15 an appropriate treatment to a Subject determined to likely 645, QC Toolbox Software Kit, Scanner 3000 7G, Scanner have acute appendicitis, where an appropriate treatment can 30007G plus Targeted Genotyping System, Scanner 30007G be determined by an ordinary physician, for example by Sur Whole-Genome Association System, GeneTitanTM Instru gical resection of the appendix (i.e. appendectomy) if the ment, GeneChip(R) Array Station, HT Array; an automated appendicitis is severe, or antibiotics if the appendicitis is not ELISA system (e.g. DSX(R) or DS2(R) form Dynax, Chantilly, SVC. Va. or the ENEASYSTEM III(R), Triturus(R, The Mago R. In one embodiment, the method is performed on a subject Plus); Densitometers (e.g. X-Rite-508-Spectro Densitom who has experienced or exhibited symptoms of acute appen eter.R, The HYRYSTM 2 densitometer); automated Fluores dicitis or one or more of the following symptoms or risk cence in situ hybridization systems (see for example, U.S. factors: pain starting centrally (periumbilical) before localiz Pat. No. 6,136,540): 2D gel imaging systems coupled with ing to the right iliac fossa (the lower right side of the abdo 2-D imaging software; microplate readers; Fluorescence acti 25 men); loss of appetite and fever, nausea or vomiting; the vated cell sorters (FACS) (e.g. Flow Cytometer FACSVantage feeling of drowsiness; the feeling of general bad health; pain SE, Becton Dickinson); and radio isotope analyzers (e.g. beginning and staying in the right iliac fossa, diarrhea and a Scintillation counters). more prolonged, Smoldering course; increased frequency of In some embodiments, the appendicitis biomarker level is urination; marked retching; tenesmus or "downward urge' the appendicitis biomarker polypeptide level of any biomar 30 (the feeling that a bowel movement will relieve discomfort); ker listed in Table 1. In some embodiments, the appendicitis positive Rovsing's sign, Psoas sign, and/or Obturator sign. biomarker level is LRG polypeptide (SEQID NO:1). In some In one embodiment, the diagnostic tool or device is used to embodiments, the appendicitis biomarker level is ORM (SEQ test a urine sample from a subject who has experienced or ID NO:3) or MASP2 (SEQ ID NO:5). exhibited symptoms of acute appendicitis or one or more of In some embodiments, the system, computer-readable the following symptoms or risk factors: pain starting centrally media and methods as disclosed herein is used to measure at 35 (periumbilical) before localizing to the right iliac fossa (the least one appendicitis biomarker level in the biological lower right side of the abdomen); loss of appetite and fever; sample such as a urine sample. nausea or vomiting; the feeling of drowsiness; the feeling of In some embodiments, the system, computer-readable general bad health; pain beginning and staying in the right media and methods as disclosed herein is used to measure at iliac fossa, diarrhea and a more prolonged, Smoldering least one appendicitis biomarker level in urine biological 40 course; increased frequency of urination; marked retching; sample which is obtained from a mammalian Subject, for tenesmus or “downward urge' (the feeling that a bowel move example a human Subject. In some embodiments, the Subject ment will relieve discomfort); positive Rovsing's sign, Psoas has at least one symptom of appendicitis as discussed herein. sign, and/or Obturator sign. In some embodiments, the system, computer-readable The device or methods as disclosed herein can be used to media and methods as disclosed herein is used to measure at assess the urine sample from a subject at one or more indi least one appendicitis biomarker level in biological sample 45 cated times following specific experienced symptoms of the obtained from a subject who has experienced one or more Subject, such as initial symptoms (e.g., at about 1 hour, 2-5 symptoms of acute appendicitis include pain starting cen hours, 10 hours, 12 hours, 24 hours, 36 hours, 48 hours, trally (periumbilical) before localizing to the right iliac fossa and/or 72 hours. (the lower right side of the abdomen); loss of appetite and It should be understood that this invention is not limited to fever, nausea or vomiting; the feeling of drowsiness; the 50 the particular methodology, protocols, and reagents, etc., feeling of general bad health; pain beginning and staying in described herein and as such may vary. The terminology used the right iliac fossa, diarrhea and a more prolonged, Smolder herein is for the purpose of describing particular embodi ing course; increased frequency of urination; marked retch ments only, and is not intended to limit the scope of the ing; tenesmus or “downward urge' (the feeling that a bowel present invention, which is defined solely by the claims. movement will relieve discomfort); positive Rovsing's sign, 55 Psoas sign, and/or Obturator sign. DEFINITIONS OF TERMS In some embodiments, the system, computer-readable media and methods as disclosed herein comprises a determi Unless otherwise explained, all technical and scientific nation module which has been configured to determine the terms used herein have the same meaning as commonly level of an additional agent in the biological sample, for understood by one of ordinary skill in the art to which this example, albumin. 60 disclosure belongs. Definitions of common terms in urology, In some embodiments, the system, computer-readable endocrinology, biochemistry and molecular biology can be media and methods as disclosed herein is used to measure at found in The Merck Manual of Diagnosis and Therapy, 18th least one appendicitis biomarker level in a urine biological Edition, published by Merck Research Laboratories, 2006 sample to indicate if a Subject has, or is at risk of acute (ISBN 0-91 1910-18-2); Robert S. Porter et al. (eds.), The appendicitis. Accordingly, in some embodiments, the system, 65 Encyclopedia of Molecular Biology, published by Blackwell computer-readable media and methods as disclosed herein is Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. used to identify if a subject is has acute appendicitis. Meyers (ed.), Molecular Biology and Biotechnology: a Com US 8,535,891 B2 43 44 prehensive Desk Reference, published by VCH Publishers, The term “specific affinity” or “specifically binds” or “spe Inc., 1995 (ISBN 1-56081-569-8); The ELISA guidebook cific binding are used interchangeably herein refers to an (Methods in Molecular Biology 149) by Crowther J. R. entity Such as a protein-binding molecule or antibody that (2000); Fundamentals of RIA and Other Ligand Assays by recognizes and binds a desired polypeptide (e.g. a specific Jeffrey Travis, 1979, Scientific Newsletters; and Immunology appendicitis biomarker protein) but that does not substan by Werner Luttmann, published by Elsevier, 2006. tially recognize and bind other molecules in the sample, i.e. a Unless otherwise Stated, the present invention was per urine sample. In some embodiments, the term “specifically formed using standard procedures, as described, for example binds” refers to binding with a K of 10 micromolar or less, in Maniatis et al., Molecular Cloning: A Laboratory Manual, preferably 1 micromolar or less, more preferably 100 nM or Cold Spring Harbor Laboratory Press, Cold Spring Harbor, less, 10 nM or less, or 1 nM or less. N.Y., USA (1982); Sambrook et al., Molecular Cloning: A 10 The term “antibody' is meant to be an immunoglobulin Laboratory Manual (2 ed.), Cold Spring Harbor Laboratory protein that is capable of binding an antigen. Antibody as used Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., herein is meant to include antibody fragments, e.g. F(ab'), Basic Methods in Molecular Biology, Elsevier Science Pub Fab', Fab, capable of binding the antigen or antigenic frag lishing, Inc., New York, USA (1986); A. R. Kimmerl Eds. ment of interest. Academic Press Inc., San Diego, USA (1987)) and Current 15 The term “humanized antibody' is used herein to describe Protocols in Immunology (CPI) (John E. Coligan, et. al., ed. complete antibody molecules, i.e. composed of two complete John Wiley and Sons, Inc.), which are all incorporated by light chains and two complete heavy chains, as well as anti reference herein in their entireties. bodies consisting only of antibody fragments, e.g. Fab., Fab'. As used herein, the term “biomarker' is a biological char F(ab'), and Fv, wherein the CDRs are derived from a non acteristic that is measured and evaluated objectively as an human source and the remaining portion of the Ig molecule or indicator of normal biological or pathogenic processes (a fragment thereof is derived from a human antibody, prefer diagnostic biomarker), or a pharmacological response to ably produced from a nucleic acid sequence encoding a therapeutic intervention (a therapeutic biomarker). A “biom human antibody. arker” can be any patient parameter that can be measured, for The terms “human antibody” and “humanized antibody' example, mRNA expression profiles, proteomic signatures, are used herein to describe an antibody of which all portions protein, hormone or lipid levels, imaging methods or electri 25 or majority (at least 80%) of the antibody molecule are cal signals. Typically, the term “biomarker” as used herein derived from a nucleic acid sequence encoding a human anti refers to a protein, polypeptide or peptide in the sample. body. Such human antibodies are most desirable for use in The term “protein binding agent' is used interchangeably antibody therapies; as such antibodies would elicit little or no herein with “protein binding molecule' or protein binding immune response in the human Subject. moiety' and refers to any entity which has specific affinity for 30 The term "chimericantibody' is used herein to describe an a protein. The term “protein-binding molecule' also includes antibody molecule as well as antibody fragments, as antibody-based binding moieties and antibodies and includes described above in the definition of the term "humanized immunoglobulin molecules and immunologically active antibody.” The term "chimeric antibody encompasses determinants of immunoglobulin molecules, e.g., molecules humanized antibodies. Chimericantibodies have at least one that contain an antigen binding site which specifically binds portion of a heavy or light chain amino acid sequence derived (immunoreacts with) to the Psap proteins. The term “anti 35 from a first mammalian species and another portion of the body-based binding moiety” is intended to include whole heavy or light chain amino acid sequence derived from a antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and second, different mammalian species. In some embodiments, includes fragments thereof which are also specifically reac a variable region is derived from a non-human mammalian tive with the Psap proteins. Antibodies can be fragmented species and the constant region is derived from a human using conventional techniques. Thus, the term includes seg 40 species. Specifically, the chimericantibody is preferably pro ments of proteolytically-cleaved or recombinantly-prepared duced from a nucleotide sequence from a non-human mam portions of an antibody molecule that are capable of selec mal encoding a variable region and a nucleotide sequence tively reacting with a certain protein. Non limiting examples from a human encoding a constant region of an antibody. of Such proteolytic and/or recombinant fragments include In the context of this invention, the term “probe' refers to Fab, F(ab')2, Fab'. Fv, dAbs and single chain antibodies a molecule which can detectably distinguish between target (scFv) containing a VL and VH domain joined by a peptide 45 molecules differing in structure. Detection can be accom linker. The scFv's can be covalently or non-covalently linked plished in a variety of different ways depending on the type of to form antibodies having two or more binding sites. Thus, probe used and the type of target molecule, thus, for example, 'antibody-base binding moiety' includes polyclonal, mono detection may be based on discrimination of activity levels of clonal, or other purified preparations of antibodies and the target molecule, but preferably is based on detection of recombinant antibodies. The term “antibody-base binding 50 specific binding. Examples of Such specific binding include moiety' is further intended to include humanized antibodies, antibody binding and nucleic acid probe hybridization. Thus, bispecific antibodies, and chimeric molecules having at least for example, probes can include enzyme substrates, antibod one antigen binding determinant derived from an antibody ies and antibody fragments, and preferably nucleic acid molecule. In a preferred embodiment, the antibody-based hybridization probes. binding moiety detectably labeled. In some embodiments, a 55 The term “label” refers to a composition capable of pro “protein-binding agent' is a co-factor or binding protein that ducing a detectable signal indicative of the presence of the interacts with the appendicitis biomarker protein to be mea target polynucleotide in an assay sample. Suitable labels Sured, for example a co-factor or binding protein or ligand to include radioisotopes, nucleotide chromophores, enzymes, the appendicitis biomarker protein. Substrates, fluorescent molecules, chemiluminescent moi The term “labeled antibody', as used herein, includes anti eties, magnetic particles, bioluminescent moieties, and the bodies that are labeled by a detectable means and include, but 60 like. As such, a label is any composition detectable by spec are not limited to, antibodies that are enzymatically, radioac troscopic, photochemical, biochemical, immunochemical, tively, fluorescently, and chemiluminescently labeled. Anti electrical, optical or chemical means. bodies can also be labeled with a detectable tag, such as The term "agent” as used herein refers to a chemical entity c-Myc, HA, VSV-G, HSV, FLAG, V5, or HIS. The detection or biological product, or combination of chemical entities or and quantification of a appendicitis biomarker protein present 65 biological products. The chemical entity or biological prod in a urine samples correlate to the intensity of the signal uct is preferably, but not necessarily a low molecular weight emitted from the detectably labeled antibody. compound, but may also be a larger compound, for example, US 8,535,891 B2 45 46 an oligomer of nucleic acids, amino acids, or carbohydrates 5-fold, at least 10-fold increase or greater as compared to a including without limitation proteins, oligonucleotides, reference level, as that term is defined herein. ribozymes, DNAZymes, glycoproteins, siRNAS, lipopro The term “high as used herein generally means a higher by teins, aptamers, and modifications and combinations thereof. a statically significant amount relative to a reference; for the The term "agent” refers to any entity selected from a group s avoidance of doubt, “high” means a statistically significant comprising; chemicals; Small molecules; nucleic acid value at least 10% higher than a reference level, for example sequences; nucleic acid analogues; proteins; peptides; aptam at least 20% higher, at least 30% higher, at least 40% higher, ers; antibodies; or fragments thereof. A nucleic acid sequence at least 50% higher, at least 60% higher, at least 70% higher, may be RNA or DNA, and may be single or double stranded, at least 80% higher, at least 90% higher, at least 100% higher, and can be selected from a group comprising; nucleic acid at least 2-fold higher, at least 3-fold higher, at least 4-fold encoding a protein of interest, oligonucleotides, nucleic acid 10 higher, at least 5-fold higher, at least 10-fold higher or more, analogues, for example peptide-nucleic acid (PNA), pseudo as compared to a reference level. complementary PNA (pc-PNA), locked nucleic acid (LNA). As used herein, the terms “treat,” “treating, and “treat etc. Such nucleic acid sequences include, for example, but not ment” refer to the alleviation or measurable lessening of one limited to, nucleic acid sequence encoding proteins, for or more symptoms or measurable markers of a disease or example that act as transcriptional repressors, antisense mol- 15 disorder; while not intending to be limited to such, disease or ecules, ribozymes, Small inhibitory nucleic acid sequences, disorders of particular interest include ischemic or ischemia/ for example but not limited to RNAi, shRNAi, siRNA, micro reperfusion injury and diabetes. Measurable lessening RNAi (mRNAi), antisense oligonucleotides etc. A protein includes any statistically significant decline in a measurable and/or peptide agent can be any protein of interest, for marker or symptom. example, but not limited to; mutated proteins; therapeutic As used herein, the terms “prevent,” “preventing and “pre proteins; truncated proteins, wherein the protein is normally vention” refer to the avoidance or delay in manifestation of absent or expressed at lower levels in the cell. Proteins can one or more symptoms or measurable markers of a disease or also be selected from a group comprising; mutated proteins, disorder. Adelay in the manifestation of a symptom or marker genetically engineered proteins, peptides, synthetic peptides, is a delay relative to the time at which Such symptom or recombinant proteins, chimeric proteins, antibodies, midi marker manifests in a control or untreated Subject with a bodies, tribodies, humanized proteins, humanized antibodies, 25 similar likelihood or susceptibility of developing the disease chimeric antibodies, modified proteins and fragments or disorder. The terms “prevent,” “preventing” and “preven thereof. In some embodiments, the agent is any chemical, tion' include not only the complete avoidance or prevention entity or moiety, including without limitation synthetic and of symptoms or markers, but also a reduced severity or degree naturally-occurring non-proteinaceous entities. In certain of any one of those symptoms or markers, relative to those embodiments the agent is a small molecule having a chemical 30 symptoms or markers arising in a control or non-treated indi moiety. For example, chemical moieties included unsubsti vidual with a similar likelihood or susceptibility of develop tuted or substituted alkyl, aromatic, or heterocyclyl moieties ing the disease or disorder, or relative to symptoms or markers including macrollides, leptomycins and related natural prod likely to arise based on historical or statistical measures of ucts or analogues thereof. Agents can be known to have a populations affected by the disease or disorder. By “reduced desired activity and/or property, or can be selected from a severity' is meant at least a 10% reduction in the severity or library of diverse compounds. 35 degree of a symptom or measurable disease marker, relative The term "support” refers to conventional supports such as to a control or reference, e.g., at least 15%, 20%, 30%, 40%, beads, particles, dipsticks, fibers, filters, membranes and 50%. 60%, 70%, 80%, 90%. 95%,99% or even 100% (i.e., no silane or silicate Supports such as glass slides. symptoms or measurable markers). The terms “reduced' or “reduce' or “decrease' as used As used herein the term “reference level is used inter herein generally means a decrease by a statistically signifi- 40 changeably herein with “reference value' and refers to a level cantamount relative to a reference. However, for avoidance of in a particular appendicitis biomarker which provides a base doubt, “reduced” means statistically significant decrease of at line against which to compare the measured appendicitis least 10% as compared to a reference level, for example a biomarker protein level from the test urine biological sample. decrease by at least 20%, at least 30%, at least 40%, at least t As an illustrative example, the reference level for a particular 50%, or least 60%, or least 70%, or least 80%, at least 90% or as appendicitis biomarker protein can be calculated as the aver more, up to and including a 100% decrease (i.e. absent level age level of that appendicitis biomarker protein level from a as compared to a reference sample), or any decrease between plurality of urine biological samples obtained from a plurality 10-100% as compared to a reference level, as that term is of Subjects with similar demographics (i.e. age, gender, defined herein. weight, ethnicity and the like) which do not have appendicitis. The term “low” as used herein generally means lower by a As another illustrative example only, a reference level for a statically significant amount; for the avoidance of doubt, 50 particular appendicitis biomarker protein can be from a plu “low” means a statistically significant value at least 10% rality of Subjects that do not have appendicitis. As another lower than a reference level, for example a value at least 20% illustrative example only, a reference level for a particular lower than a reference level, at least 30% lower than a refer appendicitis biomarker protein can be from the same subject ence level, at least 40% lower than a reference level, at least taken at an earlier timepoint. Typically, a reference level is 50% lower than a reference level, at least 60% lower than a 55 normalized to “0” value, and an increase, for example at least reference level, at least 70% lower than a reference level, at about a 2-fold increase in the particular appendicitis biomar least 80% lower thana reference level, at least 90% lower than ker protein measured by the determination module or in the a reference level, up to and including 100% lower than a system and methods as disclosed herein relative to the refer reference level (i.e. absent level as compared to a reference ence level would indicate a subject would likely have appen sample). dicitis (i.e. a positive appendicitis test result). A reference The terms “increased' or “increase' as used herein gener 60 appendicitis biomarker level can be from an individual not ally mean an increase by a statically significant amount; for affected by a given pathology (i.e. not affected with appen the avoidance of doubt, “increased' means a statistically sig dicitis or having a symptom of appendicitis), or, alternatively, nificant increase of at least 10% as compared to a reference from the same individual being tested, where the urine for the level, including an increase of at least 20%, at least 30%, at reference appendicitis biomarker level was taken at an at least least 40%, at least 50%, at least 60%, at least 70%, at least 65 one earlier time point (i.e. to t t etc) when the Subject did 80%, at least 90%, at least 100% or more, including, for not exhibit a symptom of appendicitis. A reference appendi example at least 2-fold, at least 3-fold, at least 4-fold, at least citis biomarker level can also be a pooled sample, taken from US 8,535,891 B2 47 48 a plurality of individuals not affected by appendicitis. Where molecular mass values, given for nucleic acids or polypep appropriate, a reference appendicitis biomarker level can also tides are approximate, and are provided for description. be a fixed reference level of an appendicitis biomarker level, Although methods and materials similar or equivalent to where a test appendicitis biomarker level above the fixed those described herein can be used in the practice or testing of reference level (i.e. at least about 2-fold above the fixed this disclosure, suitable methods and materials are described reference level) identifies a subject likely to have appendici below. The abbreviation, "e.g. is derived from the Latin tis. It is preferred that a reference sample be from an indi exempligratia, and is used herein to indicate a non-limiting vidual or group of individuals of similar characteristics to the example. Thus, the abbreviation “e.g. is synonymous with tested individual, e.g., that the reference be taken from indi the term “for example.” viduals of similar age, gender, rave or ethnic background, etc. As used herein, the term "comprising means that other In some embodiments, other reference levels can also be 10 elements can also be present in addition to the defined ele used, for example apositive reference appendicitis biomarker ments presented, whether essential or not. The use of “com level can be used as a positive control for a subject having a prising indicates inclusion rather than limitation. risk of acute appendicitis. Typically, where a positive refer As used herein the term “consisting essentially of refers to ence level is used, if the appendicitis biomarker level in the those elements required for a given embodiment. The term test urine biological sample is Substantially the same or close 15 permits the presence of elements that do not materially affect in the value of the positive reference appendicitis biomarker the basic and novel or functional characteristic(s) of that level, it would indicate a positive test result for acute appen embodiment of the invention. dicitis. The term “consisting of refers to kits and methods thereof The term “computer can refer to any non-human appara as described herein, which are exclusive of any element not tus that is capable of accepting a structured input, processing recited in that description of the embodiment. the structured input according to prescribed rules, and pro All patents and other publications identified are expressly ducing results of the processing as output. Examples of a incorporated herein by reference for the purpose of describ computer include: a computer, a general purpose computer; a ing and disclosing, for example, the methodologies described Supercomputer, a mainframe; a Super mini-computer; a mini in Such publications that might be used in connection with the computer, a workstation; a micro-computer; a server, an present invention. These publications are provided solely for interactive television; a hybrid combination of a computer 25 their disclosure prior to the filing date of the present applica and an interactive television; and application-specific hard tion. Nothing in this regard should be construed as an admis ware to emulate a computer and/or software. A computer can sion that the inventors are not entitled to antedate such dis have a single processor or multiple processors, which can closure by virtue of prior invention or for any other reason. All operate in parallel and/or not in parallel. A computer also statements as to the date or representation as to the contents of refers to two or more computers connected together via a 30 these documents is based on the information available to the network for transmitting or receiving information between applicants and does not constitute any admission as to the the computers. An example of such a computer includes a correctness of the dates or contents of these documents. distributed computer system for processing information via The present invention can be defined by any of the follow computers linked by a network. ing alphabetized paragraphs: The term “computer-readable medium” may refer to any AA device for detecting at least one appendicitis biom storage device used for storing data accessible by a computer, 35 arker protein in a urine sample from a subject to identify as well as any other means for providing access to data by a if the subject is likely to have acute appendicitis, the computer. Examples of a storage-device-type computer-read device comprising: (a) at least one protein-binding agent able medium include: a magnetic hard disk; a floppy disk; an which specifically binds to at least one appendicitis optical disk, such as a CD-ROM and a DVD; a magnetic tape; biomarker protein selected from the group of leucine a memory chip. 40 C-2 glycoprotein (LRG), mannan-binding lectin serine The term “software' can refer to prescribed rules to operate protease 2 (MASP2), C-1-acid glycoprotein 1 (ORM); a computer. Examples of Software include: Software; code and (b) at least one solid Support for the at least one segments; instructions; computer programs; and pro protein binding-agent in (a), wherein the protein-bind grammed logic. ing agent is deposited on the solid Support. The term a “computer system” may refer to a system hav B. The device of paragraph Al, wherein the protein ing a computer, where the computer comprises a computer 45 binding agent deposited on the Solid Support specifically readable medium embodying software to operate the com binds the polypeptide of leucine C-2 glycoprotein puter. (LRG) of SEQID NO: 1. The term “proteomics' may refer to the study of the expres C. The device of paragraph Al, wherein the protein Sion, structure, and function of proteins within cells, includ binding agent deposited on the Solid Support specifically ing the way they work and interact with each other, providing 50 binds to the polypeptide of C-1-acid glycoprotein 1 different information than genomic analysis of gene expres (ORM) of SEQID NO:3. S1O. ID The device of paragraph Al, wherein the protein As used herein the term “consisting essentially of refers to binding agent deposited on the Solid Support specifically those elements required for a given embodiment. The term binds to the polypeptide of mannan-binding lectin serine permits the presence of elements that do not materially affect 55 protease 2 (MASP2) of SEQID NO: 5. the basic and novel or functional characteristic(s) of that EThe device of paragraph A., wherein the device further embodiment of the invention. comprises at least one additional different protein-bind Other than in the operating examples, or where otherwise ing agent deposited on the solid Support, wherein the indicated, all numbers expressing quantities of ingredients or additional protein-binding agent specifically binds to an reaction conditions used herein should be understood as appendicitis biomarker protein selected from the group modified in all instances by the term “about.” The term 60 consisting of leucine-rich C-2-glycoprotein (LRG); "about when used in connection with percentages may S100-A8 (calgranulin); C.-1.-acid glycoprotein 1 meant 1%. (ORM); lasminogen (PLG); mannan-binding lectin The singular terms “a,” “an and “the include plural ref serine protease 2 (MASP2); zinc-C-2-glycoprotein erents unless context clearly indicates otherwise. Similarly, (AZGP1); apolipoprotein D (Apol)); C.-1-antichymot the word 'or' is intended to include “and” unless the context 65 rypsin (SERPINA3). clearly indicates otherwise. It is further to be understood that F.The device of paragraph Al, wherein the device further all base sizes oramino acid sizes, and all molecular weight or comprises at least one additional different protein-bind US 8,535,891 B2 49 50 ing agent deposited on the solid Support, wherein the S. The method of any of paragraphs PI-R), wherein the additional protein-binding agent specifically binds to an measuring is completed with the use of an immunoassay appendicitis biomarker protein selected from the group or an automated immunoassay. consisting of Adipocyte specific adhesion molecule: TThe method of any of paragraphs PI-S, wherein the AMBP; Amyloid-like protein 2: Angiotensin converting appendicitis biomarker protein is leucine C-2 glycopro enzyme 2: BAZ1B: Carbonic anhydrase 1: CD14; chro tein (LRG). mogranin A; FBLN7; FXR2; Hemoglobin C.; Hemoglo UThe method of any of paragraph (P-T), wherein the bin B; Interleukin-1 receptor antagonist protein; Inter appendicitis biomarker is C.-1.-acid glycoprotein 1 C-trypsin inhibitor; Lipopolysaccharide binding (ORM). protein; Lymphatic vessel endothelial hyaluronan acid 10 IV. The method of any of paragraphs P-U, wherein the receptor 1; MLKL: Nicastrin; Novel protein (Accession appendicitis biomarker protein is mannan-binding lectin No: IPIO0550644); PDZK1 interacting protein 1: serine protease 2 (MASP2) PRIC285; Prostaglandin-H2 D-isomerase: Rcl: S100 WThe method of any of paragraphs PI-S, wherein the A9; Serum amyloid A protein; SLC13A3: SLC2A1; appendicitis biomarker protein is selected from a group SLC2A2: SLC4A1 SLC9A3; SORBS1, SPRX2: 15 consisting of leucine C-2 glycoprotein (LRG), calgranu Supervillin: TGFbeta2R: TTYH3; VAOD1; Vascular lin A (S100-A8), C-1-acid glycoprotein 1 (ORM), plas adhesion molecule 1: Versican; V1P36; CL-1-acid glyco minogen (PLG), mannan-binding lectin serine protease protein 2; and B-1,3-galactosyltransferase. 2 (MASP2), Zinc-C-2-glycoprotein (AZGP1), C-1-an G|The device of paragraph Al, wherein the solid support tichymotrypsin (SERPINA3) and apolipoprotein D is in the format of a dipstick, microfluidic chip or a (Apod). cartridge. DXThe method of any of paragraphs P-W), wherein the H The device of any of paragraphs A to G, wherein the reference level is a level of the appendicitis biomarker protein-binding agent is an antibody, antibody fragment, protein in a urine sample of a healthy human not having aptamer, Small molecule or variant thereof. acute appendicitis. I The device of any of paragraphs. A to H, wherein the Y The method of any of paragraphs P-W), wherein the Subject is a human Subject. 25 reference level is an average level of the appendicitis JThe device of any of paragraphs. A to II, wherein the biomarker protein in a plurality of urine samples from a Subject is a Subject with at least one symptom of appen population of healthy humans not having acute appen dicitis. dicitis. KThe device of any of the paragraphs. A to J, wherein ZThe method of any of paragraphs P-W), wherein the the protein-binding agent deposited on the device spe 30 reference level is a normalized level of the appendicitis cifically binds to the appendicitis biomarker protein biomarker protein in a urine sample of a healthy human when the level of the appendicitis biomarker protein is at not having acute appendicitis, wherein the normaliza least 2-fold above a reference level for that biomarker tion is performed against the level of albumin in the urine protein. sample of a healthy human not having acute appendici LA device of paragraph K. wherein the reference level tis. is an average level of the appendicitis biomarker protein 35 AA. The method of any of paragraphs P-Z, wherein the in a plurality of urine samples from a population of urine sample is collected in mid-stream. healthy humans not having acute appendicitis. BBThe method of any of paragraph P-Z, wherein the M. Use of the device of any of paragraphs A to L to urine sample is obtained by depositing the urine on to a identify if a Subject to have acute appendicitis, wherein test strip. if at least one appendicitis biomarker protein specifically 40 This invention is further illustrated by the following binds to at least one protein-binding agent, the Subject is examples which should not be construed as limiting. The likely to have acute appendicitis. contents of all references, patents, and patent applications N A kit comprising: (a) a device according to any of cited throughout this application, as well as the figures and paragraphs. A to IL; and (b) a first agent, wherein the table are incorporated herein by reference. first agent produces a detectable signal in the presence of a protein-binding agent which deposited on the device is 45 Example 1 specifically bound to an appendicitis biomarker protein. OThe kit of paragraph N. further comprising a second Urine Proteomics for Profiling of Human Disease agent, wherein the second agent produces a different Using High Accuracy Mass Spectrometry detectable signal in the presence of a second protein binding agent deposited on the device which is specifi 50 Knowledge of the biologically relevant components of cally bound to a second appendicitis biomarker protein. human tissues has enabled the invention of numerous clini PA method to identify the likelihood of a subject to have cally useful diagnostic tests, as well as non-invasive ways of acute appendicitis comprising: (a) measuring the level of monitoring disease and its response to treatment. By virtue of at least one appendicitis biomarker protein selected from tissue perfusion, blood serum is the most useful material for the group listed in Table 1 in a urine sample from the 55 the discovery of such biomarkers in general. However, the human Subject; (b) comparing the level of the at least one relatively high concentration of serum proteins, as well as appendicitis biomarker protein measured in step (a) to a their wide range of concentrations, spanning at least 9 orders reference level for the measured biomarker; wherein if of magnitude, often limit the study of serum biomarkers 1. the level of the measured appendicitis biomarker protein though several recent approaches are promising 2-4. is at least 2-fold increased than the reference level for the On the other hand, of the biological fluids amenable to appendicitis biomarker protein, it identifies the Subject 60 routine clinical evaluation, urine has the advantage of being is likely to have acute appendicitis. frequently and non-invasively available, abundant, and as a QThe method of paragraph P. further comprising deter result of being a filtrate of serum, relatively simple in its mining the level of albumin in the urine sample from the composition. Consequently, detection of urinary proteins has human Subject. been used to identify markers of disease affecting the kidney R. The method of any of paragraphs P-Q, wherein the 65 and the urogenital tract 5, 6, as well as distal organs such as human exhibits at least one symptom of acute appendi the brain and the intestine 7, 8). However, the current under citis. standing of the human urinary proteome is incomplete, spe US 8,535,891 B2 51 52 cifically with respect to its overall composition and dynamics, proteins and peptides were resuspended in aqueous 50 mM not to mention the identity of variable components that may ammonium bicarbonate buffer (pH 8.5). dependent on physiologic state and disease. Protein Precipitation. Several approaches have been used to characterize the Proteins remaining in Solution after cation exchange were human urinary proteome. Initial studies using electrophoresis precipitated by adding trichloroacetic acid to 20% (w/v), with and immunoblotting were able to identify tens of abundant deoxycholate to 0.02% (w/v) and TritonX-100 to 2.5% (v/v) and rare urinary proteins 9. Recently, Pisitkun and col as carriers, and incubating the samples for 16 hours at 4°C. leagues applied ultracentrifugation and liquid chromatogra Precipitates were sedimented at 10,000 g for 15 minutes at 4 C. and pellets were washed twice with neat acetone at 4°C. phy (LC)-tandem mass spectrometry (MS/MS) to identify with residual acetone removed by air drying. Dried pellets 295 highly abundant unique proteins isolated from urinary 10 were resuspended in 0.1 ml of 1 x Laemmli buffer. exosomes 10. Sun and colleagues identified 226 soluble Gel Electrophoresis. proteins by using multidimensional LC-MS/MS 11. For an Laemmli buffer suspended fractions (from 17,000 g and overview, see Pisitkun et al 12. And most recently, Adachi 210,000 g centrifugation, and from protein precipitation) and colleagues identified more than 1,500 unique proteins were incubated at 70° C. for 15 min and separated by using from ultrafiltered urine with a high degree of accuracy by 15 NuPage 10% polyacrylamide Bis-Tris gels according to using a hybrid linear ion trap-Orbitrap (LTQ-Orbitrap) mass manufacturers instructions (Invitrogen). Gels were washed spectrometer 13. three times with distilled water, fixed with 5% (v/v) acetic The inventors herein extend the current characterization of acid in 50% (v/v) aqueous methanol for 15 minutes at room the human urinary proteome by extensively fractionating temperature, and stained with Coomassie. Each gel lane was urine using ultracentrifugation, gel electrophoresis, ion cut into 6 fragments and each fragment was cut into roughly exchange and reverse phase chromatography, effectively 1 mm particles, which were subsequently washed 3 times reducing mixture complexity while minimizing loss of mate with water and once with acetonitrile. rial. By using high accuracy mass measurements of the LTQ Protein Reduction, Alkylation and Trypsinization. Orbitrap mass spectrometer and LC-MS/MS of peptides gen Protein containing gel particles and cation exchange puri erated from Such extensively fractionated specimens, the fied proteins were reduced with 10 mM dithiotreitol in 50 mM inventors identified over 2,000 unique proteins in routinely 25 ammonium bicarbonate (pH 8.5) at 56° C. for 45 minutes. collected individual urine specimens. The inventors provide They were subsequently alkylated with 55 mM iodoaceta assessments of the physical and tissue origins of the urinary mide in 50 mM ammonium bicarbonate (pH 8.5) at room proteome, as well as dependence of its detection on instru temperature in darkness for 30 minutes. Gel particles were mental and individual variables. Finally, by using text mining washed 3 times with 50 mMammonium bicarbonate (pH 8.5) and machine learning the inventors annotate the urinary pro 30 prior to digestion. Alkylated peptides were purified by using teome with respect to 27 common and more than 500 rare PepClean C-18 spin columns as described above to remove human diseases, thereby establishing a widely useful residual iodoacetamide from the cation exchange fraction. resource for the study of human pathophysiology and biom They were then digested with 12.5 ng/ul sequencing grade arker discovery. bovine trypsin in 50 mMammonium bicarbonate (pH 8.5) at Materials and Methods for Example 1 37° C. for 16 hours. Tryptic products were purified by using Sample Collection. 35 PepClean C-18 spin columns as described above, vacuum Urine was collected as clean catch, mid stream specimens centrifuged and stored at -80°C. as part of routine evaluation of 12 children and young adults Mass Spectrometry and Liquid Chromatography. (ages 1-18 years, median 11) presenting with acute abdomi Fractions containing tryptic peptides dissolved in aqueous nal pain in the Children’s Hospital Boston's Emergency 5% (v/v) acetonitrile and 0.1% (v/v) formic acid were Department. Upon obtaining informed consent, urine was 40 resolved and ionized by using nanoflow high performance frozen at -80°C. in 12 ml aliquots in polyethylene tubes. All liquid chromatography (nanoLC, Eksigent) coupled to the samples were frozen within 6 hours of collection. LTQ-Orbitraphybrid mass spectrometer (Thermo Scientific). Reagents. Nanoflow chromatography and electrospray ionization were All reagents were of highest purity available and purchased accomplished by using a 15 cm fused silica capillary with 100 from Sigma Aldrich unless specified otherwise. HPLC-grade mm inner diameter, in-house packed with Magic C18 resin solvents were purchased from Burdick and Jackson. 45 (200 A, 5tm, Michrom Bioresources). Peptide mixtures were Urine Sedimentation. injected onto the column at a flow rate of 1000 ml/min and Aliquots were thawed and centrifuged at 17,000 g for 15 resolved at 400 ml/min using 45 minlinear acetonitrile gradi minutes at 10° C. to sediment cellular debris. Absence of ents from 5 to 40% (v/v) aqueous acetonitrile in 0.1% (v/v) intact cells in the sediment was confirmed by light micros formic acid. Mass spectrometer was operated in data depen copy (data not shown). Subsequently, Supernatant was cen 50 dent acquisition mode, recording high accuracy and high trifuged at 210,000 g for 60 minutes at 4°C. to sediment resolution Survey Orbitrap spectra using the lock mass for vesicles and high molecular weight complexes. Resultant internal mass calibration, with the resolution of 60,000 and pellets were resuspended in 0.5 ml of 0.1x Laemmli buffer, m/Z range of 350-2000. Six most intense multiply charged concentrated 10-fold to 0.05 ml by vacuum centrifugation ions were sequentially fragmented by using collision induced and stored at -80° C. 55 dissociation, and spectra of their fragments were recorded in Cation Exchange Chromatography. the linear ion trap, with the dynamic exclusion of precursor Supernatant remaining after ultracentrifugation was ions already selected for MS/MS of 60 sec. diluted 5-fold with 0.1 Macetic acid, 10% (v/v) methanol, pH Spectral Processing and Peptide Identification. 2.7 (Buffer A) and incubated with 1 ml 50% (v/v) slurry of SP Custom written software was used to extract the 200 most Sephadex (40-120.im beads, Amersham) for 30 minutes at 4 intense peaks from each MS/MS spectrum and to generate C. to adsorb peptides that are <30 kDa molecular weight. 60 mascot generic format files. Peak lists were searched against Upon washing the beads twice with Buffer A, peptides were the human International Protein Index database (version3.36. eluted by incubating the beads in 5 ml of 0.5 Mammonium at the World WideWebsite of “ebi.ac.uk/IPI) by using Mas acetate, 10% (v/v) methanol, pH 7 for 30 minutes at 4°C. cot (version 2.1.04; Matrix Science), allowing for variable Eluted peptides were purified by reverse phase chromatogra formation of N-pyroglutamate, ASn and Gln deamidation, phy by using PepClean C-18 spin columns, according to 65 N-acetylation, and methionine oxidation, requiring full manufacturers instructions (Pierce). Residual purification trypsin cleavage of identified peptides with 2 possible mis Solvents were removed by vacuum centrifugation and Small cleavages, and mass tolerances of 5 ppm and 0.8 Da for the US 8,535,891 B2 53 54 precursor and fragmentions, respectively. Searches allowing increase stringency of the protein disease literature associa semi-tryptic peptides did not affect overall search yields (data tions, the inventors estimated the odds that the number of not shown). Spectral counts were calculated by Summing the overlapping documents found between a given disease and number of fragmention spectra assigned to each unique pre protein could occur by chance, considering the number of cursor peptide. documents matching either the disease or the proteins in Data Analysis. Medline. Only protein name/disease name pairs with odds Assessment of identification accuracy was carried out by ratio greater than 2,000 were reported. Lists of overlapping searching a decoy database composed of reversed protein documents were formatted in HTML files organized in hier sequences of the target IPI database. Frequency of apparent archies of diseases or proteins. false positive identifications was calculated by merging indi List of Abbreviations. vidual target and decoy searches for each sample. An initial 10 Liquid chromatography-tandem mass spectrometry (LC estimate of the apparent false positive rate was obtained by MS/MS), linear ion trap (LTQ). dividing the number of peptide identifications with a Mascot Results score greater than the identity score obtained from the target Exhaustive Protein Capture from Routinely Collected search by the number of peptide identifications with a score Human Urine higher than the identity score threshold extracted from the 15 In order to identify medically useful urinary proteins, the decoy search 37. Only proteins identified on the basis of inventors obtained urine as routinely collected clean catch, more than 2 peptides were included in the comparison. Par mid stream urine specimens, collected at the time of clinical Simonious protein grouping was performed by remapping all evaluation. The inventors examined urine samples from 12 peptide identifications onto their corresponding proteins as children and young adults evaluated for abdominal pain in our listed in the IPI. This step was necessary to generate a mini Emergency Department, all of whom were previously mal, non-redundant list of proteins that explain all of the healthy. Also examined were the urine samples from asymp identified peptides, while excluding proteins that could not be tomatic patients evaluated 6-8 weeks after they underwent unambiguously unidentified. This parsimonious list of pro appendectomies. Allurines exhibited normal profiles without teins was used for comparisons of various samples at the evidence of renal disease or infection, as assessed by using protein level. For Gene Ontology annotation, the inventors clinical urinalysis (data not shown). Allurine specimens were used GO slim terms version 1.8, accessed by using GOfact (at 25 frozen within 6 hours of collection, consistent with earlier the WorldWideWebsite of "hupo.org.cn/GOfact”). For anno temporal analysis of whole urine specimens which indicated tation of tissue expression of detected proteins, the inventors that no detectable degradation occurred for as long as 24 used version 2 of the GNF gene expression atlas (at the World hours of 4°C. refrigerated Storage with Subsequent freezing at Wide Website of “expression.gnif.org), accessed by using -80° C. 14-16. This is expected given the fact that urine is BioMart (at the World Wide Website of “biomart.org). 30 stored in situ for many hours in the bladder, reaching a physi Disease Annotations. ologic equilibrium prior to collection. The inventors linked proteins found in the urine proteome Urine is a complex mixture with abundant proteins such as to published articles that associate a protein with a human albumin and uromodulin obscuring the identification of less disease, as well articles that associate a disease with a protein. concentrated, biologically more informative proteins such as For the former, the inventors derived sets of diseases from secreted cytokines and hormones for example. Thus, the OMIM 38, MeSH (at the World Wide Website of “nlm.ni 35 inventors adopted a fractionation method that reduced mix h.gov/mesh/), and a shortlist of common diseases of interest ture complexity while minimizing loss of material by first not described in OMIM or MeSH (Additional Files, at the ultracentrifugating to fractionate urinary exosomes and other World Wide Website of “childrenshospital.org/research/ high molecular weight complexes from Soluble peptides and steenlab'). The inventors extracted disease names from proteins, Subsequently capturing the latter by using size MeSH by selecting MeSH concepts with Descriptor Record 40 excluded cation exchange chromatography and trichloroace Descriptor Class=1, and marked by SemanticTypeName tic acid precipitation, respectively, which has been shown to Disease or Syndrome. Synonym disease names were capture more than 95% of proteins under similar conditions obtained from the content of Term or TermList elements for 17, 18. the main concept. For OMIM, documents matching an Secondary and tertiary fractionations of thus captured pro OMIM entry were obtained by searching Medline with a teins and peptides were achieved by using one dimensional query of the form (Term 1 OR Term2 . . . . Termk), where 45 SDS-PAGE of the ultracentrifugation and precipitation frac Termk include the 100 lowest frequency terms in a given tions, and liquid chromatography of the tryptic peptides of OMIM entry. These OMIM disease queries were executed by SDS-PAGE resolved proteins, respectively. As a result, high using Twease with the BM25EC scorer against abstracts in abundance proteins such as albumin and uromodulin, which Medline 39, accessedJul. 7, 2008. Documents that matched would otherwise comprise more than 99% of the mixture, can the query with a BM25EC score above a Z-score of 10 were 50 be separated effectively from the bulk of the proteome (FIG. considered matching the OMIM disease 40. Each MeSH 1). Though the composition and concentration of urine varies disease name and synonyms were expressed as a query of the with physiologic state, there was less than 10+10% form (“disease name” “alias 1” “alias 2 . . . ). Common (meant standard deviation) difference in total protein abun disease names were expressed as a single phrase query. dance among individual specimens, as ascertained by using To determine diseases that are associated with a given 55 gel image densitometry (FIG. 1), similar to earlier studies of protein, the inventors queried BioMart by using IPI identifiers urine of children 19-21. for proteins in the urine proteome to obtain corresponding Accurate and Comprehensive Identification of Urinary Pro protein descriptions and gene names. Queries of the form teones (IPI-id "description' GeneName) were generated for each In order to maximize detection sensitivity while minimiz protein, where IPI-id is the IPI identifier, and description is ing identification errors, the inventors used the recently devel the description phrase retrieved from BioMart. These queries 60 oped hybrid LTQ-Orbitrap mass spectrometer for tryptic pep were run against Medline by using Twease with the slider tide sequencing of the above fractionated proteomes. A parameter set to 0. Lists of documents matching protein representative set of tandem mass spectra is shown in FIG. 2, names were stored and overlapped with lists of documents achieving mass errors of less than 2 ppm for the majority of matching diseases. Pairs of disease associated proteins that the LC/MS runs as judged from analysis of trypsin autolysis matched less than 5 documents were discarded (manual 65 peptides (FIG. 3). Peptide sequences were identified from examination indicated that this level of overlap frequently tandem mass spectra by using probability based Mascot happens as an artifact of the search procedure). To further searches of the human IPI database (Methods). By carrying US 8,535,891 B2 55 56 out simultaneous searches of the data against a decoy data in angiogenesis and vascular homeostasis, and is expressed base containing reversed protein sequences, and rejecting by the vascular endothelium 27. (false) identifications of spectra that matched decoy Individual Urinary Proteomes sequences, as well as excluding proteins identified on the By virtue of studying individual urinary proteomes, the basis of single peptides, the inventors were able to achieve an 5 inventors assessed the extent of similarities and differences apparent false positive protein identification frequency of less among them. For the 12 specimens studied in this example, than 1%. The median number of unique peptides per identi the inventors detected 1,124+292 (meanistandard deviation) fied protein was 10. proteins per individual proteome, with the average concor As a result, the inventors identified with high degree of dance of 68%, as calculated over all binary comparisons. accuracy 12, 126 unique peptides, corresponding to 2.362 10 Highly abundant proteins common to all individual pro proteins. These proteins include 891 proteins identified in an teomes include molecules involved in renotubular trafficking earlier high accuracy study of the human urine proteome 13. (uromodulin, cubilin, and megalin (LRP2)), serum filtered and more than 1,000 additional proteins identified for the first enzymes and carriers (bikunin (AMBP), aminopeptidase N. time (FIG. 4). These data are provided as Additional Files, ceruloplasmin, apolipoproteins, and immunoglobulins), and can be accessed publicly from the inventors server (at the 15 extracellular structural components (perlecan, glial fibrillary World Wide Website of “childrenshospital.org/research/ acidic proteins), as well as a variety of other secreted mol steenlab'). ecules Such as CD44, tetraspanin, and lysosomal associated Origin of the Human Urinary Proteome membrane proteins (LAMPs). Many of these have been The composition of the identified proteomes was charac detected in human urine previously, and many were identified terized with respect to Gene Ontology (GO) annotated bio for the first time. Examples of the latter include claudin, a logical function, apparent physical origin, and predicted tis regulator of tight junctions involved in the maintenance of sue expression. As compared with the entire list of IPI entries, glomerular and tubular integrity 28, collectrin, a novel analysis of GO annotated biological function revealed Satu homolog of the angiotensin converting enzyme related car ration of cellular components such as the cytoplasm, endo boxypeptidease implicated in renal failure and the pathogen plasmic reticulum, golgi, lysosome, and the plasma mem esis of polycystic kidney disease 29, SLC5A2, a tubular brane. Proteins from the nucleus were relatively under 25 Sodium-glucose transporter which causes autosomal reces represented, consistent with the general absence of intact sive renal glucosuria when defective 30, and numerous cells in human urine. Similar to 13, the inventors observed other proteins with poorly understood functions such as peflin a relative enrichment of hydrolases, peptidases, carbohydrate and trefoil factor 2. and lipid binding proteins, and a relative under-representation In large part, the variability observed among individual of nucleic acid binding proteins. 30 proteomes appears to be multifactorial in origin, as Suggested By comparing whether identified proteins sedimented in by the multimodal distribution of the coefficients of variation the 17,000 g versus 210,000 g ultracentrifugation fractions, of proteins apparent detectability, as measured by using were adsorbed onto size exclusion ion exchange resin or were spectral counting 31 (FIG. 5; representative proteins are TCA precipitated, the inventors defined them as large or small labeled). Proteins with high degree of apparent variability complexes, and soluble peptides or proteins, respectively. included complement factors, C.1-anti-trypsin, protein C The fractions of proteins identified uniquely from these 35 inhibitor, galectin (LGALS3BP), CD59, CD14, C-enolase, physical states were 14, 20, 3 and 9%, respectively, demon C.2-macroglobulin, gelsolin, haptoglobin, hemopexin, strating that individual proteins or their variants exist in mul intelectin, fibrinogen, arylsulfatase, serum amyloid A2, cys tiple physical states. For example, components of the urinary tatin C, angiotensin, and resistin, among others. Many of exosomes including the endosomal sorting complex (ESCRT these proteins are components of the acute phase response I), BRO1/ALIX, and VPS4, were detected as both small com 40 32, consistent with the collection of some of the studied plexes and soluble proteins. Similarly, insulin-like growth specimens from patients with acute abdominal pain. Other factor binding proteins (IGFBPs) which are low molecular differences among proteomes included components of semi weight circulating hormones were detected as soluble pro nal fluid and other sex specific proteins such as semenogelin. teins, peptides, and in Small complexes. Though the size Urine Proteomics for Profiling of Human Disease excluded ion exchange fraction contributed only 3% to the The inventors annotated the identified urinary proteins total unique protein identifications, it was substantially 45 with respect to possible associations with human disease by enriched for biomedically significant molecules which would using machine learning and text mining of Medline abstracts. not be detected otherwise, including circulating hormones Annotations identified for the 26 common and more than 200 Such as hepcidin and chromogranin 22, 23, and shed cell rare examined diseases are available in hypertext documents Surface molecules such as Ly-6 and platelet glycoproteins (Additional Files, http://www.childrenshospital.org/re 24, 25. 50 search/steenlab), with links to information about the identi The inventors assessed the probable tissue origin of the fied proteins and original studies about their role in disease. identified proteome by comparing it to published tissue They include common kidney diseases such as nephrotic expression atlases. As expected, 90% of the proteins detected syndrome (72 proteins) and nephritis (139), systemic ill in the urinary proteome have tissue expression profiles that nesses such as sepsis (42), diseases of distal organs Such as include organs of the urogenital tract, Such as the kidneys and 55 pneumonia (34), meningitis (22), and colitis (45). In addition, the bladder, from which they likely originate. In addition to the proteome was annotated with respect to more than 500 these proximal organs, the urinary proteome contains a Sub rare diseases, including storage diseases such as Niemann stantial number of proteins that appear to originate from distal Pick disease, immune system disorders such as Wiskott-Al tissues. Among them are 336 proteins that are uniquely drich syndrome, and diseases of the nervous system such as expressed in distal tissues such as the nervous system, heart spinocerebellar ataxia. 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Acta Paediatr 2005, 94:1732-7. inspired double mutant recovery experiment. In: TREC: 20. EF De Palo, R Gatti, F Lancerin, E Cappellin, A Sartorio, 2006: Gaithersburg, Md., USA. 836-850. P Spinella: The measurement of insulin-like growth fac 65 40. F Campagne: Objective and automated protocols for the tor-I (IGF-I) concentration in random urine samples. Clin evaluation of biomedical search engines using No Title Chem Lab Med 2002, 40:574-8. Evaluation protocols. BMC Bioinformatics 2008, 9:132. US 8,535,891 B2 59 60 Example 2 The expected number of patients was estimated by using the Pearson 2 test to detect a difference at a two-sided statistical Appendicitis is among many human diseases, for which the significance level of 5% and power of 90% that requires 6 diagnosis is complicated by the heterogeneity of its clinical patients in each group, assuming that 80% of the positive presentation and shortage of diagnostic markers. As such, it samples (5 patients) will contain at least one protein unique to remains the most common Surgical emergency of children, the appendicitis as compared to the non-appendicitis group with initial diagnosis accuracy additionally challenged (21). This study was approved by the Children’s Hospital because of non-specific but similar symptoms of many other childhood conditions (1). Delays inaccurate diagnosis lead to Boston Committee on Clinical Investigation, began in increased mortality, morbidity, and costs associated with the November of 2006, and ended in May of 2008. complications of appendicitis. 10 Discovery Urine Proteome Profiling and Validation Target The use of high resolution computed tomography (CT) to Mass Spectrometry. identify appendiceal inflammation was hoped to improve For the discovery of markers, thawed 10 ml urine aliquots both the diagnosis and treatment of acute appendicitis. were fractionated by using ultracentrifugation, cation Though variable, these improvements have been modest at exchange chromatography, protein precipitation, polyacryla best, with rates of unnecessary appendectomies and ruptures 15 mide gel electrophoresis, and reverse phase liquid chroma of 3-30% and 30-45%, respectively (2-4). In addition, avail tography. Their protein composition was discovered by using ability of and experience with CT limit the usefulness of this liquid chromatography tandem mass spectrometry (LC-MS/ approach. Furthermore, recently its use has been re-evaluated MS) using a nanoflow HPLC system (Eksigent) coupled to a due to concerns of cancer risk (5). hybrid linear ion trap-Orbitrap (LTQ-Orbitrap) mass spec Thus, several studies sought to identify laboratory markers trometer (Thermo Scientific). The LTQ-Orbitrap enables an of acute appendicitis, by studying both markers of the acute unprecedented combination of high detection sensitivity in phase response, as well as specific inflammatory mediators. the attomolar (10-18M) range, and high mass accuracy of less The performance of both appeared to be limited (6-11), likely than 2 parts per million (0.001 Da for a typical 500 Da because of the non-specific and unrelated mechanisms of peptide), as described in detail in the accompanying manu their elevation during acute appendicitis which is character Script (22). Validation of markers was performed using 1 ml ized specifically by the infiltration of neutrophils and release 25 aliquots of coded specimens that were blinded to the final of distinct cytokines (12, 13). outcome. The entire experimental procedure is schematized As disclosed herein, the inventors using an unbiased in FIG. 7. approach, have profiled the molecular alterations on a pro Analysis. teomic scale, including molecules that are being secreted Urine markers were ranked by calculating relative enrich locally by the diseased tissues themselves or produced sys 30 ment ratios (RER) of detection in appendicitis versus non temically in response to local disease. The inventors have appendicitis groups by Summing individual protein spectral identified various urinary markers for appendicitis. Because counts normalized to the spectral counts of albumin to urine is abundant, obtained frequently and non-invasively, account for small differences in total protein abundance, (23) and as a result of being a serum filtrate, is relatively simple in where RER (appendicitis) XC/C/(non-appendicitis) XC/ its composition, the inventors have discovered urinary mark C, with C, and C denoting spectral counts of protein mark ers for the use in an simple and rapid method to identify a 35 ers and albumin, respectively. Urinary markers were addi Subject with appendicitis. tionally ranked by assessing the prevalence of their detection Recently, advanced mass spectrometry (MS) has been used among different specimens by using a uniformity parameter effectively to discover the protein composition of human (U), calculated by dividing the number of appendicitis cases urine, (14-16) and to identify markers of diseases affecting in which they were detected by the total number of appendi the kidney (17) and the urogenital tract (18). Similarly, MS 40 citis cases. Urinary markers were filtered to have U>0.7 and studies of urine have been used to study proteins produced by RER >5 to identify those that were variably detected or insuf distal organs such as the brain (19) and the intestine, (20) and ficiently enriched, respectively. Support vector machine to relate them to braininjury and inflammatory bowel disease, analysis and comparison of urine protein markers with tissue respectively. gene expression profiles of diseased appendices were carried Here, the inventors demonstrate the use of urine proteome as described herein. The latter was based on a previous study profiling and have discovered urinary markers of acute 45 (24). Receiver operating characteristics were calculated using appendicitis. By using high accuracy mass spectrometry, the standard methods. inventors identified more than 2,000 unique proteins in urine Outcome Measures. specimens routinely collected from children and young adults Final diagnosis was determined by the presence or absence evaluated for acute abdominal pain in the emergency depart of appendicitis on gross and histological examination. All ment (ED). Statistical comparisons of individual urine pro 50 appendectomy specimens were reviewed by a clinical teomes, pattern recognition class prediction, and gene expres pathologist, and their disease assignments were confirmed by sion profiling of diseased appendices were used to discover an independent, blinded review. One patient with perforated diagnostic markers. By carrying out a blinded, prospective appendicitis underwent an interval appendectomy, and was study of these markers, the inventors assessed their diagnostic not included in the histologic review. Assessment of the his performance. 55 tologic severity of appendicitis was done by classifying the Methods Use in Example 2 specimens as having: no inflammatory changes (normal); foci Study Population. of neutrophilic infiltration in mucosa or wall (focal); scattered The inventors studied 67 children and young adults who transmural infiltration (mild); dense transmural infiltration presented to the EDSuspected of having acute appendicitis. with tissue distortion (moderate); dense transmural infiltra Patients were excluded if they had pre-existing autoimmune, tion with tissue necrosis or wall perforation (severe). For neoplastic, renal or urologic disease or were pregnant. Urine 60 patients who did not undergo appendectomies, the outcome was collected as clean catch, mid stream samples as part of was confirmed via telephone 6-8 weeks after the ED evalua routine ED evaluation of abdominal pain. Additional intra tion. All patients enrolled in the study received a final out individual control specimens were collected from selected COC. patients with appendicitis after undergoing appendectomies. Tissue Immunohistochemistry and Urine Immunoblotting. Informed consent was obtained prior to knowledge of final 65 Immunohistochemical staining of formalin fixed, paraffin diagnosis and the urine remaining in the laboratory was embedded appendices was performed by using the rabbit retrieved and stored at -80° C. within 6 hours of collection. anti-LRG polyclonal antibody at 1:750 dilution (Atlas Anti US 8,535,891 B2 61 62 bodies), OmniMap DAB anti-rabbit HRP detection kit and TABLE 3-continued the Ventana Discovery XT automated slide processing plat form, according to the manufacturers instructions (Ventana Final diagnosis of the 67 study patients Medical Systems). Staining specificity was confirmed by Number of patients using liver and muscle as the positive and negative controls, 5 respectively (data not shown). Influenza or scarlet fever For immunoblotting of urine, specimens were precipitated Intussusception and resolved by SDS-PAGE as described for target mass Inflammatory bowel disease spectrometry. Western blotting was done blinded to final out Diverticulitis come, as described previously (25), using the rabbitanti-LRG 10 polyclonal antibody at 1:2000 dilution, and the SuperSignal Discovery of diagnostic markers by using urine proteomic West Pico chemiluminescent reagent (Thermo). Equal total protein loading was assessed by Coomassie staining (22). profiling urine markers of appendicitis were identified from Results the analysis of 12 specimens, collected at the onset of the Study Population study, and distributed equally between patients with and with Over the 18 month course of this study, 67 patients were 15 out appendicitis. Table 4 lists the 32 markers, identified by enrolled who presented to our Emergency department (ED) ranking their relative enrichment ratios (RER). These pro and underwent evaluation for possible acute appendicitis. In teins include known components of the acute phase response agreement with earlier studies of the epidemiology and pre Such as C-1-acid glycoprotein (orosomucoid), plasminogen, sentation of acute appendicitis in pediatric EDS, the meanage carbonic anhydrase, angiotensin converting enzyme, and of our study population was 11 years, with presenting signs lipopolysaccharide binding protein, consistent with the sys and symptoms described in Table 2. Twenty five patients temic inflammatory response that accompanies acute appen (37%) received a final diagnosis of appendicitis. All patients dicitis. with appendicitis underwent appendectomies, 16% of which were found to have a perforation. One patient (4%) who TABLE 4 received a pre-operative diagnosis of appendicitis was found to have no gross or histologic evidence of appendicitis upon 25 urine marker proteins identified using undergoing appendectomy. Twenty four percent of patients relative enrichment ratio analysis. were found to have no specific cause of their abdominal pain, Protein Accession Number Uf RERf with the remaining patients found to have a variety of com Adipocyte specific adhesion molecule PIOOO24929 1.O 18 mon and rare mimicking conditions (Table 3). Leucine-rich C-2-glycoprotein PIOOO22417 1.0 9.5 30 Zinc-C-2-glycoprotein PIOO 166729 1.O 7.3 TABLE 2 C-1-acid glycoprotein 2 PIOOO2O091 1.O 5.8 MLKL PIOO180781 1.O 5.5 Presenting signs, symptoms and diagnostic studies C-1-acid glycoprotein 1 PIOOO22429 1.O S.3 of 67 patients with acute abdominal pain. Plasminogen PIOOO1958O 1.O 5.1 Carbonic anhydrase 1 PIOO2 15983 O.8 15 Final Diagnosis 35 Angiotensin converting enzyme 2 PIOO46S 187 O.8 12 Nicastrin PIOOO21983 O.8 12 Appendicitis Non-appendicitis Lipopolysaccharide binding protein PIOOO32311 O.8 11 Vascular adhesion molecule 1 PIOOO18136 O.8 10 Number 25 42 PDZK1 interacting protein 1 PIOOO1 1858 O.8 7.5 Gender (% male) 56 40 SLC9A3 PIOOO11184 O.8 7.5 Age (years) 113.5 11 + 4.2 40 Lymphatic vessel endothelial PIOO290856 O.8 6.9 Duration of symptoms (days) 2.72.O 2.2 1.7 hyaluronan receptor 1 Nausea or vomiting (%) 72 52 FXR2 PIOOO 162SO O.7 NAA Fever (%) 52 48 SORBS1 PIOOOO2491 O.7 NAA Pain migration (%) 36 14 SLC4A1 PIOOO22361 O.7 44 RLQ pain or tenderness (%) 100 95 PRIC285 PIOO2493 OS O.7 14.9 Temperature at triage (C.) 36.9 O.6 36.6 0.9 45 TGFbeta2R PIOO383479 O.7 11.3 Peripheral white blood cell count 15.7 S.2 11.O 6.4 SLC2A1 PIOO22O194 0.7 10.7 (K cells/mm) Rc PIOOOO7926 0.7 9.7 Absolute neutrophil count (K 1285.4 8.5 - 6.6 VAOD1 PIOOO34159 O.7 8.9 cells/mm) SLC13A3 PIOO103426 0.7 7.8 US imaging (%) 88 74 TTYH3 PIOO749.429 0.7 7.3 US diagnosis of appendicitis (%) 64 O SPRX2 PIOOOO4446 O.7 6.4 CT imaging (%) 60 64 50 BAZ1B PIOO216695 O.7 6.1 CT diagnosis of appendicitis (%) 93 7.4 3-1,3-galactosyltransferase PIOOO32O34 O.7 6.1 chromogramin A PIOO383975 0.7 5.9 Walues are reported as mean standard deviation, where appropriate, RLQ (right lower Novel protein PIOOSSO644 0.7 5.5 quadrant), US (ultrasound), CT (computer tomography), SLC2A2 PIOOOO3905 0.7 5.2 FBLN7 PIOO167710 0.7 5.1 55 TABLE 3 Values of U = 1 indicate markers detected in all appendicitis specimens, whereas values of relative enrichment ration (RER) = 1 indicate markers that exhibit no apparent enrichment in appendicitis as compared to non-appendicitis groups, Final diagnosis of the 67 study patients NiA (not detected) identifies markers not detected in non-appendicitis specimens), Number of patients (International Protein Index (version3.36, at the WorldWideWebsite of “ebi.ac.uk/IPI).) Appendicitis 25 60 The markers also include a number of cell adhesion pro Non specific abdominal pain 1 teins such as adipocyte specific adhesion molecule, a compo Ovarian cyst or torsion Constipation nent of the epithelial and endothelial tight junctions, leucine Pyelonephritis or Urinary Tract Infection rich C-2-glycoprotein (LRG), a marker of neutrophil Renal calculus differentiation involved in cell trafficking, vascular adhesion Mesentericadenitis 65 molecule 1, which mediates lymphocyte-endothelial adhe Gastroenteritis or gastritis Sion, and lymphatic vessel endothelial hyaluronan acid recep tor 1 involved in cell migration, consistent with earlier find US 8,535,891 B2 63 64 ings of leukocyte trafficking and infiltration into mucosal TABLE 5 tissue that accompanies acute appendicitis. Remaining top ranking markers do not appear to share any Urine marker proteins validated by target mass Spectrometry. known functional or structural similarities, though some of ROC AUC 95% confidence them such as B-1,3-galactosyltransferase and VAOD1 have 5 Protein AUC interval been shown to function specifically in the colonic epithelium, Leucine-rich C-2-glycoprotein (LRG) 0.97 O.93-1.O and therefore, may include components of the local and sys calgranulin A (S100-A8) O.84 O.72-0.95 temic appendicitis response. Additional markers were iden C-1-acid glycoprotein 1 (ORM) O.84 O.72-0.95 tified by using Support vector machine (SVM) learning, as Plasminogen (PLG) 0.79 O.67-0.91 well as comparisons with tissue gene expression profiles of 10 Mannan-binding lectin serine O.74 O.61-0-88 protease 2 (MASP2) diseased appendices (Tables 6 and 7). In total, 49 markers Zinc-C-2-glycoprotein (AZGP1) O.74 O60-088 were identified. C-1-antichymotrypsin (SERPINA3) O.84 O.73-0.94 Apollipoprotein D (ApoD) O.S3 O38-0.69 TABLE 6 15 ROC (receiver operating characteristic), AUC (area under the curve), urine marker proteins identified using SVM analysis Indeed, receiver operating characteristic (ROC) curves for Protein Accession Number these markers exhibited excellent performance, with LRG Serum amyloid A protein PIOO552.578 and S100-A8 having area under the curve (AUC) values of C-1-antichymotrypsin PIOO550991 0.97 and 0.84, respectively (FIG.9, Table 5). Other prospec Supervillin PIOO412650 tively validated markers with apparently good performance Mannan-binding lectin serine protease 2 PIOO306378 included orosomucoid and C-1-antichymotrypsin (Serpin Inter-C-trypsin inhibitor PIOO218.192 A3); plasminogen, mannan-binding lectin serine protease 2 VIP36 PIOOOO995O Prostaglandin-H2 D-isomerase PIOOO13179 (MASP2), zinc-C-2-glycoprotein (AZGP) exhibited interme C-1-acid glycoprotein 2 PIOOO2O091 diate performance, and apolipoprotein D exhibited poor per AMBP PIOOO22426 25 formance. These findings are consistent with most of these C-1-acid glycoprotein 1 PIOOO22429 proteins being components of the general acute phase CD14 PIOOO29260 Hemoglobin C. PIOO41071.4 response, during which they may be upregulated by a variety Apollipoprotein D PIOOOO6.662 of infectious and inflammatory conditions, including some Hemoglobin B PIOO654755 that are represented in the non-appendicitis group (Table 3). Leucine-rich C-2-glycoprotein PIOOO22417 30 The inventors assessed the relationship between apparent Zinc-C-2-glycoprotein PIOO166729 urine protein abundance of markers and the apparent severity of appendicitis by classifying appendectomy specimens with respect to the degree of neutrophil infiltration (12). As can be TABLE 7 seen from FIG. 10, LRG appears to be a marker of focal 35 appendicitis, whereas S100-A8 appears to be a marker of Urine marker proteins identified by comparisons progressive disease, reaching a peak level with moderate with corresponding tissue gene overexpression. appendicitis. In addition to exhibiting excellent diagnostic Fold gene performance, LRG was detected strongly in diseased as com Accession Affymetrix Ower pared to normal appendices by using tissue immunohis Protein Number gene ID* expression* 40 tochemistry (FIG. 10), consistent with its biological function and proposed role in appendicitis. Its enrichment in urine of S100-A8 IPIOOOO7047 214370 at 67 S100-A9 IPIOOO27462 203535 at 45 patients with appendicitis relative to those with other condi Amyloid-like protein 2 IPIO0031030 214456 X at 38 tions was confirmed by using Western immunoblotting (FIG. Versican IPIOOOO9802 211571 S. at 11 9B), demonstrating that clinical diagnostic immunoassays SPRX2 IPIOOOO4446 205499 at 8.1 45 can be used as a method to identify the urinary markers C-1-acid glycoprotein 1 IPIOOO22429 205041 S. at 7.8 disclosed herein. Interleukin-1 receptor IPIOOOOOO45 212657 s at 4.3 antagonist protein As disclosed herein, the inventors used urine proteome Lymphatic vessel IPIOO290.856 220037 s at 2.0 profiling to discover urinary markers of acute appendicitis. endothelial hyaluronan Usage of exhaustive protein capture and fractionation acid receptor 1 coupled with high accuracy mass spectrometry allowed the 50 inventors to detect more than 2,000 unique proteins in rou *From Murphy CG, et al. Mucosal Immunol. 2008; 1:297-308. tinely collected urine specimens, constituting the largest and Validation of Urine Protein Markers by Using Target Mass most comprehensive characterization of protein composition Spectrometry of human urine to date (22). The discovered urinary diagnos tic markers (Tables 4, 6, and 7) were subsequently validated in In order to assess their diagnostic performance, the inven 55 a prospective, blinded study of children Suspected of having tors determined their concentrations in urine of all enrolled acute appendicitis, identifying several with statistically sig patients in a prospective fashion, with experimental measure nificant enrichment in the urine of children with histologi ments blinded to the patients outcomes. Proteins detected cally proven appendicitis as compared to those without (Table with Sufficient uniformity among the 67 specimens examined 5). are listed in Table 5. The remaining proteins were detected in 60 The use of high resolution CT and US has led to substantial less than half of specimens, likely as a result of differences in improvements in the diagnosis of acute appendicitis, with processing of the discovery and validation specimens. Com respect to both the rates of complications and unnecessary parison of differences in urinary concentration between the appendectomies (2-4). However, significant diagnostic chal appendicitis and non-appendicitis patient groups revealed lenges remain, largely because of the non-specific nature of LRG, S100-A8, and C-1-acid glycoprotein 1 (orosomucoid) 65 signs and symptoms of many conditions that can mimic acute as exhibiting Substantial apparent enrichment in the urines of appendicitis. Similarly, CT and US findings can often be patients with appendicitis (FIG. 8). indeterminate or equivocal (26). Finally, limited availability US 8,535,891 B2 65 66 and experience with dedicated CT protocols for appendicitis, LRG is expressed by differentiating neutrophils, liver, and as well as future risk of cancer, can often limit its usefulness high endothelial Venules of the mesentery, including the (5). meso-appendix, functioning in leukocyte activation and Numerous studies have sought to identify biomarkers to chemotaxis, respectively (29, 30). Its enrichment in the urine aid the diagnosis of appendicitis, with the absolute blood 5 of patients with acute appendicitis demonstrates that it may neutrophil count and serum C-reactive protein levels being be shed by locally activated neutrophils and/or local inflam most useful, but still limited with respect to their sensitivity matory sites such as the meso-appendix through which they and specificity (27, 28). Recent attempts to identify new and improved diagnostic likely traffic (FIG. 10). As such, it is likely a specific marker markers, such as CD44, interleukin-6, interleukin-8, and of local inflammatory processes such as those that specifi 5-hydroxy indole acetate, produced limited improvements as 10 cally characterize acute appendicitis, as opposed to general compared to the existing ones (6-11), likely as a result of markers of systemic response Such as the acute phase reac being closely correlated with the existing markers of the tants, and macroscopic markers of local inflammation Such as general acute phase response, or not specific for the distinct those observed using US and CT imaging. immune mechanisms that characterize acute appendicitis. LRG appears to be enriched in the urine of patients with By taking advantage of the latest generation of mass spec 15 appendicitis in the absence of macroscopic inflammatory trometers that combine high accuracy with high sensitivity, changes, as evidenced by its accurate diagnosis of appendi and carrying out exhaustive protein capture and fractionation citis of 2 patients who exhibited normal imaging findings but of routinely collected urine specimens, the inventors devel had evidence of acute appendicitis on histologic examination, oped a method that enables unbiased discovery and validation as well as its accurate diagnosis of the absence of appendicitis of multiple diagnostic markers, thereby overcoming the limi in a patient without histologic evidence of appendicitis, but tations of conventional approaches based on single hypoth who underwent appendectomy as a result of findings of esis testing. Because of the depth of discovery achieved, appendiceal enlargement on CT. Lastly, LRG appears to be identifying more than 2,000 unique proteins in total, urine enriched in the urine of patients with pyelonephritis, consis proteomic profiling, like gene expression profiling, may be tent with its proposed role in local inflammatory processes. Susceptible to noise and selection bias. In order to minimize Consequently, LRG will be useful to diagnose acute appen these potential problems (12), discovery urine proteomes 25 dicitis following ruling out other local tissue infections, such were compared not only between patients with histologically as pyelonephritis, abscesses, and pelvic inflammatory disease proven appendicitis and those without, but also with the same (31). Importantly, LRG appears to be strongly expressed in patients after they recovered from appendectomies, thereby diseased appendices, demonstrating that it may underlie a minimizing individual differences due to age, gender, physi principal pathway of appendiceal inflammation by localizing ologic state or genetic variation. High Stringency identifica 30 or Sustaining the local neutrophilic infiltration that specifi tion criteria were used, essentially eliminating false identifi cally characterizes acute appendicitis (12, 13, 24). cations (22). The discriminatory power of diagnostic markers The inventors have not tested urine protein markers of was assessed by examining the level and uniformity of their acute appendicitis in patients evaluated in settings other than enrichment in patients with appendicitis (Table 4), by using the emergency department, as well as in older adult patients, pattern recognition class prediction learning algorithms who may include other causes of abdominal pain from those (Table 6), and by comparing discovered urine protein markers 35 observed in the study cohort. The inventors’ demonstration of with tissue gene expression profiles of diseased appendices urinary markers for appendicitis establishes a useful para (Table 7) (24). digm for the identification of other clinically useful urinary As a result, the 49 discovered urinary markers constitute an markers of human disease, including infectious, endocrine, extensive characterization of the molecular response that autoimmune and neoplastic diseases. accompanies acute appendicitis, including both systemically 40 References cited in Example 2 and disclosed in italicized and locally produced molecules. Among the former are brackets (i.e. “(i)') are below and each are incorporated known components of the acute phase response. Such as herein in their entirety by reference. orosomucoid, plasminogen, angiotensin converting enzyme, 1. Addiss D G, Shaffer N, Fowler B S, Tauxe RV. The carbonic anhydrase, TGF B, lipopolysaccharide binding pro epidemiology of appendicitis and appendectomy in the tein, serum amyloid A. C.-1-antichymotrypsin, AMBP (biku 45 United States. Am J Epidemiol 1990; 132:910-25. nin), and mannan-binding lectin serine protease (2). Numer 2. Rao PM, Rhea JT, Novelline RA, MostafaviAA, McCabe ous cell adhesion molecules that may participate in the local CJ. Effect of computed tomography of the appendix on generation of the systemic inflammatory response or its local treatment of patients and use of hospital resources. NEngl ization to the appendiceal tissue were identified, including the J Med 1998; 338:141-6. vascular adhesion molecule 1, lymphatic vessel endothelial 3. Peck J. Peck A, Peck C. The clinical role of noncontrast hyaluronan acid receptor 1, adipocyte specific adhesion mol 50 helical computed tomography in the diagnosis of acute ecule, Supervillin, CD14, and leucine-rich C-2-glycoprotein. appendicitis. Am J Surg 2000; 18.0:133-6. Likewise, several potential local inflammatory mediators and 4. Partrick DA, Janik JE, Janik JS, Bensard DD, Karrer FM. cytokines were identified such as chromogranin A, B-1,3- Increased CT scan utilization does not improve the diag galactosyltransferase, interleukin-1 receptor antagonist pro nostic accuracy of appendicitis in children. J Pediatr Surg tein, and S100-A8. 55 2003: 38:659-62. The discovered urinary diagnostic markers were validated 5. Brenner DJ. Hall E.J. Computed tomography—an increas in their ability to accurately diagnose acute appendicitis by ing source of radiation exposure. N Engl J Med 2007: measuring their urinary concentrations in a prospective and 357:2277-84. blinded study of 67 patients who were suspected to have acute 6. Taha AS, Grant V. Kelly RW. Urinalysis for interleukin-8 appendicitis, with the final diagnosis verified by blinded his in the non-invasive diagnosis of acute and chronic inflam tologic examination of removed appendices. 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HEV glycoprotein as a HEV marker. J Immunol 2002: 13. MaZZucchelli L. Hauser C. Zgraggen K, et al. Expression 168:1050-9. of interleukin-8 gene in inflammatory bowel disease is 33. Bini L. Magi B, Marzocchi B, et al. Two-dimensional related to the histological grade of active inflammation. 15 electrophoretic patterns of acute-phase human serum pro Am J Pathol 1994: 144:997-1007. teins in the course of bacterial and viral diseases. Electro 14. Rai AJ. Stemmer PM, Zhang Z. et al. Analysis of Human phoresis 1996; 17:612-6. Proteome Organization 15. Plasma Proteome Project (HUPO PPP) reference speci Example 3 mens using Surface enhanced laser desorption/ionization time of flight (SELDI-TOF) mass spectrometry: multi Discovery and Validation of Urine Markers of Acute institution correlation of spectra and identification of Appendicitis. Using High Accuracy Mass biomarkers. Proteomics 2005; 5:3467-74. Spectrometry 16. Pisitkun T. Johnstone R, Knepper M A. Discovery of appendicitis biomarkers. Mol Cell Proteomics 2006. 17. Adachi J. Kumar C, Zhang Y. Olsen J. V. Mann M. The 25 Discovery of Diagnostic Markers by Using Urine Proteomic human urinary proteome contains more than 1500 pro Profiling teins, including a large proportion of membrane proteins. In order to identify candidate urinary markers of acute Genome Biol 2006: 7:R80. appendicitis, the inventors assembled a discovery urine pro 18. Woroniecki RP Orlova TN, Mendelev N, et al. Urinary teome dataset, derived from the analysis of 12 specimens, proteome of steroid-sensitive and steroid-resistant idio 30 without any clinical urinalysis abnormalities, collected at the pathic nephrotic syndrome of childhood. Am J Nephrol onset of the study, and distributed equally between patients 2006; 26:258-67. with and without appendicitis. Six of these specimens were 19. Oetting W. S. Rogers T B, Krick TP. Matas AJ, Ibrahim collected from patients who were found to have histologic HN. Urinary beta2-microglobulin is associated with acute evidence of appendicitis (2 mild, 3 moderate, 1 severe). Three renal allograft rejection. Am J Kidney Dis 2006: 47:898 specimens were collected from patients without appendicitis 904. 35 (1 with non-specific abdominal pain, 1 with constipation, 1 20. Berger R P. Kochanek PM. Urinary S100B concentra with mesenteric adenitis). From the 3 patients with appendi tions are increased after brain injury in children: A prelimi citis, the inventors collected additional control specimens at nary study. Pediatr Crit. Care Med 2006: 7:557-61. their routine post-Surgical evaluation 6-8 weeks after under 21. Propst A, Propst T. Herold M, Vogel W. Judmaier G. going appendectomies, at which time they were asymptom Interleukin-1 receptor antagonist in differential diagnosis 40 atic and in their usual state of health. These specimens were of inflammatory bowel diseases. Eur JGastroenterol Hepa included in the analysis in order to minimize the potential tol 1995; 7:1031-6. effect of individual variability in urinary composition that 22. Campbell M J. Estimating sample sizes for binary, may arise due to age, gender, physiologic state or possible ordered categorical, and continuous outcomes in two group genetic variation. comparisons. British Medical Journal 1995; 3 11:1145-48. The urine proteome composition of these 12 specimens 23. Kentsis A, Monigatti F. Dorff K, Campagne F. Bachur R 45 was discovered by using protein capture and fractionation G, Steen H. Urine proteomics for profiling of human dis coupled with high accuracy mass spectrometry, as described ease using high accuracy mass spectrometry. Submitted in detail in the accompanying study, and schematized in FIG. 2008. 7. As urine is a complex mixture with abundant proteins such 24. Carvalho PC, Hewel J. Barbosa V C, Yates J R, 3rd. as albumin obscuring the detection of less concentrated, Identifying differences in protein expression levels by 50 potentially diagnostic proteins such as secreted cytokines and spectral counting and feature selection. Genet Mol Res mediators of the inflammatory response, the inventors 2008; 7:342-56. devised a fractionation method that reduced mixture com 25. Murphy C G, Glickman J. N. Tomczak K, et al. Acute plexity while minimizing loss of material (FIG. 7). Appendicitis is Characterized by a Uniform and Highly As a result, the inventors were able to identify 2,362 pro Selective Pattern of Inflammatory Gene Expression. 55 teins in routinely collected urine specimens with the apparent Mucosal Immunol 2008; 1:297-308. rate of false identifications of less than 1%, as ascertained 26. Kentsis A, Topisirovic I, Culjkovic B, Shao L, Borden K from decoy database searching. More than 1,200 identified L. Ribavirin Suppresses eIF4E-mediated oncogenic trans proteins have not been detected in previous proteomic studies formation by physical mimicry of the 7-methylguanosine ofurine, and more than 300 proteins appear to be filtered from mRNA cap. Proc Natl AcadSci USA 2004: 101:18105-10. serum and expressed in distal tissues, including the intestine. 27. Kharbanda A. B. Taylor G. A. Bachur R. G. Suspected 60 For the discovery of candidate appendicitis markers, the appendicitis in children: rectal and intravenous contrast inventors further increased the stringency of peptide identifi enhanced versus intravenous contrast-enhanced CT. Radi cations to less than 0.1% false identifications, yielding essen ology 2007: 243:520-6. tially no false protein identifications for proteins identified on 28. Okamoto T. Sano K. Ogasahara K. Receiver-operating the basis of multiple peptides. For example, proteins identi characteristic analysis of leukocyte counts and serum C-re 65 fied on the basis of 10 unique peptides (median for the entire active protein levels in children with advanced appendici dataset), have an approximate identification error frequency tis. Surg. Today 2006:36:515-8. of 10-19. US 8,535,891 B2 69 70 In order to identify candidate markers of appendicitis, the in an inclusion list dependent acquisition mode, searching inventors took advantage of the quantitative information pro detected precursor ions against m/z values of candidate vided by tandem mass spectrometry by recording the number marker peptides with a tolerance of 0.05 Da, using an inclu of fragment ion spectra assigned to each unique precursor sion list of masses and charges of candidate marker peptides, peptide, which are proportional to peptide abundance, and derived from the analysis of the discovery proteomes. Six have been used for relative quantification of components of 5 most intense matched ions were sequentially fragmented by complex protein mixtures.' Though the composition and con using collision induced dissociation, and spectra of their frag centration of urine varies with physiologic state, there was ments were recorded in the linear ion trap, with the dynamic less than 10+10% (meanistandard deviation) difference in exclusion of precursor ions already selected for MS/MS of 60 total protein abundance among individual specimens, similar Sec. Such an approach is Superior to conventional data depen to earlier studies of urine of children.” Individual protein 10 dent acquisition methods by minimizing the detection of non spectral counts, calculated by Summing spectral counts of target peptides. Differences in apparent protein abundance unique peptides assigned to distinct proteins, were normal were normalized relative to exogenously added SSB refer ized relative to the spectral counts of albuminto account for ence standard to account for instrumental variability. Absence these small differences in total protein abundance." of SSB from urine specimens without its addition was con In order to maximize the depth of candidate marker dis 15 firmed by searching the data against database of E. coli pro covery, the inventors Subjected the discovery urine proteome teins (data not shown). to support vector machine (SVM) learning in order to identify Recorded mass spectra were processed and identified, as candidate urine markers that may be enriched as a group but described." The accuracy of peptide identification was not necessarily individually, as required by the relative assessed by decoy database searching, enforcing a false pep expression ration (RER) analysis above. This approach is tide discovery rate of less than 1%, which corresponds to implemented in a biomarker discovery program BDVAL that essentially Zero false protein discovery rate, given that all of uses cross-validation to identify predictive biomarkers (Fa the candidate diagnostic marker proteins were identified on bien Campagne, unpublished results, at the WorldWideWeb the basis of at least 9 peptides, which corresponds to an site of “icb.med.cornell.edu/wiki/index.php/BDVAL), simi apparent false identification frequency of less than 10-18. For lar to established methods for microarray class discovery. example, leucine-rich C-2-glycoprotein (LRG) was identified Because of the low number of samples, the inventors per 25 on the basis of 55 unique peptides. formed cross-validation with four folds, repeated 5 times with Urine Markers of Appendiceal Inflammatory Response random fold assignments (12 Samples total, 6 cases, 6 con Because acute appendicitis is characterized by the trols). In this setting, 20 individual evaluation models (5x4) increased expression of distinct chemoattractants in the gut were trained. Each model was trained with a set of 50 features mucosa," and specific infiltration of neutrophils,' the inven (normalized protein abundance levels). In each split, consist 30 tors wondered if markers of acute appendicitis identified from ing of 9 training samples and 3 test samples, a Student t-test studies of appendiceal tissue may be detected in the urine of pre-filtering step prioritized up to 400 features whose average patients with appendicitis. To this end, the inventors com value differed the most between cases and controls in the pared candidate urine protein markers as identified by using training set. The 400 intermediate features were ranked by urine proteome profiling (Table 4) with tissue markers iden decreasing Support vector machine weights and the top 50 tified in a different study by using microarray gene expression features were used to train the evaluation model (models were 35 of diseased appendices.' FIG. 6 plots RER values of the 40 implemented as a Support vector machine, implemented in most uniformly detected (UD-0.7) candidate urine markers as libSVM with linear kernel, and margin parameter C=1). At a function of the tissue overexpression of their respective the end of the evaluation, the lists of features were inspected microarray profiled genes. Of these, more than 50% exhibit a to determine how many times a given feature has been used in positive correlation between tissue overexpression and urine any one of the 20 evaluation models. The inventors consid 40 enrichment (FIG. 6) demonstrating that tissue gene expres ered features for validation only if they were found in at least sion profiles are useful to identify disease markers. However, 50% of the evaluation models generated (10 models in this only 3 of the genes that are overexpressed in diseased as case). opposed to normal appendices were also identified as candi Table 6 lists 17 proteins identified by SVManalysis, which date markers by urine proteome profiling: SPRX2, lymphatic include several proteins that were identified by RER analysis, vessel endothelial hyaluronan acid receptor 1 (LYVE1), and as well as many that were not, including additional compo 45 C-1-acid glycoprotein 1 (orosomucoid 1), demonstrating that nents of the acute phase response, such as serum amyloid A. detection of markers of local disease in the urine is not solely C-1-antichymotrypsin, and bikunin (AMBP). Notably, exclu dependent on tissue overexpression, but likely also requires sion of control specimens collected from asymptomatic other factors, such as shedding, circulation in blood, and patients after they underwent appendectomies increased the accumulation in urine. Table 7 lists urine protein markers that number of candidate markers to 273 by additionally including 50 were enriched in the urines of patients with appendicitis with a variety of proteins unlikely to be related to the appendicitis corresponding genes that were overexpressed in diseased response, such as the universal tyrosine kinase Src for appendices. example, Suggesting that individually variant factors such as In contrast to LRG which is expressed exclusively by the those that influence protein filtration and urine production neutrophils, liver and the mesentery, S100-A8 is a cytokine may significantly affect biomarker discovery studies. 55 expressed by diverse tissues, including a variety of endothe Candidate Validation Target Mass Spectrometry lial and epithelial cells.' ' It is upregulated specifically in Thawed 1 ml urine aliquots were precipitated by adding inflammatory states, including the processes of neutrophil trichloroacetic acid to 20% (w/v), and incubating the samples activation and migration. Findings of its overexpression in for 1 hour at 4°C. Precipitates were sedimented at 10,000 g appendiceal tissue during acute appendicitis,' and enrich for 15 minutes at 4° C. and pellets were washed twice with ment in the urine of appendicitis patients demonstrate that neat acetone at 4°C., with residual acetone removed by air 60 like LRG, it is also a marker of local inflammation, though its drying. Dried pellets were resuspended in Laemmli buffer, expression in a wide variety of tissues may affect its diagnos resolved by SDS-PAGE, alkylated and digested with trypsin tic specificity, consistent with its slightly reduced dynamic as described." To each sample, 0.4 ug of single stranded range and performance as compared to those of LRG (Table 5, binding (SSB) protein purified from Escherichia coli (USB) FIG.9). Accordingly, it has been found to be upregulated in a was added to serve as a reference standard. Target nanoLC 65 wide variety of conditions, including inflammatory bowel MS/MS was accomplished by using the LTQ-Orbitrap mass disease.'arthritis.'Kawasakivasculitis' cancer, and sep spectrometer, using the parameters described, but operated sis.' US 8,535,891 B2 71 72 References cited in Example 3 and disclosed as superscript Example 4 (i.e. ") are listed below and each are incorporated herein in their entirety by reference. Diagnostic Lateral Flow Immunoassay Test 1. Kentsis A, Monigatti F. Dorff K, Campagne F. Bachur RG, Strips-Design 1 Steen H. Urine proteomics for profiling of human disease using high accuracy mass spectrometry. Submitted 2008. The levels of biomarker proteins described herein can be 2. Elias J. E. Gygi S. P. Target-decoy search strategy for determined using lateral flow immunoassay (LFIA) test Strips increased confidence in large-scale protein identifications as illustrated in FIG. 11-12. This test strip can be used in by mass spectrometry. Nat Methods 2007; 4:207-14. point-of-care testing (POCT). The test strip has a sample (S) 3. Old W M, Meyer-Arendt K, Aveline-Wolf L, et al. Com 10 position at one end of the test strip and a control (C) position parison of label-free methods for quantifying human pro found at the opposite end the test strip (FIG. 11A). There is a teins by shotgun proteomics. Mol Cell Proteomics 2005; test (T) position located at the middle of the test strip, between 4:1487-5O2. S and T. For this embodiment of a test strip, the solid support 4. Carvalho PC, Hewel J, Barbosa V C, Yates J R, 3rd. 101 can be made of plastic or other non porous material, Identifying differences in protein expression levels by 15 supporting the matrix 103. Located at S is a defined quantity spectral counting and feature selection. Genet Mol Res of dehydrated anti-biomarker protein antibody. The defined 2008; 7:342-56. quantity of dehydrated anti-biomarker protein antibody, 5. Cindik N. Baskin E. Agras PI, Kinik ST. Turan M. Saatci when rehydrated, will bind at saturation a fixed amount of U. Effect of obesity on inflammatory markers and renal biomarker antigen, meaning that this fixed amount of biom functions. Acta Paediatr 2005: 94:1732-7. arker protein will completely occupy all of the Fv binding 6. De Palo E. F. Gatti R. Lancerin F. Cappellin E. Sartorio A, sites of that defined quantity of antibody. If there is additional Spinella P. The measurement of insulin-like growth fac biomarker protein in excess of the fixed amount of biomarker tor-I (IGF-I) concentration in random urine samples. Clin that is required to bind all of the amount of antibody from Chem Lab Med 2002:40:574-8. position S, the excess biomarker proteins will be free and are 7. Skinner AM, Clayton PE, Price DA. Addison GM, Mui not bound to any antibody in the form of an antibody-biom CY. Variability in the urinary excretion of growth hormone 25 arker complex. The fixed amount of biomarker protein is the in children: a comparison with other urinary proteins. J predetermined reference level of biomarker protein which is Endocrinol 1993: 138:337-43. the level found in healthy individuals who do not have acute 8. Radmacher MD, McShane LM, Simon R. A paradigm for appendicitis. The antibody at position S can be conjugated to class prediction using gene expression profiles. J Comput colloidal gold beads or colored latex beads for visualization Biol 2002; 9:505-11. 30 purposes. At position T, there is a defined quantity of biom 9. Jaffe J. D. Keshishian H. Chang B. Addona TA, Gillette M arker protein immobilized on the test strip. This is the same A, Can SA. Accurate inclusion mass Screening: a bridge biomarker protein that binds the antibody deposited at posi from unbiased discovery to targeted assay development for tion S. At position C, there is another immobilized protein, an biomarker verification. Mol Cell Proteomics 2008. antibody immunoreactive to the anti-biomarker protein anti 10. MaZZucchelli L. Hauser C. Zgraggen K, et al. Expression body located at the S position (FIG. 11). of interleukin-8 gene in inflammatory bowel disease is 35 The following is a description on how to use and interpret related to the histological grade of active inflammation. the results obtained for the test strip shown in FIG. 11. A Am J Pathol 1994: 144:997-1007. sample of urine is applied at S. The water in the urine rehy 11. Tsuji M. Puri P. Reen DJ. Characterisation of the local drates the dehydrated anti-biomarker protein antibody that inflammatory response in appendicitis. J Pediatr Gastroen has been deposited at S. The dehydrated anti-biomarker pro terol Nutr 1993; 16:43-8. 40 tein antibody can be labeled with colloidal gold beads or 12. Murphy C G, Glickman J. N. Tomczak K, et al. Acute colored latex beads. The biomarker protein in the urine binds Appendicitis is Characterized by a Uniform and Highly to this rehydrated anti-biomarker protein antibody to forman Selective Pattern of Inflammatory Gene Expression. antibody-biomarker complex. Any biomarker protein in the Mucosal Immunol 2008; 1:297-308. urine that is in excess of the rehydrated anti-biomarker pro 13. Passey RJ, Xu K, Hume DA, Geczy C L. S100A8: tein antibody deposited at S will be free and is not bound to emerging functions and regulation. J Leukoc Biol 1999; 45 any antibody. A mixture of antibody-biomarker complex and 66:549-56. free antibody or free biomarker will move by capillary action 14. Foell D. Wittkowski H, Vogl T. Roth J. S100 proteins away from position Sandwill move toward the Tposition and expressed in phagocytes: a novel group of damage-associ subsequently to the C position. When the biomarker protein ated molecular pattern molecules. J Leukoc Biol 2007; of interest is below the reference level, the mixture of anti 81:28-37. 50 body and biomarker protein will contain free anti-biomarker 15. Fagerberg UL, Loof L. Lindholm J. Hans son LO, Finkel protein antibody and antibody-biomarker protein complexes. Y. Fecal calprotectin: a quantitative marker of colonic At position T, any free anti-biomarker protein antibody will inflammation in children with inflammatory bowel dis bind to the immobilized biomarker protein at T. The localized ease. J Pediatr Gastroenterol Nutr 2007;45:414-20. concentration of free anti-biomarker protein antibody that is 16. de Seny D, Fillet M, Ribbens C, et al. Monomeric cal 55 colloidal gold or latex bead labeled will become visible as a granulins measured by SELDI-TOF mass spectrometry colored line at the T position (FIG. 12B). There is free anti and calprotectin measured by ELISA as biomarkers in body only when the biomarker protein in the urine is below arthritis. Clin Chem 2008:54: 1066-75. the threshold reference value found in healthy humans, which 17. Hirono K, Foell D, Xing Y, et al. Expression of myeloid is the predetermined reference level of biomarker protein. related protein-8 and -14 in patients with acute Kawasaki When the protein of interest is at or above the predetermined disease. JAm Coll Cardiol 2006: 48:1257-64. 60 reference level, the mixture of antibody and biomarker pro 18. Hiratsuka S. Watanabe A, Aburatani H, Maru Y. Tumour tein will contain all antibody-biomarker protein complexes mediated upregulation of chemoattractants and recruit and no free anti-biomarker protein antibody. At the T posi ment of myeloid cells predetermines lung metastasis. Nat tion, there will be no anti-biomarker protein antibody cap Cell Biol 2006; 8:1369-75. tured by the immobilized biomarker protein. Thus there will 19. Payen D. Lukaszewicz AC, Belikova I, et al. Gene pro 65 be no colloidal gold or latex bead labeled anti-protein anti filing in human blood leucocytes during recovery from body accumulation, and the area remains clear (FIG. 12A). At septic shock. Intensive Care Med 2008:34:1371-6. position C, the antibody-biomarker complex formed initially US 8,535,891 B2 73 74 at S will be bound and captured by the immobilized antibody the same test strip. The positions of the expected results in the immunoreactive against the anti-protein antibody coming T and C positions for each biomarker are indicated 141. from the S position. This will in turn result in a concentration The test strip can be designed in a form of a dipstick test of a colloidal gold or latex bead labeled anti-protein antibody strip (FIG. 11B). As a dipstick test strip, the strip is dipped accumulated at the C position and will become visible as into a sample (e.g. urine) at the S position end with sample colored line at the C position. The C position result serves as level not to exceed the boundary limit. The strip is then laid a test control to indicate that there is functional anti-protein horizontally with the membrane Surface facing up on a flat antibody in the test material and should always be present surface. A fixed amount of time is given for the antibody (FIGS. 12A and 12B). When sufficient amount of labeled re-hydration, capillary action, and antibody biomarker pro anti-biomarker protein antibody from the complex accumu 10 tein binding reaction to take place. At the end of the fixed lates at C, aband becomes visible here. A band at C indicates time, there should be visible bands at the C position and that labeled antibody from S had moved to C. Therefore, a depending on the level of the protein of interest, there may or band at C indicates that the band at T is not a false positive. may not be a visible band at the Tposition (FIG. 12). FIG. 13 Arrowheads indicate the boundary limit that a urine sample shows a method of using three separate dipstick test strips to should not cross on the test strip. 15 test for the three biomarkers of interest. Each dipstick test FIG. 12A-12D show the possible outcomes and interpre strip is labeled 131 to indicate which biomarker protein is tations of the results for such a test strip. FIG. 12A shows no being tested. A diagnostic kit can comprise multiple types of band at position T but a distinct band at position C, indicating single biomarker test strips, a type for each biomarker of that the biomarker protein level is above predetermined ref interest. erence level. Acute appendicitis is indicated. FIG.12B shows a band at position T and a distinct band at position C, indi Example 5 cating that the biomarker protein level is below predeter mined reference level. Acute appendicitis is not indicated. Diagnostic Lateral Flow Immunoassay Test FIG. 12C shows a band at position T but no band at position Strips-Design 2 C, indicating that the data at T may be a false positive. FIG. 12D shows no band at either positions T and C, indicating the 25 An alternative embodiment of the lateral flow immunoas data at T may be a false negative. Both FIGS. 12C and 12D say (LFIA) test strips for determining the level of biomarker indicate invalid data and the lateral flow immunoassay should protein level is illustrated in FIG.15A-D. This test strip can be be repeated with a new test strip. used in point-of-care testing. Here the test strip contains two The defined quantity of dehydrated anti-protein antibody at different anti-biomarker protein antibodies specific for the Sposition is such that there is just enough antibody to bind the 30 same biomarker, each antibody binds the biomarker at a dif biomarker protein from the sample (e.g. urine) when the ferent epitope. This is a double sandwich LFIA test strip. The biomarker protein is at the reference/control level. The refer first antibody is labeled (e.g. colored latex beads), deposited ence/control level can be the level of the biomarker found in on the Solid Support matrix but is not immobilized on it, (i.e. the samples of healthy individuals. Therefore, when the the antibody is mobile), and is deposited in excess at the S biomarker protein is at or above the reference level, all of the position. The second anti-biomarker protein antibody is not anti-biomarker antibody at the S position will be bound to the 35 labeled but is immobilized and is in excess at position T. This biomarker protein in the form of biomarker protein-antibody second anti-biomarker protein antibody binds an epitope on complex; there will be no free anti-biomarker protein anti the biomarker that is not affected by the binding of the first body present. antibody. At position C, there is an excess of non-labeled The choice of the anti-biomarker protein antibody placed antibody against the anti-biomarker antibody deposited at the at the S position can be any antibody that is specifically 40 S position. The antibody at C serves to capture any free immunoreactive to any of the proteins of interest, e.g. biom labeled anti-biomarker antibody migrating from S. When arker described herein. The antibody can be monoclonal, sufficient free labeled anti-biomarker antibody is accumu polyclonal, or a mixture of both monoclonal and polyclonal lated at C, a visible band appears. The band is a control to antibodies. Antibody-based moiety can also be used. confirm that the band(s) observed on the test strip at T are due When only one biomarker protein is studied, the S position to the mobile antibody at the S position. should have only one anti-biomarker protein antibody that 45 Initially before use, there is no visible band at position T specifically immunoreactive with just that one biomarker of and C of the test strip (FIG. 15B). When a fluid sample (e.g. interest (FIG. 13). A kit comprising test strips for use as urine) is place at the S position, the water in the urine rehy POCT can have several single biomarker protein test strips. drates the dehydrated anti-biomarker protein antibody that The kit can test for only one biomarker or more then one has been deposited at S. The dehydrated anti-biomarker pro biomarker proteins. In this embodiment, the test strip can be 50 tein antibody can be labeled with colloidal gold beads or labeled 131 on one end to identify the biomarker protein the colored latex beads. The biomarker protein in the urine binds test strip is used for, e.g. the label “L” represents leucine C-2 to this rehydrated anti-biomarker protein antibody to forman glycoprotein (LRG); “M” represents mannan-binding lectin antibody-biomarker complex. A mixture of free anti-biomar serine protease 2 (MASP2); and “O'” represents C-1-acid ker antibody and biomarker protein:antibody complexes is glycoprotein 1 (ORM) (see FIG. 13). On the other hand, if 55 formed. The mixture migrates by capillary action towards the more than one, e.g. three biomarker proteins are to be studied T and the C positions. The second anti-biomarker antibody simultaneously, the S position can have three different types immobilized at T will capture all the biomarker protein: anti of anti-biomarker protein antibodies, each type specifically body complexes but not the free anti-biomarker protein anti immunoreactive to one biomarker protein and does not body. The localized concentration of anti-biomarker protein: exhibit cross-reactivity with the other two non-ligand pro antibody complexes that is colloidal gold or latex bead teins (FIG. 14). Arrowheads indicate the boundary limit that 60 labeled will become visible as a colored line at the T position sample should not cross on the membrane. At positions T or (FIG. 15C). Only when the biomarker protein is at or above C, up to three bands can be visible, each band corresponding the reference level will sufficient labeled antibody be cap to each of the biomarker protein that is being tested. When tured at T to produce a visible band (FIG. 15C). When the three proteins are to be studied simultaneously, all three pro biomarker is below the reference level, no visible band should tein types can be represented at the T position and at their 65 appear at the T position (FIG. 15D). respective quantities (FIG. 14). FIG. 14 shows an alternative At position C, free anti-biomarker antibody initially from S design where three proteins can be studied simultaneously on will be bound and captured by the immobilized antibody US 8,535,891 B2 75 76 immunoreactive against the antibody coming from the Sposi expands and develops into a band. The greater the level of tion. This will in turn result in a concentration of a colloidal biomarker in the sample, the wider the colored band at the T gold or latex bead labeled anti-protein antibody accumulated position (FIGS. 17A and B). at the C position and will become visible as colored line at the When excess free anti-biomarker protein antibody from C position. The C position result serves as a test control to the S position arrives to the C position and bind to the immo indicate that there is functional anti-protein antibody in the bilized reference amount of biomarker protein there, another test material and should always be present. A band at C color line become visible. Since there is a reference amount of indicates that labeled antibody from Shad moved to C. There immobilized biomarker protein at the C position, the thick fore, a band at C indicates that the band at T is not a false ness of the visible colored line at the C position defines the positive or that the absence of a band at T is a false negative. 10 reference value of protein. By comparing the thickness of the color band at the Tand C positions on the same test strip, one Example 6 can estimate whether the biomarker protein level is below or greater than the reference value of the protein. When the Diagnostic Lateral Flow Immunoassay Test biomarker protein level is equal or greater than the reference Strips-Design 3 15 value, the color band at the T position will be equal or larger than the color band at the C position respectively (FIGS. 17A An alternative embodiment of the lateral flow immunoas and B). Acute appendicitis is indicated. When the biomarker say (LFIA) test strips for determining the level of biomarker protein level is below the threshold level, the color band at the protein level is illustrated in FIG. 16. This test strip can be T position will be smaller or even absent than the color band used in point-of-care testing. The test strip is as described in at the C position (FIGS. 17C and D). Acute appendicitis is not indicated. The C position band also serves as a test control to FIG. 11 having a sample (S), a test (T), and a control (C) confirm that there is functional anti-protein antibody at the S positions, all three spatially arranged as shown in FIG. 11 and position and that the functional anti-biomarker protein anti FIG. 15. For this embodiment of a test strip, the solid support body is derived from the S position (FIGS. 17E and F). FIG. 161 can be made of plastic or other non porous material, 17E shows a band at position T but no band at position C, supporting the matrix 163. In this embodiment, the S position 25 indicating that the data at T may be a false positive. FIG. 17F contain an excess amount of dehydrate anti-biomarker pro shows no band at either positions Tand C, indicating the data tein antibody (first antibody) that can be labeled (e.g. colloi at T may be a false negative. Both FIGS. 17E and 17F indicate dal gold or color latex bead). Similar to the embodiments in invalid data and that the lateral flow immunoassay should be FIG. 11-14, the anti-biomarker protein antibody at S is repeated with a new test strip. mobile; once the antibody is re-hydrated, the antibody moves 30 When only one biomarker protein is studied, the S position by capillary action towards the T and C positions. should have only one anti-biomarker protein antibody that The T position contains a second anti-biomarker protein specifically immunoreactive with just that one biomarker of antibody that is also immunoreactive to the biomarker protein interest. Akit comprising test strips for use as POCT can have of interest, but to a different epitope on the biomarker (FIG. several single biomarker protein test strips. The kit can test for 16). This second antibody is in excess and is immobilized on only one biomarker or more then one biomarker proteins. In the matrix. This second anti-biomarker protein antibody 35 this embodiment, the test strip can be labeled 181 on one end binds a part of the biomarker protein that is different from the to identify the biomarker protein the test strip is used for, e.g. part of the protein that is bound by the first anti-biomarker the label “L” represents leucine C-2 glycoprotein (LRG); protein antibody found at the S position. In this embodiment, “M” represents mannan-binding lectin serine protease 2 the second antibody at the T position will bind and capture (MASP2): “O'” represents C-1-acid glycoprotein 1 (ORM) 40 and “S” represents C-1-antichymotrypsin (SERPINA3) (see both free unbound biomarker protein and biomarker protein FIG. 18). On the other hand, if more than one, e.g. three antibody complexes, and concentrate them at the T position. biomarker proteins are to be studied simultaneously, the S The C position contains a defined quantity of biomarker position can have three different types of anti-biomarker pro protein immobilized on the membrane (FIG. 16B). The tein antibodies, each type specifically immunoreactive to one defined quantity is the predetermined reference value of the biomarker protein and does not exhibit cross-reactivity with biomarker protein being analyzed on the test strip. The refer 45 the other two non-ligand proteins (FIG. 19). FIG. 19 shows an ence/control level can be the level of the biomarker found in alternative embodiment of a test strip where three biomarker the samples of healthy men. When the excess free anti-biom proteins can be studied simultaneously on the same test strip. arker protein antibody from the Sposition arrives and bind the The positions for each biomarker on the single strip are indi immobilized biomarker protein at C, gradually accumulation cated 191. at C produces a concentration of labeled first antibody will 50 The test strip can be designed in a form of a dipstick test become visible as a colored line at the C position (FIG. 17A, strip (FIG. 16B). As a dipstick test strip, the strip is dipped B, D). into a sample (e.g. urine) at the S position end with sample An application of a fluid sample (e.g. urine) at the S posi level not to exceed the boundary limit. The strip is then laid tion will re-hydrate the excess amount of anti-biomarker pro horizontally with the membrane Surface facing up on a flat tein antibody there. All of the biomarker protein of interest 55 surface. A fixed amount of time is given for the antibody should be bound to the excess anti-biomarker protein anti re-hydration, capillary action, and antibody biomarker pro body. A fluid mixture of free biomarker protein antibody and tein binding reaction to take place. At the end of the fixed biomarker protein-antibody complex is formed and will move time, there should be visible bands at the C position and along the membrane by capillary action towards the T posi depending on the levels of the biomarker protein(s) of inter tion and then Subsequently to the C position. At the Tposition, est, there may or may not be a visible band at the T position all of the biomarker protein-antibody complex will be cap 60 (FIGS. 18 and 19) and the bands can be may different thick tured and immobilized by the second anti-biomarker protein ness. FIG. 18 shows a method of using four separate dipstick antibody. The localized concentration of biomarker protein test strips to test for the four biomarkers of interest. Such test antibody complexes, wherein the anti-biomarker protein anti strip can be the component of a diagnostic kit. Each dipstick body that is colloidal gold or latex bead labeled, will become test strip is labeled 181 to indicate which biomarker protein is visible as a colored line at the T position (FIG. 17A, B, D). 65 being tested. A diagnostic kit can comprise multiple types of With increasing amount of biomarker protein-antibody com single biomarker test strips, a type for each biomarker of plexes and concentrated at the T position, the colored line interest. US 8,535,891 B2 77

SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 52

<210s, SEQ ID NO 1 &211s LENGTH: 347 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 1 Met Ser Ser Trp Ser Arg Glin Arg Pro Llys Ser Pro Gly Gly Ile Glin 1. 5 1O 15 Pro His Val Ser Arg Thr Lieu. Phe Lieu. Lieu. Lieu Lleu Lieu Ala Ala Ser 2O 25 3O Ala Trp Gly Val Thir Lieu. Ser Pro Lys Asp Cys Glin Val Phe Arg Ser 35 4 O 45 Asp His Gly Ser Ser Ile Ser Cys Gln Pro Pro Ala Glu Ile Pro Gly SO 55 6 O Tyr Lieu Pro Ala Asp Thr Val His Lieu Ala Val Glu Phe Phe Asn Lieu. 65 70 7s 8O Thir His Lieu Pro Ala Asn Lieu. Lieu. Glin Gly Ala Ser Llys Lieu. Glin Glu 85 90 95 Lieu. His Lieu. Ser Ser Asn Gly Lieu. Glu Ser Lieu. Ser Pro Glu Phe Lieu. 1OO 105 11 O Arg Pro Val Pro Gln Lieu. Arg Val Lieu. Asp Lieu. Thir Arg Asn Ala Lieu 115 12 O 125 Thr Gly Lieu Pro Pro Gly Lieu Phe Glin Ala Ser Ala Thr Lieu. Asp Thr 13 O 135 14 O Lieu Val Lieu Lys Glu Asn Gln Lieu. Glu Val Lieu. Glu Val Ser Trp Lieu. 145 150 155 160 His Gly Lieu Lys Ala Lieu. Gly His Lieu. Asp Lieu. Ser Gly Asn Arg Lieu. 1.65 17O 17s Arg Llys Lieu Pro Pro Gly Lieu. Lieu Ala Asn. Phe Thr Lieu. Lieu. Arg Thr 18O 185 19 O Lieu. Asp Lieu. Gly Glu Asn Gln Lieu. Glu Thir Lieu Pro Pro Asp Lieu. Lieu. 195 2OO 2O5 Arg Gly Pro Lieu Gln Lieu. Glu Arg Lieu. His Lieu. Glu Gly Asn Llys Lieu 21 O 215 22O Glin Val Lieu. Gly Lys Asp Lieu. Lieu. Lieu Pro Glin Pro Asp Lieu. Arg Tyr 225 23 O 235 24 O Lieu. Phe Lieu. Asn Gly Asn Llys Lieu Ala Arg Val Ala Ala Gly Ala Phe 245 250 255 Glin Gly Lieu. Arg Glin Lieu. Asp Met Lieu. Asp Lieu. Ser Asn. Asn. Ser Lieu. 26 O 265 27 O Ala Ser Val Pro Glu Gly Lieu. Trp Ala Ser Lieu. Gly Glin Pro Asn Trp 27s 28O 285 Asp Met Arg Asp Gly Phe Asp Ile Ser Gly ASn Pro Trp Ile Cys Asp 29 O 295 3 OO Glin Asn Lieu. Ser Asp Lieu. Tyr Arg Trp Lieu. Glin Ala Gln Lys Asp Llys 3. OS 310 315 32O Met Phe Ser Glin Asn Asp Thr Arg Cys Ala Gly Pro Glu Ala Wall Lys 3.25 330 335 Gly Glin Thir Lieu. Lieu Ala Wall Ala Lys Ser Glin 34 O 345

<210s, SEQ ID NO 2 US 8,535,891 B2 79 - Continued

&211s LENGTH: 93 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 2 Met Lieu. Thr Glu Lieu. Glu Lys Ala Lieu. Asn. Ser Ile Ile Asp Val Tyr 1. 5 1O 15 His Llys Tyr Ser Lieu. Ile Lys Gly Asn. Phe His Ala Val Tyr Arg Asp 2O 25 3O Asp Lieu Lys Llys Lieu. Lieu. Glu Thr Glu. Cys Pro Glin Tyr Ile Arg Llys 35 4 O 45 Lys Gly Ala Asp Val Trp Phe Lys Glu Lieu. Asp Ile Asn. Thir Asp Gly SO 55 6 O Ala Val Asn. Phe Glin Glu Phe Lieu. Ile Lieu Val Ile Llys Met Gly Val 65 70 7s 8O Ala Ala His Llys Llys Ser His Glu Glu Ser His Lys Glu 85 90

<210s, SEQ ID NO 3 &211s LENGTH: 2O1 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 3 Met Ala Lieu. Ser Trp Val Lieu. Thr Val Lieu. Ser Lieu. Lieu Pro Lieu. Lieu. 1. 5 1O 15 Glu Ala Glin Ile Pro Leu. Cys Ala Asn Lieu Val Pro Val Pro Ile Thr 2O 25 3O Asn Ala Thir Lieu. Asp Glin Ile Thr Gly Lys Trp Phe Tyr Ile Ala Ser 35 4 O 45 Ala Phe Arg Asn. Glu Glu Tyr Asn Llys Ser Val Glin Glu Ile Glin Ala SO 55 6 O Thr Phe Phe Tyr Phe Thr Pro Asn Llys Thr Glu Asp Thr Ile Phe Leu 65 70 7s 8O Arg Glu Tyr Glin Thr Arg Glin Asp Gln Cys Ile Tyr Asn. Thir Thr Tyr 85 90 95 Lieu. Asn Val Glin Arg Glu Asn Gly. Thir Ile Ser Arg Tyr Val Gly Gly 1OO 105 11 O Glin Glu. His Phe Ala His Lieu. Lieu. Ile Lieu. Arg Asp Thir Lys Thr Tyr 115 12 O 125 Met Lieu Ala Phe Asp Wall Asn Asp Glu Lys Asn Trp Gly Lieu. Ser Val 13 O 135 14 O Tyr Ala Asp Llys Pro Glu Thir Thr Lys Glu Gln Leu Gly Glu Phe Tyr 145 150 155 160 Glu Ala Lieu. Asp Cys Lieu. Arg Ile Pro Llys Ser Asp Val Val Tyr Thr 1.65 17O 17s Asp Trp Llys Lys Asp Llys Cys Glu Pro Lieu. Glu Lys Gln His Glu Lys 18O 185 19 O Glu Arg Lys Glin Glu Glu Gly Glu Ser 195 2OO

<210s, SEQ ID NO 4 &211s LENGTH: 810 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 4 Met Glu. His Lys Glu Val Val Lieu. Lieu. Lieu. Lieu Lleu Phe Lieu Lys Ser US 8,535,891 B2 81 82 - Continued

1O 15

Gly Glin Gly Glu Pro Lell Asp Asp Tyr Wall ASn Thir Glin Gly Ala Ser 2O 25

Lell Phe Ser Wall Thir Glin Luell Gly Ala Gly Ser Ile Glu Glu 35 4 O 45

Ala Ala Cys Glu Glu Asp Glu Glu Phe Thir Arg Ala Phe SO 55 6 O

His Ser Lys Glu Glin Glin Wall Ile Met Ala Glu Asn Arg 70

Ser Ser Ile Ile Ile Arg Met Arg Asp Wall Wall Lell Phe Glu Lys 85 90 95

Wall Luell Ser Glu Thir Gly ASn Gly Asn 105 11 O

Gly Thir Met Ser Lys Thir Asn Gly Ile Thir Glin Trp Ser 115 12 O 125

Ser Thir Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thir His Pro Ser 13 O 135 14 O

Glu Gly Luell Glu Glu Asn Arg Asn Pro Asp Asn Asp Pro Glin 145 150 155 160

Gly Pro Trp Tyr Thir Thir Asp Pro Glu Arg Asp Tyr Cys 1.65 17O 17s

Asp Ile Luell Glu Cys Glu Glu Glu Cys Met His Ser Gly Glu Asn 18O 185 19 O

Asp Gly Ile Ser Thir Met Ser Gly Lell Glu Glin Ala 195

Trp Asp Ser Glin Ser Pro His Ala His Gly Ile Pro Ser Phe 21 O 215 22O

Pro Asn Asn Lell Lys Asn Arg Asn Pro Asp Arg Glu 225 23 O 235 24 O

Lell Arg Pro Trp Cys Phe Thir Thir Asp Pro ASn Arg Trp Glu Luell 245 250 255

Asp Ile Pro Arg Thir Thir Pro Pro Pro Ser Ser Gly Pro Thir 26 O 265 27 O

Glin Cys Luell Lys Gly Thir Gly Glu Asn Arg Gly Asn Wall Ala 27s 285

Wall Thir Wall Ser Gly His Thir Glin His Trp Ser Ala Glin Thir Pro 29 O 295 3 OO

His Thir His Asn Arg Thir Pro Glu Asn Phe Pro Asn Luell Asp 3. OS 310 315

Glu Asn Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His 3.25 330 335

Thir Thir Asn Ser Glin Wall Arg Trp Glu Ile Pro Ser Cys 34 O 345 35. O

Asp Ser Ser Pro Wall Ser Thir Glu Glin Luell Ala Pro Thir Ala Pro Pro 355 360 365

Glu Luell Thir Pro Wall Wall Glin Asp His Gly Asp Gly Glin Ser 37 O 375 38O

Tyr Arg Gly Thir Ser Ser Thir Thir Thir Thir Gly Glin Ser 385 390 395 4 OO

Trp Ser Ser Met Thir Pro His Arg His Glin Thir Pro Glu Asn Tyr 4 OS 41O 415

Pro Asn Ala Gly Lell Thir Met Asn Tyr Arg Asn Pro Asp Ala Asp 42O 425 43 O US 8,535,891 B2 83 - Continued Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr 435 44 O 445 Cys Asn Lieu Lys Lys Cys Ser Gly Thr Glu Ala Ser Val Val Ala Pro 450 45.5 460 Pro Pro Val Val Lieu. Leu Pro Asp Val Glu Thr Pro Ser Glu Glu Asp 465 470 47s 48O Cys Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Arg Ala Thir Thr 485 490 495 Val Thr Gly Thr Pro Cys Glin Asp Trp Ala Ala Glin Glu Pro His Arg SOO 505 51O His Ser Ile Phe Thr Pro Glu Thir Asn Pro Arg Ala Gly Lieu. Glu Lys 515 52O 525 Asn Tyr Cys Arg ASn Pro Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr 53 O 535 54 O Thir Thr Asn Pro Arg Llys Lieu. Tyr Asp Tyr Cys Asp Val Pro Gln Cys 5.45 550 555 560 Ala Ala Pro Ser Phe Asp Cys Gly Llys Pro Glin Val Glu Pro Llys Llys 565 st O sts Cys Pro Gly Arg Val Val Gly Gly Cys Val Ala His Pro His Ser Trp 58O 585 59 O Pro Trp Glin Val Ser Lieu. Arg Thr Arg Phe Gly Met His Phe Cys Gly 595 6OO 605 Gly Thr Lieu. Ile Ser Pro Glu Trp Val Lieu. Thir Ala Ala His Cys Lieu. 610 615 62O Glu Lys Ser Pro Arg Pro Ser Ser Tyr Llys Val Ile Lieu. Gly Ala His 625 630 635 64 O Glin Glu Val Asn Lieu. Glu Pro His Val Glin Glu Ile Glu Val Ser Arg 645 650 655 Lieu. Phe Lieu. Glu Pro Thr Arg Lys Asp Ile Ala Lieu Lleu Lys Lieu. Ser 660 665 67 O Ser Pro Ala Val Ile Thr Asp Llys Val Ile Pro Ala Cys Lieu. Pro Ser 675 68O 685 Pro Asn Tyr Val Val Ala Asp Arg Thr Glu. Cys Phe Ile Thr Gly Trp 69 O. 695 7 OO Gly Glu Thr Glin Gly Thr Phe Gly Ala Gly Lieu Lleu Lys Glu Ala Glin 7 Os 71O 71s 72O Lieu Pro Val Ile Glu Asn Llys Val Cys Asn Arg Tyr Glu Phe Lieu. Asn 72 73 O 73 Gly Arg Val Glin Ser Thr Glu Lieu. Cys Ala Gly His Lieu Ala Gly Gly 740 74. 7 O Thr Asp Ser Cys Glin Gly Asp Ser Gly Gly Pro Leu Val Cys Phe Glu 7ss 760 765 Lys Asp Llys Tyr Ile Lieu. Glin Gly Val Thir Ser Trp Gly Lieu. Gly Cys 770 775 78O Ala Arg Pro Asn Llys Pro Gly Val Tyr Val Arg Val Ser Arg Phe Val 78s 79 O 79. 8OO Thir Trp Ile Glu Gly Val Met Arg Asn Asn 805 810

<210s, SEQ ID NO 5 &211s LENGTH: 185 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 5 US 8,535,891 B2 85 86 - Continued

Met Arg Luell Lel Lell Lell Gly Luell Luell Cys Gly Ser Wall Ala Thir 15

Pro Luell Gly Pro Trp Pro Glu Pro Wall Phe Gly Arg Luell Ala Ser 2O 25 3O

Pro Gly Phe Gly Glu Ala Asn Asp Glin Glu Arg Arg Trp Thir 35 4 O 45

Lell Thir Ala Pro Gly Tyr Arg Luell Arg Luell Tyr Phe Thir His Phe SO 55 6 O

Asp Luell Glu Lel Ser His Lell Glu Tyr Asp Phe Wall Luell Ser 65 70

Ser Gly Ala Wall Lell Ala Thir Luell Cys Gly Glin Glu Ser Thir Asp 85 90 95

Thir Glu Arg Ala Pro Gly Asp Thir Phe Ser Lell Gly Ser Ser 1OO 105 11 O

Lell Asp Ile Thir Phe Arg Ser Asp Ser ASn Glu Lys Pro Phe Thir 115 12 O 125

Gly Phe Glu Ala Phe Ala Ala Glu Asp Ile Asp Glu Glin Wall 13 O 135 14 O

Ala Pro Gly Glu Ala Pro Thir Asp His His His Asn His Luell 145 150 155 160

Gly Gly Phe Cys Ser Arg Ala Gly Tyr Wall Lell His Arg Asn 1.65 17O 17s

Arg Thir Cys Ser Glu Glin Ser Luell 18O 185

<210s, SEQ ID NO 6 &211s LENGTH: 298 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 6

Met Val Arg Met Wall Pro Wall Luell Luell Ser Luell Lell Lell Luell Luell Gly 1. 15

Pro Ala Wall Pro Glin Glu Asn Glin Asp Gly Arg Ser Luell Thir 2O 25

Ile Thir Gly Lell Ser His Wall Glu Asp Wall Pro Ala Phe Glin 35 4 O 45

Ala Luell Gly Ser Lell Asn Asp Luell Glin Phe Phe Arg Asn Ser SO 55 6 O

Asp Arg Ser Glin Pro Met Gly Luell Trp Arg Glin Wall Glu Gly Met 65 70 7s 8O

Glu Asp Trp Glin Asp Ser Glin Luell Glin Ala Arg Glu Asp Ile 85 90 95

Phe Met Glu Thir Lell Asp Ile Wall Glu Asn Asp Ser Asn 105 11 O

Gly Ser His Wall Lell Glin Gly Arg Phe Gly Glu Ile Glu Asn Asn 115 12 O 125

Arg Ser Ser Gly Ala Phe Trp Tyr Asp Gly Asp 13 O 135 14 O

Ile Glu Phe Asn Glu Ile Pro Ala Trp Wall Pro Phe Pro Ala 145 150 155 160

Ala Glin Ile Thir Lys Glin Trp Glu Ala Glu Pro Wall Wall Glin 1.65 17O 17s

Arg Ala Ala Tyr Lell Glu Glu Glu Pro Ala Thir Luell Arg 18O 185 19 O US 8,535,891 B2 87 - Continued Tyr Lieu Lys Tyr Ser Lys Asn. Ile Lieu. Asp Arg Glin Asp Pro Pro Ser 195 2OO 2O5 Val Val Val Thir Ser His Glin Ala Pro Gly Glu Lys Llys Llys Lieu Lys 21 O 215 22O Cys Lieu Ala Tyr Asp Phe Tyr Pro Gly Lys Ile Asp Val His Trp Thr 225 23 O 235 24 O Arg Ala Gly Glu Val Glin Glu Pro Glu Lieu. Arg Gly Asp Val Lieu. His 245 250 255 Asn Gly Asn Gly Thr Tyr Glin Ser Trp Val Val Val Ala Val Pro Pro 26 O 265 27 O Gln Asp Thr Ala Pro Tyr Ser Cys His Val Gln His Ser Ser Leu Ala 27s 28O 285 Gln Pro Leu Val Val Pro Trp Glu Ala Ser 29 O 295

<210s, SEQ ID NO 7 &211s LENGTH: 189 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OO > SEQUENCE: 7 Met Val Met Lieu Lleu Lleu Lleu Lieu. Ser Ala Lieu Ala Gly Lieu. Phe Gly 1. 5 1O 15 Ala Ala Glu Gly Glin Ala Phe His Lieu. Gly Lys Cys Pro Asn Pro Pro 2O 25 3O Val Glin Glu Asn. Phe Asp Val Asn Llys Tyr Lieu. Gly Arg Trp Tyr Glu 35 4 O 45 Ile Glu Lys Ile Pro Thir Thr Phe Glu Asn Gly Arg Cys Ile Glin Ala SO 55 6 O Asn Tyr Ser Lieu Met Glu Asn Gly Lys Ile Llys Val Lieu. Asn Glin Glu 65 70 7s 8O Lieu. Arg Ala Asp Gly Thr Val Asin Glin Ile Glu Gly Glu Ala Thr Pro 85 90 95 Val Asn Lieu. Thr Glu Pro Ala Lys Lieu. Glu Val Llys Phe Ser Trp Phe 1OO 105 11 O Met Pro Ser Ala Pro Tyr Trp Ile Leu Ala Thr Asp Tyr Glu Asn Tyr 115 12 O 125 Ala Leu Val Tyr Ser Cys Thr Cys Ile Ile Gln Leu Phe His Val Asp 13 O 135 14 O Phe Ala Trp Ile Lieu Ala Arg Asin Pro Asn Lieu Pro Pro Glu Thr Val 145 150 155 160 Asp Ser Lieu Lys Asn. Ile Lieu. Thir Ser Asn. Asn. Ile Asp Wall Lys Llys 1.65 17O 17s Met Thr Val Thr Asp Glin Val Asn Cys Pro Llys Lieu Ser 18O 185

<210s, SEQ ID NO 8 &211s LENGTH: 448 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 8 Met Lys Ile His Tyr Ser Arg Glin Thr Ala Lieu. Glu Ser Thr Ser Tyr 1. 5 1O 15 Ile Glin Lieu Pro Glu Ala Glu Lieu. Arg Met Glu Arg Met Lieu Pro Lieu. 2O 25 3O

Lieu Ala Lieu. Gly Lieu. Lieu Ala Ala Gly Phe Cys Pro Ala Val Lieu. Cys US 8,535,891 B2 89 90 - Continued

35 4 O 45

His Pro Asn Ser Pro Lell Asp Glu Glu Asn Luell Thir Glin Glu Asn Glin SO 55 6 O

Asp Arg Gly Thir His Wall Asp Luell Gly Luell Ala Ser Ala Asn Wall Asp 65 70

Phe Ala Phe Ser Lell Tyr Glin Luell Wall Luell Ala Pro Asp 85 90 95

Asn Wall Ile Phe Ser Pro Lell Ser Ile Ser Thir Ala Lell Ala Phe Luell 105 11 O

Ser Luell Gly Ala His Asn Thir Thir Luell Thir Glu Ile Lell Gly Luell 115 12 O 125

Phe Asn Luell Thir Glu Thir Ser Glu Ala Glu Ile His Glin Ser Phe 13 O 135 14 O

Glin His Luell Luell Arg Thir Lell Asn Glin Ser Ser Asp Glu Luell Glin Luell 145 150 155 160

Ser Met Gly Asn Ala Met Phe Wall Glu Glin Lell Ser Luell Luell Asp 1.65 17O 17s

Arg Phe Thir Glu Asp Ala Arg Luell Tyr Gly Ser Glu Ala Phe Ala 18O 185 19 O

Thir Asp Phe Glin Asp Ser Ala Ala Ala Lell Ile Asn Asp 195

Wall Lys Asn Gly Thir Arg Gly Lys Ile Thir Asp Lell Ile Asp Luell 21 O 215

Asp Ser Glin Thir Met Met Wall Luell Wall Asn Tyr Ile Phe Phe Ala 225 23 O 235 24 O

Trp Glu Met Pro Phe Asp Pro Glin Asp Thir His Glin Ser Arg Phe 245 250 255

Luell Ser Lys Lys Trp Wall Met Wall Pro Met Met Ser Luell His 26 O 265 27 O

His Luell Thir Ile Pro Phe Arg Asp Glu Glu Lell Ser Thir Wall 27s 285

Wall Glu Luell Tyr Thir Gly Asn Ala Ser Ala Lell Phe Ile Luell Pro 29 O 295 3 OO

Asp Glin Asp Met Glu Glu Wall Glu Ala Met Lell Lell Pro Glu Thir 3. OS 310 315

Lell Arg Trp Arg Asp Ser Luell Glu Phe Arg Glu Ile Gly Glu Luell 3.25 330 335

Luell Pro Lys Phe Ser Ile Ser Arg Asp Asn Lell Asn Asp Ile 34 O 345 35. O

Lell Luell Glin Luell Gly Ile Glu Glu Ala Phe Thir Ser Lys Ala Asp Luell 355 360 365

Ser Gly Ile Thir Gly Ala Arg Asn Luell Ala Wall Ser Glin Wall Wall His 37 O 375

Lys Ala Wall Luell Asp Wall Phe Glu Glu Gly Thir Glu Ala Ser Ala Ala 385 390 395 4 OO

Thir Ala Wall Ile Thir Lell Luell Ser Ala Luell Wall Glu Thir Arg Thir 4 OS 415

Ile Wall Arg Phe Asn Arg Pro Phe Luell Met Ile Ile Wall Pro Thir Asp 42O 425 43 O

Thir Glin Asn Ile Phe Phe Met Ser Wall Thir Asn Pro Glin Ala 435 44 O 445

<210s, SEQ ID NO 9 &211s LENGTH: 373 US 8,535,891 B2 91 - Continued

212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 9 Met Ser Lieu. Lieu Lleu Lleu Lleu Lieu. Lieu Val Ser Tyr Tyr Val Gly. Thir 1. 5 1O 15 Lieu. Gly Thr His Thr Glu Ile Lys Arg Val Ala Glu Glu Lys Val Thr 2O 25 3O Lieu Pro Cys His His Glin Lieu. Gly Lieu Pro Glu Lys Asp Thir Lieu. Asp 35 4 O 45 Ile Glu Trp Lieu. Lieu. Thir Asp Asn. Glu Gly Asn Gln Llys Val Val Ile SO 55 6 O Thr Tyr Ser Ser Arg His Val Tyr Asn Asn Lieu. Thr Glu Glu Gln Lys 65 70 7s 8O Gly Arg Val Ala Phe Ala Ser Asn. Phe Lieu Ala Gly Asp Ala Ser Lieu. 85 90 95 Glin Ile Glu Pro Lieu Lys Pro Ser Asp Glu Gly Arg Tyr Thr Cys Llys 1OO 105 11 O Val Lys Asn. Ser Gly Arg Tyr Val Trp Ser His Val Ile Lieu Lys Val 115 12 O 125 Lieu Val Arg Pro Ser Llys Pro Llys Cys Glu Lieu. Glu Gly Glu Lieu. Thir 13 O 135 14 O Glu Gly Ser Asp Lieu. Thir Lieu Gln Cys Glu Ser Ser Ser Gly Thr Glu 145 150 155 160 Pro Ile Val Tyr Tyr Trp Glin Arg Ile Arg Glu Lys Glu Gly Glu Asp 1.65 17O 17s Glu Arg Lieu Pro Pro Llys Ser Arg Ile Asp Tyr Asn His Pro Gly Arg 18O 185 19 O Val Lieu. Leu Glin Asn Lieu. Thir Met Ser Tyr Ser Gly Lieu. Tyr Glin Cys 195 2OO 2O5 Thir Ala Gly Asn. Glu Ala Gly Lys Glu Ser Cys Val Val Arg Val Thr 21 O 215 22O Val Glin Tyr Val Glin Ser Ile Gly Met Val Ala Gly Ala Val Thr Gly 225 23 O 235 24 O Ile Val Ala Gly Ala Lieu. Lieu. Ile Phe Lieu. Lieu Val Trp Lieu. Lieu. Ile 245 250 255 Arg Arg Lys Asp Llys Glu Arg Tyr Glu Glu Glu Glu Arg Pro Asn. Glu 26 O 265 27 O Ile Arg Glu Asp Ala Glu Ala Pro Lys Ala Arg Lieu Val Llys Pro Ser 27s 28O 285 Ser Ser Ser Ser Gly Ser Arg Ser Ser Arg Ser Gly Ser Ser Ser Thr 29 O 295 3 OO Arg Ser Thir Ala Asn. Ser Ala Ser Arg Ser Glin Arg Thr Lieu. Ser Thr 3. OS 310 315 32O Asp Ala Ala Pro Gln Pro Gly Lieu Ala Thr Glin Ala Tyr Ser Lieu Val 3.25 330 335 Gly Pro Glu Val Arg Gly Ser Glu Pro Llys Llys Val His His Ala Asn 34 O 345 35. O Lieu. Thir Lys Ala Glu Thir Thr Pro Ser Met Ile Pro Ser Glin Ser Arg 355 360 365

Ala Phe Glin. Thir Wall 37 O

<210s, SEQ ID NO 10 &211s LENGTH: 352 US 8,535,891 B2 93 - Continued

212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 10 Met Arg Ser Lieu. Gly Ala Lieu. Lieu. Lieu. Lieu. Lieu. Ser Ala Cys Lieu Ala 1. 5 1O 15 Val Ser Ala Gly Pro Val Pro Thr Pro Pro Asp Asn Ile Glin Val Glin 2O 25 3O Glu Asn. Phe Asn. Ile Ser Arg Ile Tyr Gly Lys Trp Tyr Asn Lieu Ala 35 4 O 45 Ile Gly Ser Thr Cys Pro Trp Leu Lys Lys Ile Met Asp Arg Met Thr SO 55 6 O Val Ser Thir Lieu Val Lieu. Gly Glu Gly Ala Thr Glu Ala Glu Ile Ser 65 70 7s 8O Met Thr Ser Thr Arg Trp Arg Lys Gly Val Cys Glu Glu Thir Ser Gly 85 90 95 Ala Tyr Glu Lys Thr Asp Thr Asp Gly Llys Phe Lieu. Tyr His Llys Ser 1OO 105 11 O Lys Trp Asn Ile Thr Met Glu Ser Tyr Val Val His Thr Asn Tyr Asp 115 12 O 125 Glu Tyr Ala Ile Phe Lieu. Thir Lys Llys Phe Ser Arg His His Gly Pro 13 O 135 14 O Thir Ile Thr Ala Lys Lieu. Tyr Gly Arg Ala Pro Glin Lieu. Arg Glu Thir 145 150 155 160 Lieu. Lieu. Glin Asp Phe Arg Val Val Ala Glin Gly Val Gly Ile Pro Glu 1.65 17O 17s Asp Ser Ile Phe Thr Met Ala Asp Arg Gly Glu. Cys Val Pro Gly Glu 18O 185 19 O Glin Glu Pro Glu Pro Ile Lieu. Ile Pro Arg Val Arg Arg Ala Val Lieu. 195 2OO 2O5 Pro Glin Glu Glu Glu Gly Ser Gly Gly Gly Gln Leu Val Thr Glu Val 21 O 215 22O Thir Lys Lys Glu Asp Ser Cys Glin Lieu. Gly Tyr Ser Ala Gly Pro Cys 225 23 O 235 24 O Met Gly Met Thr Ser Arg Tyr Phe Tyr Asn Gly Thr Ser Met Ala Cys 245 250 255 Glu Thr Phe Glin Tyr Gly Gly Cys Met Gly Asn Gly Asn Asn Phe Val 26 O 265 27 O Thr Glu Lys Glu. Cys Lieu. Glin Thr Cys Arg Thr Val Ala Ala Cys Asn 27s 28O 285 Lieu Pro Ile Val Arg Gly Pro Cys Arg Ala Phe Ile Glin Lieu. Trp Ala 29 O 295 3 OO Phe Asp Ala Wall Lys Gly Lys Cys Val Lieu. Phe Pro Tyr Gly Gly Cys 3. OS 310 315 32O Glin Gly Asn Gly Asn Llys Phe Tyr Ser Glu Lys Glu. Cys Arg Glu Tyr 3.25 330 335 Cys Gly Val Pro Gly Asp Gly Asp Glu Glu Lieu. Lieu. Arg Phe Ser Asn 34 O 345 35. O

<210s, SEQ ID NO 11 &211s LENGTH: 763 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 11 Met Ala Ala Thr Gly Thr Ala Ala Ala Ala Ala Thr Gly Arg Lieu. Lieu. US 8,535,891 B2 95 96 - Continued

1O 15

Luell Luell Luell Wall Gly Lell Thir Ala Pro Ala Lell Ala Luell Ala Gly 2O 25 3O

Ile Glu Ala Lell Ala Ala Asn Ala Gly Thir Gly Phe Ala Wall Ala 35 4 O 45

Glu Pro Glin Ile Ala Met Phe Gly Luell Asn Met His Wall Asn SO 55 6 O

Ile Glin Thir Gly Trp Glu Pro Asp Pro Thir Gly Thir Ser Cys 65 70

Phe Glu Thir Glu Glu Wall Luell Glin Tyr Cys Glin Glu Met Tyr Pro 85 90 95

Glu Luell Glin Ile Thir Asn Wall Met Glu Ala ASn Glin Arg Wall Ser Ile 1OO 105 11 O

Asp Asn Trp Arg Asp Lys Glin Cys Ser Arg Phe Wall 115 12 O 125

Thir Pro Phe Cys Lell Wall Gly Glu Phe Wall Ser Asp Wall Luell Luell 13 O 135 14 O

Wall Pro Glu Cys Glin Phe Phe His Glu Arg Met Glu Wall Cys 145 150 155 160

Glu Asn His Glin His Trp His Thir Wall Wall Glu Ala Luell Thir 1.65 17O 17s

Glin Gly Met Thir Lell Ser Gly Met Luell Lell Pro Cys Gly Wall 18O 185 19 O

Asp Glin Phe His Gly Thir Glu Tyr Wall Pro Glin Thir Ile 195

Ile Gly Ser Wall Ser Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu 21 O 215

Glu Glu Glu Asp Glu Glu Glu Asp Asp Wall Ser Glu Phe 225 23 O 235 24 O

Pro Thir Glu Ala Asp Lell Glu Asp Phe Thir Glu Ala Wall Asp Glu 245 250 255

Asp Asp Glu Asp Glu Glu Glu Gly Glu Glu Wall Wall Asp Arg Asp 26 O 265 27 O

Tyr Asp Thir Phe Gly Asp Asp Tyr Asn Glu Asn Pro 27s

Thir Glu Pro Gly Ser Asp Gly Thir Met Ser Asp Lys Ile Thir His 29 O 295 3 OO

Asp Wall Ala Wall Cys Ser Glin Glu Ala Met Thir Pro Arg 3. OS 310 315

Ala Wall Met Pro Arg Trp Phe Asp Luell Ser Cys Wall 3.25 330 335

Arg Phe Ile Tyr Gly Gly Gly Gly Asn Arg Asn Asn Phe Glu Ser 34 O 345 35. O

Glu Asp Tyr Met Ala Wall Cys Ala Met Ile Pro Pro Thir Pro 355 360 365

Lell Pro Thir Asn Asp Wall Asp Wall Phe Glu Thir Ser Ala Asp Asp 37 O 375

Asn Glu His Ala Arg Phe Glin Ala Glu Glin Lell Glu Ile Arg 385 390 395 4 OO

His Arg Asn Arg Met Asp Arg Wall Lys Glu Trp Glu Glu Ala Glu 4 OS 41O 415

Lell Glin Ala Lys Asn Lell Pro Ala Glu Arg Glin Thir Luell Ile Glin 42O 425 43 O US 8,535,891 B2 97 - Continued His Phe Glin Ala Met Wall Lys Ala Lieu. Glu Lys Glu Ala Ala Ser Glu 435 44 O 445 Lys Glin Glin Lieu Val Glu Thir His Lieu Ala Arg Val Glu Ala Met Lieu. 450 45.5 460 Asn Asp Arg Arg Arg Met Ala Lieu. Glu Asn Tyr Lieu Ala Ala Lieu. Glin 465 470 47s 48O Ser Asp Pro Pro Arg Pro His Arg Ile Lieu. Glin Ala Lieu. Arg Arg Tyr 485 490 495 Val Arg Ala Glu Asn Lys Asp Arg Lieu. His Thir Ile Arg His Tyr Glin SOO 505 51O His Val Lieu Ala Val Asp Pro Glu Lys Ala Ala Glin Met Lys Ser Glin 515 52O 525 Val Met Thir His Lieu. His Val Ile Glu Glu Arg Arg Asn. Glin Ser Lieu 53 O 535 54 O Ser Lieu. Lieu. Tyr Llys Val Pro Tyr Val Ala Glin Glu Ile Glin Glu Glu 5.45 550 555 560 Ile Asp Glu Lieu. Lieu. Glin Glu Glin Arg Ala Asp Met Asp Glin Phe Thr 565 st O sts Ala Ser Ile Ser Glu Thr Pro Val Asp Val Arg Val Ser Ser Glu Glu 58O 585 59 O

Ser Glu Glu. Ile Pro Pro Phe His Pro Phe His Pro Phe Pro Ala Lieu. 595 6OO 605 Pro Glu Asn Glu Asp Thr Gln Pro Glu Lieu. Tyr His Pro Met Lys Lys 610 615 62O Gly Ser Gly Val Gly Glu Gln Asp Gly Gly Lieu. Ile Gly Ala Glu Glu. 625 630 635 64 O Llys Val Ile Asn. Ser Lys Asn Llys Val Asp Glu Asn Met Val Ile Asp 645 650 655 Glu Thir Lieu. Asp Wall Lys Glu Met Ile Phe Asn Ala Glu Arg Val Gly 660 665 67 O Gly Lieu. Glu Glu Glu Arg Glu Ser Val Gly Pro Lieu. Arg Glu Asp Phe 675 68O 685 Ser Lieu. Ser Ser Ser Ala Lieu. Ile Gly Lieu. Lieu Val Ile Ala Val Ala 69 O. 695 7 OO Ile Ala Thr Val Ile Val Ile Ser Lieu Val Met Lieu. Arg Lys Arg Glin 7 Os 71O 71s 72O Tyr Gly. Thir Ile Ser His Gly Ile Val Glu Val Asp Pro Met Lieu. Thr 72 73 O 73 Pro Glu Glu Arg His Lieu. Asn Llys Met Glin Asn His Gly Tyr Glu Asn 740 74. 7 O Pro Thr Tyr Lys Tyr Lieu. Glu Gln Met Glin Ile 7ss 760

<210s, SEQ ID NO 12 &211s LENGTH: 805 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 12 Met Ser Ser Ser Ser Trp Lieu. Lieu. Lieu. Ser Lieu Val Ala Val Thir Ala 1. 5 1O 15 Ala Glin Ser Thir Ile Glu Glu Glin Ala Lys Thr Phe Lieu. Asp Llys Phe 2O 25 3O Asn His Glu Ala Glu Asp Lieu. Phe Tyr Glin Ser Ser Lieu Ala Ser Trip 35 4 O 45 US 8,535,891 B2 99 100 - Continued

Asn Tyr Asn Thir Asn Ile Thir Glu Glu Asn Wall Glin Asn Met Asn Asn SO 55 6 O

Ala Gly Asp Trp Ser Ala Phe Luell Glu Glin Ser Thir Luell Ala 65 70 8O

Glin Met Pro Lell Glin Glu Ile Glin Asn Luell Thir Wall Luell Glin 85 90 95

Lell Glin Ala Luell Glin Glin Asn Gly Ser Ser Wall Lell Ser Glu Asp 105 11 O

Ser Arg Luell Asn Thir Ile Luell Asn Thir Met Ser Thir Ile Ser 115 12 O 125

Thir Gly Wall Cys Asn Pro Asp Asn Pro Glin Glu Luell Luell Luell 13 O 135 14 O

Glu Pro Gly Luell Asn Glu Ile Met Ala Asn Ser Lell Asp Asn Glu 145 150 155 160

Arg Luell Trp Ala Trp Glu Ser Trp Arg Ser Glu Wall Gly Glin Luell 1.65 17O 17s

Arg Pro Luell Tyr Glu Glu Tyr Wall Wall Luell Lys Asn Glu Met Ala Arg 18O 185 19 O

Ala Asn His Glu Asp Gly Asp Trp Arg Gly Asp Glu 195

Wall Asn Gly Wall Asp Gly Tyr Asp Ser Arg Gly Glin Luell Ile Glu 21 O 215 22O

Asp Wall Glu His Thir Phe Glu Glu Ile Pro Lell Tyr Glu His Luell 225 23 O 235 24 O

His Ala Wall Arg Ala Luell Met Asn Ala Pro Ser Tyr Ile 245 250 255

Ser Pro Ile Gly Cys Lell Pro Ala His Luell Luell Gly Asp Met Trp Gly 26 O 265 27 O

Arg Phe Trp Thir Asn Lell Tyr Ser Luell Thir Wall Pro Phe Gly Glin 28O 285

Pro Asn Ile Asp Wall Thir Asp Ala Met Wall Asp Glin Ala Trp Asp Ala 29 O 295 3 OO

Glin Arg Ile Phe Glu Ala Glu Phe Phe Wall Ser Wall Gly Luell 3. OS 310 315

Pro Asn Met Thir Glin Gly Phe Trp Glu Asn Ser Met Lell Thir Asp Pro 3.25 330 335

Gly Asn Wall Glin Ala Wall His Pro Thir Ala Trp Asp Luell Gly 34 O 345 35. O

Gly Asp Phe Ile Lell Met Thir Wall Thir Met Asp Asp 355 360 365

Phe Luell Thir Ala His His Glu Met Gly His Ile Glin Tyr Asp Met Ala 37 O 375

Tyr Ala Ala Glin Pro Phe Lell Luell Arg Asn Gly Ala Asn Glu Gly Phe 385 390 395 4 OO

His Glu Ala Wall Gly Glu Ile Met Ser Luell Ser Ala Ala Thir Pro 4 OS 415

His Luell Ser Ile Gly Lell Luell Ser Pro Asp Phe Glin Glu Asp Asn 425 43 O

Glu Thir Glu Ile Asn Phe Lell Luell Glin Ala Lell Thir Ile Wall Gly 435 44 O 445

Thir Luell Pro Phe Thir Met Luell Glu Trp Arg Trp Met Wall Phe 450 45.5 460

Lys Gly Glu Ile Pro Lys Asp Glin Trp Met Lys Trp Trp Glu Met 465 470 47s 48O US 8,535,891 B2 101 102 - Continued

Arg Glu Ile Wall Gly Wall Wall Glu Pro Wall Pro His Asp Glu Thir 485 490 495

Asp Pro Ala Ser Lell Phe His Wall Ser Asn Asp Tyr Ser Phe SOO 505 51O

Ile Arg Tyr Thir Arg Thir Luell Tyr Glin Phe Glin Phe Glin Glu Ala 515 525

Lell Cys Glin Ala Ala His Glu Gly Pro Luell His Asp Ile 53 O 535 54 O

Ser Asn Ser Thir Glu Ala Gly Glin Llys Lieu. Phe Asn Met Luell Arg Luell 5.45 550 555 560

Gly Ser Glu Pro Trp Thir Luell Ala Lieu Glu Asn Wall Wall Gly Ala 565 st O sts

Asn Met Asn Wall Arg Pro Luell Luell Asn Tyr Phe Glu Pro Luell Phe 585 59 O

Thir Trp Luell Asp Glin Asn Lys Asn. Ser Phe Wall Gly Trp Ser Thir 595 605

Asp Trp Ser Pro Tyr Ala Asp Glin Ser Ile Lys Wall Arg Ile Ser Luell 610 615

Lys Ser Ala Luell Gly Asp Ala Tyr Glu Trp Asn Asp Asn Glu Met 625 630 635 64 O

Luell Phe Arg Ser Ser Wall Ala Met Arg Glin Phe Luell 645 650 655

Wall Asn Glin Met Ile Luell Phe Gly Glu Glu Asp Wall Arg Wall 660 665 67 O

Ala Asn Luell Pro Arg Ile Ser Phe Asn Phe Phe Wall Thir Ala Pro 675 685

Asn Wall Ser Asp Ile Ile Pro Arg Thr Glu Wall Glu Ala Ile 69 O. 695 7 OO

Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Lell Asn Asp Asn 7 Os

Ser Luell Glu Phe Lell Gly Ile Glin Pro Thir Luell Gly Pro Pro Asn Glin 72 73 O 73

Pro Pro Wall Ser Ile Trp Lell Ile Wall Phe Gly Wall Wall Met Gly Wall 740 74. 7 O

Ile Wall Wall Gly Ile Wall Ile Luell Ile Phe Thir Gly Ile Arg Asp Arg 760 765

Lys Asn Ala Arg Ser Gly Glu ASn Pro Ala Ser Ile 770 775

Asp Ile Ser Gly Glu Asn Asn Pro Gly Phe Glin Asn Thir Asp Asp 78s 79 O 79. 8OO

Wall Glin Thir Ser Phe 805

SEQ ID NO 13 LENGTH: 1479 TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 13 Met Ala Pro Lieu. Lieu. Gly Arg Llys Pro Phe Pro Leu Wall Llys Pro Lieu. 1. 5 15

Pro Gly Glu Glu Pro Leu Phe Thr Ile Pro His Thr Glin Glu Ala Phe 25

Arg Thr Arg Glu Glu Tyr Glu Ala Arg Lieu. Glu Arg Tyr Ser Glu Arg 35 4 O 45 US 8,535,891 B2 103 104 - Continued

Ile Trp Thir Lys Ser Thir Gly Ser Ser Glin Lell Thir His Lys Glu SO 55 6 O

Ala Trp Glu Glu Glu Glin Glu Wall Ala Glu Luell Lell Glu Glu Phe 65 70 8O

Pro Ala Trp Glu Lell Wall Luell Glu Met Wall His His Asn Thir 85 90 95

Ala Ser Luell Glu Lell Wall Asp Thir Ala Trp Lell Glu Ile Met Thir 105 11 O

Ala Wall Gly Glu Glu Cys Asp Phe Glu Wall Gly Lys Glu 115 12 O 125

Met Luell Wall Lys Ile Wall Ile His Pro Lell Glu Wall Asp 13 O 135 14 O

Glu Glu Ala Thir Glu Lys Ser Asp Gly Ala Asp Ser Pro Ser 145 150 155 160

Ser Asp Glu Asn Ser Ser Glin Ile Ala Glin Asp His Glin Lys 1.65 17O 17s

Glu Thir Wall Wall Lys Glu Asp Glu Gly Arg Arg Glu Ser Ile Asn Asp 18O 185 19 O

Arg Ala Arg Arg Ser Pro Arg Lys Luell Pro Thir Ser Lell Gly 195

Glu Arg Trp Ala Pro Pro Phe Luell Pro His Asp Wall 21 O 215 22O

Lys Luell Glin Asn Glu Asp Ile Ile Ser ASn Wall Pro Ala Asp Ser 225 23 O 235 24 O

Lell Ile Arg Thir Glu Arg Pro Pro Asn Lys Glu Ile Wall Arg Tyr Phe 245 250 255

Ile Arg His Asn Ala Lell Arg Ala Gly Thir Gly Glu Asn Ala Trp 26 O 265 27 O

Wall Wall Glu Asp Glu Lell Wall Lys Ser Lell Pro Ser Phe 285

Ser Asp Phe Luell Lell Asp Pro Met Thir Lell Asn Ser 29 O 295 3 OO

Thir Arg Asn Thir Gly Ser Pro Asp Arg Pro Ser Lys 3. OS 310 315

Ser Thir Asp Asn Ser Ser Luell Ser Ser Pro Lell Asn Pro Lys Luell 3.25 330 335

Trp His Wall His Lell Ser Luell Ser Gly Ser Pro Lel 34 O 345 35. O

Wall Asn Ser Asn Ser Lys Ser Pro Glu Glu His Luell Glu Glu 355 360 365

Met Met Met Met Ser Pro Asn Luell His Thir Asn Phe His Ile 37 O 375

Pro Gly Pro Pro Ala Pro Gly Lys His Ser Asp Lys 385 390 395 4 OO

Pro Luell Ala Lys Gly Arg Ser Gly Ile Lell Asn Gly Glin 4 OS 415

Ser Thir Gly Asn Ser Ser Pro Lys Gly Lell Thir Pro 425 43 O

Thir Met Glin Met Thir Luell Luell Asp Met Ala Lys Gly Thir Glin 435 44 O 445

Met Thir Arg Ala Pro Arg Asn Ser Gly Gly Thir Pro Arg Thir Ser 450 45.5 460

Ser Pro His His Lell Pro Pro Ala Ala Lell His Luell Ile Ala US 8,535,891 B2 105 106 - Continued

465 470

Glu Asn Asp Arg Glu Asp Arg Ser Ala Luell Ser 485 490 495

Wall Ile Ser Thir Ala Arg Luell Luell Ser Ser Glu Asp Arg Ala SOO 505

Arg Luell Pro Glu Glu Lell Arg Ser Luell Wall Glin Arg Glu Luell 515 525

Lell Glu His Lys Arg Trp Ala Ser Met Ser Glu Glu Glin Arg 53 O 535 54 O

Glu Tyr Luell Lys Lys Arg Glu Glu Luell Lys Luell Glu 5.45 550 555 560

Ala Glu Arg Arg Glu Glu Met Luell Glu Arg Luell Glu 565 st O sts

Glin Arg Tyr Glu Asp Glin Glu Luell Thir Gly Asn Luell Pro Ala 585 59 O

Phe Arg Luell Wall Asp Thir Pro Glu Gly Luell Pro Asn Thir Luell Phe Gly 595 605

Asp Wall Ala Met Wall Wall Glu Phe Luell Ser Tyr Ser Gly Luell Luell 610 615

Lell Pro Asp Ala Glin Tyr Pro Ile Thir Ala Wall Ser Lell Met Glu Ala 625 630 635 64 O

Lell Ser Ala Asp Lys Gly Gly Phe Luell Tyr Luell Asn Arg Wall Luell Wall 645 650 655

Ile Luell Luell Glin Asp Glu Ile Ala Glu Asp Gly Glu Luell Gly Met 660 665 67 O

Luell Ser Glu Ile Pro Lell Thir Luell His Ser Wall Ser Glu Luell Wall 675 685

Arg Luell Luell Arg Arg Ser Asp Wall Glin Glu Glu Ser Glu Gly Ser 69 O. 695 7 OO

Asp Thir Asp Asp Asn Lys Asp Ser Ala Ala Phe Glu Asp Asn Glu Wall 7 Os

Glin Asp Glu Phe Lell Glu Luell Glu Thir Ser Glu Phe Phe Glu Luell 72 73 O 73

Thir Ser Glu Glu Lell Glin Ile Luell Thir Ala Lell His Arg Ile 740 74. 7 O

Lell Met Thir Ser Wall Glin Asp His Met Glu Thir Arg Glin Met 760 765

Ser Ala Glu Luell Trp Glu Arg Luell Ala Wall Lell Glu Asn 770 775

Asp Arg Ala Glu Glin Arg Lys Glu Met Glu Lys 79 O 79.

Asn Glu Asn Gly Lys Wall Glu Asn Gly Luell Gly Thir Arg 805 810

Glu Ile Wall Phe Glu Pro Glin Wall Asp Thir Glu Glu 825 83 O

Met Ile Ser Ala Wall Ser Arg Arg Luell Lell Ala Ile Ala 835 84 O 845

Lys Glu Arg Glu Ile Glin Glu Arg Glu Met Lys Wall Luell Glu 850 855 860

Arg Glin Ala Glu Glu Glu Arg Ile Arg His Ala Ala Ala Glu 865 87s

Ala Phe Glin Glu Gly Ile Ala Ala Lell Wall Met Arg Arg 885 890 895 US 8,535,891 B2 107 108 - Continued

Thir Pro Ile Gly Thr Asp Arg Asn His Asn Arg Tyr Trp Lieu. Phe Ser 9 OO 905 91 O

Asp Glu Wall Pro Gly Lieu. Phe Ile Glu Lys Gly Trp Val His Asp Ser 915 92 O 925

Ile Asp Arg Phe Asn His His Cys Lys Asp His Thr Val Ser Gly 93 O 935 94 O

Asp Glu Asp Tyr Cys Pro Arg Ser Lys Lys Ala Asn Lieu. Gly Lys Asn 945 950 955 96.O

Ala Ser Met Asn Thr Gln His Gly Thr Ala Thr Glu Val Ala Val Glu 965 97O 97.

Thir Thir Thir Pro Llys Glin Gly Glin Asn Lieu. Trp Phe Lieu. Cys Asp Ser 98O 985 99 O

Glin Glu Lieu. Asp Glu Lieu. Lieu. Asn. Cys Lieu. His Pro Gln Gly Ile 995 1OOO 1005

Arg Ser Glin Lieu Lys Glu Arg Lieu. Glu Lys Arg Tyr Glin Asp O15 O2O

Ile His Ser Ile His Lieu Ala Arg Llys Pro Asn Lieu. Gly Lell O25 O3 O O35

Ser Asp Gly Asn. Glin Glu Lieu. Lieu. Asn. Phe Lieu. Arg Ser O4 O O45 OSO

Ile Glu Val Ala Thr Arg Lieu. Glin Lys Gly Gly Lieu. Gly O6 O O65

Glu Glu Thir Ser Glu Phe Glu Ala Arg Val Ile Ser Lell O7 O8O

Lell Lys Asp Phe Gly Glu. CyS Val Ile Ala Lieu Gln Ala O9 O O95

Ser Ile Llys Llys Phe Lieu. Glin Gly Phe Met Ala Pro Llys Glin O5 10

Arg Llys Lieu. Glin Ser Glu Asp Ser Ala Lys Thr Glu Glu 2O 25

Wall Glu Glu Lys Llys Met Val Glu Glu Ala Lys Val Ala Ser 35 4 O

Ala Glu Llys Trp Llys Thir Ala Ile Arg Glu Ala Glin Thr Phe SO 55

Ser Met His Val Lieu. Lieu. Gly Met Lieu. Asp Ala Cys Ile 65 70

Trp Met Ser Ala Glu Asn Ala Arg Cys Llys Val Cys Arg 8O 85

Glu Asp Asp Llys Lieu. Ile Lieu. Cys Asp Glu. Cys Asn 95 2OO

Ala His Lieu. Phe Cys Lieu. Arg Pro Ala Lieu. Tyr Glu Val Pro 21 O 215

Asp Glu Trp Gln Cys Pro Ala Cys Gln Pro Ala Thr Ala Arg 225 23 O

Arg Asn Ser Arg Gly Arg Asn Tyr Thr Glu Glu Ser Ala Ser Glu 235 24 O 245

Asp Ser Glu Asp Asp Glu Ser Asp Glu Glu Glu Glu Glu Glu Glu 250 255 26 O

Glu Glu Glu Glu Glu Glu Asp Tyr Glu Val Ala Gly Lieu. Arg Lell 265 27 O 27s

Arg Pro Arg Lys. Thir Ile Arg Gly Lys His Ser Val Ile Pro Pro 28O 285 29 O

Ala Arg Ser Gly Arg Arg Pro Gly Lys Llys Pro His Ser Thir 295 3OO 305 US 8,535,891 B2 109 110 - Continued

Arg Arg Ser Glin Pro Lys Ala Pro Pro Val Asp Asp Ala Glu Val 310 315 32O

Asp Glu Lieu Val Lieu. Glin Thir Lys Arg Ser Ser Arg Arg Glin Ser 3.25 33 O 335

Lell Glu Lieu. Glin Lys Cys Glu Glu Ile Lieu. His Lys Ile Val Lys 34 O 345 350 Phe Ser Trp Pro Phe Arg Glu Pro Val Thr Arg Asp Glu 355 360 365

Ala Glu Asp Tyr Tyr Asp Val Ile Thr His Pro Met Asp Phe Glin 37O 375 38O

Thir Glin Asn Lys Cys Ser Cys Gly Ser Tyr Arg Ser Val Glin 385 390 395

Glu Phe Lieu. Thir Asp Met Lys Glin Val Phe Thr Asn Ala Glu Val 4 OO 405 41 O

Asn Cys Arg Gly Ser His Val Lieu Ser Cys Met Val Lys Thr 415 42O 425

Glu Glin Cys Lieu Val Ala Lieu. Lieu. His Llys His Lieu Pro Gly His 43 O 435 44 O

Pro yr Val Arg Arg Lys Arg Llys Llys Phe Pro Asp Arg Lieu Ala 445 450 45.5

Glu Glu Gly Asp Ser Glu Pro Glu Ala Val Gly Glin Ser Arg 465 47 O Gly Arg Glin Llys Llys

<210s, SEQ ID NO 14 &211s LENGTH: 261 212. TYPE : PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 14 Met Ala Ser Pro Asp Trp Gly Tyr Asp Asp Lys Asn Gly Pro Glu Glin 1. 5 1O 15

Trp Ser Llys Lieu. Tyr Pro Ile Ala Asn Gly Asn. Asn Glin Ser Pro Val 2O 25 3O

Asp Ile Lys. Thir Ser Glu Thir Lys His Asp Thr Ser Lieu Lys Pro Ile 35 4 O 45

Ser Wall Ser Tyr Asn Pro Ala Thr Ala Lys Glu Ile Ile Asin Val Gly SO 55 6 O

His Ser Phe His Val Asn. Phe Glu Asp Asin Asp Asn Arg Ser Val Lieu 65 70 7s 8O Gly Gly Pro Phe Ser Asp Ser Tyr Arg Lieu Phe Glin Phe His Phe 85 90 95

His Trp Gly Ser Thr Asn Glu. His Gly Ser Glu. His Thr Val Asp Gly 1OO 105 11 O

Wall Tyr Ser Ala Glu Lieu. His Val Ala His Trp Asn. Ser Ala Lys 115 12 O 125

Ser Ser Lieu Ala Glu Ala Ala Ser Lys Ala Asp Gly Lieu Ala Val 13 O 135 14 O

Ile Gly Val Lieu Met Llys Val Gly Glu Ala ASn Pro Llys Lieu. Glin Lys 145 150 155 160

Wall Luell Asp Ala Lieu. Glin Ala Ile Llys Thir Lys Gly Lys Arg Ala Pro 1.65 17O 17s

Phe Thir Asin Phe Asp Pro Ser Thr Lieu. Leu Pro Ser Ser Lieu. Asp Phe 18O 185 19 O US 8,535,891 B2 111 112 - Continued

Trp Thir Tyr Pro Gly Ser Lieu. Thr His Pro Pro Lell Tyr Glu Ser Wall 195 2OO 2O5

Thir Trp Ile Ile Cys Lys Glu Ser Ile Ser Wall Ser Ser Glu Glin Luell 21 O 215 22O

Ala Glin Phe Arg Ser Lieu. Luell Ser Asn Wall Glu Gly Asp Asn Ala Wall 225 23 O 235 24 O

Pro Met Glin His Asn Asn Arg Pro Thir Glin Pro Lell Gly Arg Thir 245 250 255

Wall Arg Ala Ser Phe 26 O

<210s, SEQ ID NO 15 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 15

Met Glu Arg Ala Ser Cys Lieu. Lieu. Luell Luell Luell Lell Pro Luell Wall His 1. 5 15

Wall Ser Ala Thir Thr Pro Glu Pro Cys Glu Luell Asp Asp Glu Asp Phe 25

Arg Wall Cys Asn. Phe Ser Glu Pro Glin Pro Asp Trp Ser Glu Ala 35 4 O 45

Phe Glin Wall Ser Ala Wall Glu Wall Glu Ile His Ala Gly Gly Luell SO 55 6 O

Asn Luell Glu Pro Phe Lieu Lys Arg Wall Asp Ala Asp Ala Asp Pro Arg 65 70

Glin Ala Asp Thr Val Lys Ala Luell Arg Wall Arg Arg Luell Thir Wall 85 90 95

Gly Ala Ala Glin Wall Pro Ala Glin Luell Luell Wall Gly Ala Luell Arg Wall 105 11 O

Lell Ala Tyr Ser Arg Lieu Lys Glu Luell Thir Luell Glu Asp Luell Ile 115 12 O 125

Thir Gly Thir Met Pro Pro Leu Pro Luell Glu Ala Thir Gly Luell Ala Luell 13 O 135 14 O

Ser Ser Luell Arg Lieu. Arg Asn. Wall Ser Trp Ala Thir Gly Arg Ser Trp 145 150 155 160

Lell Ala Glu Luell Glin Gln Trp Lieu Pro Gly Lell Wall Luell Ser 1.65 17O 17s

Ile Ala Glin Ala His Ser Pro Ala Phe Ser Glu Glin Wall Arg Ala 18O 185 19 O

Phe Pro Ala Luell Thir Ser Lieu. Asp Luell Ser Asp Asn Pro Gly Luell Gly 195 2OO

Glu Arg Gly Luell Met Ala Ala Lieu Pro His Lys Phe Pro Ala Ile 21 O 215 22O

Glin Asn Luell Ala Lieu. Arg Asn. Thir Gly Met Glu Thir Pro Thir Gly Wall 225 23 O 235 24 O

Ala Ala Luell Ala Ala Ala Gly Wall Glin Pro His Ser Luell Asp Luell 245 250 255

Ser His Asn Ser Lieu. Arg Ala Thr Wall Asn Pro Ser Ala Pro Arg 26 O 265 27 O

Met Trp Ser Ser Ala Lieu. Asn. Ser Luell Asn Luell Ser Phe Ala Gly Luell 27s 28O 285

Glu Glin Wall Pro Lys Gly Lieu Pro Ala Luell Arg Wall Luell Asp Luell 29 O 295 3 OO US 8,535,891 B2 113 114 - Continued

Ser Asn Arg Lieu. Asn Arg Ala Pro Glin Pro Asp Glu Lieu Pro Glu 3. OS 310 315 32O

Wall Asp Asn Luell Thir Lieu. Asp Gly Asn Pro Phe Lell Wall Pro Gly Thir 3.25 330 335

Ala Luell Pro His Glu Gly Ser Met Asn Ser Gly Wall Wall Pro Ala 34 O 345 35. O

Ala Arg Ser Thir Lieu. Ser Val Gly Wall Ser Gly Thir Lell Wall Luell Luell 355 360 365

Glin Gly Ala Arg Gly Phe Ala 37 O 375

<210s, SEQ ID NO 16 &211s LENGTH: 367 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 16

Glu Pro Arg Ala Gly Glu Lieu Ala Gly Gly Ser Thir Asp Arg Arg Thir 1. 5 15

His Ala Glu Ala Lieu. Arg Pro Glin Pro Arg Ala Gly Ala Thir Ala Ala 25

Arg Pro Arg Pro Pro Wall Glin Pro Pro Luell Ala Arg Cys Luell Gly Ala 35 4 O 45

Arg Pro His Thir Ala Ser Cys Ser Ala Pro Gly Ser Ala Met Arg Ser SO 55 6 O

Ala Ala Wall Luell Ala Lieu. Lieu. Lieu. Ala Gly Glin Wall Thir Ala Luell 65 70

Pro Wall Asn Ser Pro Met Asn Lys Gly Asp Thir Glu Wall Met Lys Cys 85 90 95

Ile Wall Glu Wall Ile Ser Asp Thr Lel Ser Pro Ser Pro Met Pro 105 11 O

Wall Ser Glin Glu Cys Phe Glu Thr Lel Arg Gly Asp Glu Arg Ile Luell 115 12 O 125

Ser Ile Luell Arg His Glin Asn Lieu. Lel Glu Lell Glin Asp Luell Ala 13 O 135 14 O

Lell Glin Gly Ala Lys Glu Arg Ala His Glin His Ser Gly 145 150 155 160

Phe Glu Asp Glu Lieu. Ser Glu Wall Lel ASn Glin Ser Ser Glin Ala 1.65 17s

Glu Luell Gly Arg Ser Glu Ala Lel Wall Asp Gly Ala Gly 18O 185 19 O

Pro Gly Ala Glu Glu Ala Glin Asp Gly Gly Glu Glin Glu 195 2OO

His Ser Glin Glin Lys Glu Glu Glu Glu Met Ala Wall Wall Pro Glin 21 O 215

Gly Luell Phe Arg Gly Gly Llys Ser Gly Luell Glu Glin Glu Glu Glu 225 23 O 235 24 O

Arg Luell Ser Glu Trp Glu Asp Ser Lys Arg Trp Ser Met Asp 245 250 255

Glin Luell Ala Lys Glu Lieu. Thir Ala Glu Arg Lell Glu Gly Glin Glu 26 O 265 27 O

Glu Glu Glu Asp Asn Arg Asp Ser Ser Met Lys Lell Ser Phe Arg Ala 27s 28O 285

Arg Ala Gly Phe Arg Gly Pro Gly Pro Glin Lell Arg Arg Gly Trp 29 O 295 3 OO US 8,535,891 B2 115 116 - Continued

Arg Pro Ser Ser Arg Glu Asp Ser Luell Glu Ala Gly Lell Pro Luell Glin 3. OS 310 315 32O

Wall Arg Gly Tyr Pro Glu Glu Lys Glu Glu Glu Gly Ser Ala Asn 3.25 330 335

Arg Arg Pro Glu Asp Glin Glu Lieu Glu Ser Luell Ser Ala Ile Glu Ala 34 O 345 35. O

Glu Lieu. Glu Lys Val Ala His Glin Luell Glin Ala Lell Arg Arg Gly 355 360 365

<210s, SEQ ID NO 17 &211s LENGTH: 439 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 17

Met Val Pro Ser Ser Pro Arg Ala Luell Phe Luell Lell Lell Luell Ile Luell 1. 5 15

Ala Cys Pro Glu Pro Arg Ala Ser Glin Asn Cys Lell Ser Lys Glin Glin 2O 25

Lell Lieu. Ser Ala Ile Arg Glin Lieu. Glin Glin Luell Lell Lys Gly Glin Glu 35 4 O 45

Thir Arg Phe Ala Glu Gly Ile Arg His Met Ser Arg Luell Ala Ala SO 55 6 O

Lell Glin Asn. Ser Val Gly Arg Val Gly Pro Asp Ala Lell Pro Wall Ser 65 70 7s

Pro Ala Lieu. Asn. Thr Pro Ala Asp Gly Arg Phe Gly Ser 85 90 95

Lieu Val Asp His Glu Val His Phe Thir Asn Pro Gly Phe Arg 1OO 105 11 O

Lell Val Gly Pro Ser Ser Val Val Luell Pro Asn Gly Thir Trp Thir 115 12 O 125

Gly Glu Glin Pro His Cys Arg Gly Ile Ser Glu Cys Ser Ser Glin Pro 13 O 135 14 O

Cys Glin Asn Gly Gly Thr Cys Val Glu Gly Wall Asn Glin Arg Cys 145 150 155 160

Ile Cys Pro Pro Gly Arg Thr Gly Asn Arg Glin His Glin Ala Glin 1.65

Thir Ala Ala Pro Glu Gly Ser Val Ala Gly Asp Ser Ala Phe Ser Arg 18O 185 19 O

Ala Pro Arg Cys Ala Glin Val Glu Arg Ala Glin His Cys Ser Glu 195 2OO

Ala Gly Phe His Leu Ser Gly Ala Ala Gly Asp Ser Wall Glin Asp 21 O 215 22O

Wall Asin Glu. Cys Glu Lieu. Tyr Gly Glin Glu Gly Arg Pro Arg Luell Cys 225 23 O 235 24 O

Met His Ala Cys Val Asn Thr Pro Gly Ser Arg Thir Cys Pro 245 250 255

Gly Gly Tyr Arg Thr Lieu Ala Asp Gly Lys Ser Glu Asp Wall Asp 26 O 265 27 O

Glu Cys Val Gly Lieu Gln Pro Val Pro Glin Gly Thir Thir Ile 27s 28O 285

Asn Thr Gly Gly Ser Phe Glin Cys Wall Ser Pro Glu Pro Glu Gly 29 O 295 3 OO

Ser Gly Asn Val Ser Tyr Val Lys Thir Ser Pro Phe Glin Glu Arg 3. OS 310 315 32O US 8,535,891 B2 117 118 - Continued

Asn Pro Cys Pro Met Asp Ser Arg Pro Cys Arg His Lell Pro Lys Thir 3.25 330 335

Ile Ser Phe His Tyr Lieu. Ser Lieu. Pro Ser ASn Lell Thir Pro Ile 34 O 345 35. O

Thir Lieu. Phe Arg Met Ala Thr Ala Ser Ala Pro Gly Arg Ala Gly Pro 355 360 365

Asn Ser Lieu. Arg Phe Gly Ile Val Gly Gly ASn Ser Arg Gly His Phe 37 O 375

Wall Met Glin Arg Ser Asp Arg Glin Thir Gly Asp Lell Ile Luell Wall Glin 385 390 395 4 OO

Asn Lieu. Glu Gly Pro Gln Thr Lieu. Glu Wall Asp Wall Asp Met Ser Glu 4 OS 415

Lieu. Asp Arg Ser Phe Glin Ala Asn His Wall Ser Wall Thir Ile 42O 425 43 O

Phe Val Ser Pro Tyr Asp Phe 435

<210s, SEQ ID NO 18 &211s LENGTH: 698 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 18

Met Gly Gly Lieu Ala Ser Gly Gly Asp Wall Glu Pro Gly Luell Pro Wall 1. 5 15

Glu Val Arg Gly Ser ASn Gly Ala Phe Gly Phe Wall Asp 2O 25 3O

Wall His Glu Asp Ser Val Thr Ile Phe Phe Glu Asn Asn Trp Glin Ser 35 4 O 45

Glu Arg Glin Ile Pro Phe Gly Asp Wall Arg Luell Pro Pro Pro Ala Asp SO 55 6 O

Tyr Asn Lys Glu Ile Thr Glu Gly Asp Glu Wall Glu Wall Ser Arg 65 70

Ala Asin Glu Gln Glu Pro Cys Gly Trp Trp Luell Ala Arg Wall Arg Met 85 90 95

Met Lys Gly Asp Phe Tyr Val Ile Glu Tyr Ala Ala Asp Ala Thir 1OO 105 11 O

Asn. Glu Ilie Wall. Thir Lieu. Glu Arg Luell Arg Pro Wall Asn Pro Asn 115 12 O 125

Pro Leu Ala Thr Lys Gly Ser Phe Phe Wall Thir Met Ala Wall Pro 13 O 135 14 O

Glu Asp Lieu. Arg Glu Ala Cys Ser Asn Glu ASn Wall His Glu Phe 145 150 155 160

Lys Ala Lieu. Gly Ala Asn. Cys Ile Phe Luell Asn Ile Thir Asn Ser 1.65 17O 17s

Glu Lieu. Phe Ile Leu Ser Thir Thr Glu Ala Pro Wall Arg Ala Ser 18O 185 19 O

Lell Lieu. Gly Asp Met His Phe Arg Ser Luell Arg Thir Lys Luell Luell Luell 195 2OO 2O5

Met Ser Arg Asn. Glu Glu Ala Thr His Luell Glu Thir Ser Glin 21 O 215

Lell Ala Ala Ala Phe Glin Glu Glu Phe Thir Wall Arg Glu Asp Luell Met 225 23 O 235 24 O

Gly Lieu Ala Ile Gly Thr His Gly Ala Asn Ile Glin Glin Ala Arg 245 250 255 US 8,535,891 B2 119 120 - Continued

Wall Pro Gly Wall Thir Ala Ile Glu Luell Gly Glu Glu Thir Cys Thir Phe 26 O 265 27 O

Arg Ile Tyr Gly Glu Thir Pro Glu Ala Cys Arg Glin Ala Arg Ser 27s 28O 285

Lell Glu Phe Ser Glu Asp Ser Wall Glin Wall Pro Arg Asn Luell Wall Gly 29 O 295 3 OO

Lys Wall Ile Gly Lys Asn Gly Wall Ile Glin Glu Ile Wall Asp Lys 3. OS 310 315

Ser Gly Wall Wall Arg Wall Arg Wall Glu Gly Asp Asn Asp Lys Asn 3.25 330 335

Pro Arg Glu Glu Gly Met Wall Pro Phe Ile Phe Wall Gly Thir Arg Glu 34 O 345 35. O

Asn Ile Ser Asn Ala Glin Ala Luell Luell Glu His Lell Ser Luell 355 360 365

Glin Glu Wall Glu Glin Lell Arg Luell Glu Arg Luell Glin Ile Asp Glu Glin 37 O 375

Lell Arg Glin Ile Gly Lell Gly Phe Arg Pro Pro Gly Ser Gly Arg Gly 385 390 395 4 OO

Ser Gly Gly Ser Asp Ala Gly Ser Thir Asp Glu Ser Ser Ser 4 OS 415

Ser Ser Luell His Ala Thir Arg Thir Tyr Gly Gly Ser Gly Gly Arg 425 43 O

Gly Arg Gly Arg Arg Thir Gly Gly Pro Ala Gly Pro Ser Ser Asp 435 44 O 445

Wall Ser Thir Ala Ser Glu Thir Glu Ser Glu Arg Glu Glu Pro Asn 450 45.5 460

Arg Ala Gly Pro Gly Asp Arg Asp Pro Pro Thir Arg Gly Glu Glu Ser 465 470

Arg Arg Arg Pro Thir Gly Gly Arg Gly Arg Gly Pro Pro Pro Ala Pro 485 490 495

Arg Pro Thir Ser Arg Asn Ser Ser Ser Ile Ser Ser Wall Luell SOO 505

Asp Pro Asp Ser Asn Pro Ser Luell Luell Asp Thir Ser Glu Pro Glu 515 525

Pro Pro Wall Asp Ser Glu Pro Gly Glu Pro Pro Pro Ala Ser Ala Arg 53 O 535 54 O

Arg Arg Arg Ser Arg Arg Arg Arg Thir Asp Glu Asp Arg Thir Wall Met 5.45 550 555 560

Asp Gly Gly Luell Glu Ser Asp Gly Pro Asn Met Thir Glu Asn Gly Luell 565 st O sts

Glu Asp Glu Ser Arg Pro Glin Arg Arg Asn Arg Ser Arg Arg Arg Arg 585 59 O

Asn Arg Gly Asn Arg Thir Asp Gly Ser Ile Ser Gly Asp Arg Glin Pro 595 605

Wall Thir Wall Ala Asp Ile Ser Arg Ala Glu Ser Glin Ser Arg Glin 610 615

Arg Pro Pro Luell Glu Arg Thir Pro Ser Glu Asp Ser Luell Ser Gly 625 630 635 64 O

Glin Gly Asp Ser Wall Ser Luell Pro Gly Pro Ser Glu Asn 645 650 655

Gly Glu Luell Ser Ala Pro Lell Glu Luell Gly Ser Met Wall Glu Trp Gly 660 665 67 O

Phe Ile Pro Pro Thir Thir Pro Pro Phe Ser Ile Ser Luell Ala US 8,535,891 B2 121 122 - Continued

675 685 Ala Glin His His Gly Pro His Arg Pro Asn 69 O. 695

<210s, SEQ ID NO 19 &211s LENGTH: 142 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 19

Met Wall Leu Ser Pro Ala Asp Llys Thir Asn Wall Ala Ala Trp Gly 1. 5 15

Lys Wall Gly Ala His Ala Gly Glu Tyr Gly Ala Glu Ala Luell Glu Arg 25 3O

Met Phe Luell Ser Phe Pro Thir Thr Thir Phe Pro His Phe Asp 35 4 O 45

Lell Ser His Gly Ser Ala Glin Wall Gly His Gly Wall Ala SO 55 6 O

Asp Ala Luell Thir Asn Ala Wall Ala His Wall Asp Asp Met Pro Asn Ala 65 70

Lell Ser Ala Luell Ser Asp Lieu. His Ala His Lell Arg Wall Asp Pro 85 90 95

Wall Asn Phe Lys Lieu Lleu Ser His Cys Luell Luell Wall Thir Luell Ala Ala 105 11 O

His Luell Pro Ala Glu Phe Thr Pro Ala Wall His Ala Ser Luell Asp 115 12 O 125

Phe Luell Ala Ser Wall Ser Thir Wall Luell Thir Ser Lys Arg 13 O 135 14 O

<210s, SEQ ID NO 2 O &211s LENGTH: 147 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2O

Met Wal His Lieu. Thr Pro Glu Glu Ser Ala Wall Thir Ala Luell Trp 1. 5 15

Gly Lys Wall Asn Val Asp Glu Val Gly Gly Glu Ala Lell Gly Arg Luell 25 3O

Lell Wall Wall Tyr Pro Trp Thr Glin Arg Phe Phe Glu Ser Phe Gly Asp 35 4 O 45

Lell Ser Thir Pro Asp Ala Wal Met Gly Asn Pro Lys Wall Ala His SO 55 6 O

Gly Wall Lieu. Gly Ala Phe Ser Asp Gly Lell Ala His Luell Asp 65 70

Asn Luell Gly Thir Phe Ala Thr Luell Ser Glu Lell His Asp 85 90 95

Lell His Wall Asp Pro Glu Asn. Phe Arg Luell Luell Gly Asn Wall Luell Wall 105 11 O

Wall Luell Ala His His Phe Gly Glu Phe Thir Pro Pro Wall Glin 115 12 O 125

Ala Ala Glin Llys Val Val Ala Gly Wall Ala Asn Ala Luell Ala His 13 O 135 14 O

Lys His 145

<210s, SEQ ID NO 21 US 8,535,891 B2 123 124 - Continued

&211s LENGTH: 177 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 21

Met Glu Ile Cys Arg Gly Lieu. Arg Ser His Lieu. Ile Thr Lieu. Lieu. Luell 1. 5 1O 15

Phe Leu Phe His Ser Glu Thir Ile Cys Arg Pro Ser Gly Arg Lys Ser 2O 25 3O

Ser Lys Met Glin Ala Phe Arg Ile Trp Asp Val Asn Gln Lys Thr Phe 35 4 O 45

Tyr Lieu. Arg Asn. Asn Glin Lieu Val Ala Gly Tyr Lieu. Glin Gly Pro Asn SO 55 6 O

Val Asn Lieu. Glu Glu Lys Ile Asp Val Val Pro Ile Glu Pro His Ala 65 70 7s Lieu. Phe Lieu. Gly Ile His Gly Gly Lys Met Cys Lieu. Ser Cys Val 85 90 95 Ser Gly Asp Glu Thir Arg Lieu. Glin Lieu. Glu Ala Val Asn. Ile Thr Asp 1OO 105 11 O

Lieu. Ser Glu Asn Arg Lys Glin Asp Lys Arg Phe Ala Phe Ile Arg Ser 115 12 O 125 Asp Ser Gly Pro Thr Thr Ser Phe Glu Ser Ala Ala Cys Pro Gly Trp 13 O 135 14 O

Phe Lieu. Cys Thr Ala Met Glu Ala Asp Glin Pro Val Ser Lieu. Thr Asn 145 150 155 160 Met Pro Asp Glu Gly Val Met Val Thr Llys Phe Tyr Phe Glin Glu Asp 1.65 17O 17s

Glu

<210s, SEQ ID NO 22 &211s LENGTH: 914 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 22

Met Llys Pro Pro Arg Pro Val Arg Thr Cys Ser Lys Val Lieu Val Luell 1. 5 1O 15 Lieu. Ser Lieu. Lieu Ala Ile His Glin Thir Thir Thr Ala Glu Lys Asn Gly 2O 25 3O

Ile Asp Ile Tyr Ser Lieu. Thr Val Asp Ser Arg Val Ser Ser Arg Phe 35 4 O 45

Ala His Thr Val Val Thr Ser Arg Val Val Asn Arg Ala Asn Thr Wall SO 55 6 O

Gln Glu Ala Thr Phe Glin Met Glu Lieu Pro Llys Lys Ala Phe Ile Thir 65 70 7s Asn Phe Ser Met Ile Ile Asp Gly Met Thr Tyr Pro Gly Ile Ile 85 90 95 Glu Lys Ala Glu Ala Glin Ala Glin Tyr Ser Ala Ala Val Ala Lys Gly 1OO 105 11 O

Llys Ser Ala Gly Lieu Val Lys Ala Thr Gly Arg Asn Met Glu Glin Phe 115 12 O 125

Glin Val Ser Val Ser Val Ala Pro Asn Ala Lys Ile Thr Phe Glu Luell 13 O 135 14 O

Val Tyr Glu Glu Lieu Lleu Lys Arg Arg Lieu. Gly Val Tyr Glu Lieu. Luell 145 150 155 160

Lieu Lys Val Arg Pro Glin Gln Lieu Val Llys His Lieu Gln Met Asp Ile US 8,535,891 B2 125 126 - Continued

1.65 17O 17s

His Ile Phe Glu Pro Glin Gly Ile Ser Phe Luell Glu Thir Glu Ser Thir 18O 185 19 O

Phe Met Thir Asn Glin Lell Wall Asp Ala Luell Thir Thir Trp Glin Asn 195

Thir Lys Ala His Ile Arg Phe Pro Thir Luell Ser Glin Glin Glin 21 O 215 22O

Ser Pro Glu Glin Glin Glu Thir Wall Luell Asp Gly Asn Lell Ile Ile Arg 225 23 O 235 24 O

Asp Wall Asp Arg Ala Ile Ser Gly Gly Ser Ile Glin Ile Glu Asn 245 250 255

Phe Wall His Phe Ala Pro Glu Gly Lell Thir Thir Met Pro 26 O 265 27 O

Asn Wall Wall Phe Wall Ile Asp Ser Gly Ser Met Ser Gly Arg 285

Ile Glin Glin Thir Arg Glu Ala Luell Ile Ile Lell Asp Asp Luell 29 O 295 3 OO

Ser Pro Arg Asp Glin Phe Asn Luell Ile Wall Phe Ser Thir Glu Ala Thir 3. OS 310 315

Glin Trp Arg Pro Ser Lell Wall Pro Ala Ser Ala Glu Asn Wall Asn 3.25 330 335

Ala Arg Ser Phe Ala Ala Gly Ile Glin Ala Luell Gly Gly Thir Asn Ile 34 O 345 35. O

Asn Asp Ala Met Lell Met Ala Wall Glin Luell Luell Asp Ser Ser Asn Glin 355 360 365

Glu Glu Arg Luell Pro Glu Gly Ser Wall Ser Luell Ile Ile Luell Luell Thir 37 O 375

Asp Gly Asp Pro Thir Wall Gly Glu Thir Asn Pro Arg Ser Ile Glin Asn 385 390 395 4 OO

Asn Wall Arg Glu Ala Wall Ser Gly Arg Tyr Ser Lell Phe Luell Gly 4 OS 415

Phe Gly Phe Asp Wall Ser Ala Phe Luell Glu Lell Ala Luell Asp 425 43 O

Asn Gly Gly Luell Ala Arg Arg Ile His Glu Asp Ser Asp Ser Ala Luell 435 44 O 445

Glin Luell Glin Asp Phe Glin Glu Wall Ala ASn Pro Lell Luell Thir Ala 450 45.5 460

Wall Thir Phe Glu Tyr Pro Ser Asn Ala Wall Glu Glu Wall Thir Glin Asn 465 470

Asn Phe Arg Luell Lell Phe Gly Ser Glu Met Wall Wall Ala Gly Lys 485 490 495

Lell Glin Asp Arg Gly Pro Asp Wall Luell Thir Ala Thir Wall Ser Gly SOO 505

Lell Pro Thir Glin Asn Ile Thir Phe Glin Thir Glu Ser Ser Wall Ala Glu 515 525

Glin Glu Ala Glu Phe Glin Ser Pro Ile Phe His Asn Phe Met 53 O 535 54 O

Glu Arg Luell Trp Ala Tyr Lell Thir Ile Glin Glin Lell Lell Glu Glin Thir 5.45 550 555 560

Wall Ser Ala Ser Asp Ala Asp Glin Glin Ala Luell Arg Asn Glin Ala Luell 565 st O sts

Asn Luell Ser Luell Ala Ser Phe Wall Thir Pro Lell Thir Ser Met Wall 58O 585 59 O US 8,535,891 B2 127 128 - Continued Wall Thir Lys Pro Asp Asp Glin Glu Glin Ser Glin Wall Ala Glu Lys Pro 595 6OO 605

Met Glu Gly Glu Ser Arg Asn Arg Asn Wall His Ser Ala Gly Ala Ala 610 615

Gly Ser Arg Met Asn Phe Arg Pro Gly Wall Luell Ser Ser Arg Glin Luell 625 630 635 64 O

Gly Leu Pro Gly Pro Pro Asp Val Pro Asp His Ala Ala His Pro 645 650 655

Phe Arg Arg Lieu Ala Ile Lieu Pro Ala Ser Ala Pro Pro Ala Thir Ser 660 665 67 O

Asn Pro Asp Pro Ala Val Ser Arg Wall Met ASn Met Lys Ile Glu Glu 675 68O 685

Thir Thir Met Thir Thr Gin. Thir Pro Ala Pro Ser Ser Arg Ser 69 O. 695 7 OO

Arg Ala Pro Ala Wall Pro Ala Pro Ile Glin Ala Pro Ser Ala Ile Luell 7 Os 71O

Pro Leu Pro Gly Glin Ser Val Glu Arg Luell Cys Wall Asp Pro Arg His 72 73 O 73

Arg Glin Gly Pro Val Asn Lieu. Lieu. Ser Asp Pro Glu Glin Gly Wall Glu 740 74. 7 O

Wall Thr Gly Glin Tyr Glu Arg Glu Ala Gly Phe Ser Trp Ile Glu 7ss 760 765

Wall Thr Phe Lys Asn Pro Leu Val Trp Wall His Ala Ser Pro Glu His 770 775

Wall Val Val Thr Arg ASn Arg Arg Ser Ser Ala Trp Glu 79 O 79.

Thir Leu Phe Ser Val Met Pro Gly Luell Lys Met Thir Met Asp Lys Thir 805 810 815

Gly Lieu. Lieu. Lieu Lleu Ser Asp Pro Asp Wall Thir Ile Gly Luell Luell 82O 825 83 O

Phe Trp Asp Gly Arg Gly Glu Gly Luell Arg Luell Lell Lell Arg Asp Thir 835 84 O 845

Asp Arg Phe Ser Ser His Val Gly Gly Thir Luell Gly Glin Phe Glin 850 855 860

Glu Val Lieu. Trp Gly Ser Pro Ala Ala Ser Asp Asp Gly Arg Arg Thir 865 87O

Lell Arg Val Glin Gly Asn Asp His Ser Ala Thir Arg Glu Arg Arg Luell 885 890 895

Asp Tyr Glin Glu Gly Pro Pro Gly Wall Glu Ile Ser Trp Ser Wall 9 OO 905 91 O

Glu Luell

<210s, SEQ ID NO 23 &211s LENGTH: 481 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 23

Met Gly Ala Lieu Ala Arg Ala Lieu. Pro Ser Ile Lell Lell Ala Luell Luell 1. 5 1O 15

Lell Thir Ser Thr Pro Glu Ala Lieu. Gly Ala ASn Pro Gly Luell Wall Ala 2O 25 3O

Arg Ile Thr Asp Llys Gly Lieu. Glin Ala Ala Glin Glu Gly Luell Luell 35 4 O 45

Ala Lieu. Glin Ser Glu Lieu. Lieu. Arg Ile Thir Luell Pro Asp Phe Thir Gly US 8,535,891 B2 129 130 - Continued

SO 55 6 O

Asp Luell Arg Ile Pro His Wall Gly Arg Gly Arg Glu Phe His Ser 65 70

Lell Asn Ile His Ser Glu Luell Luell His Ser Ala Lell Arg Pro Wall 85 90 95

Pro Gly Glin Gly Lell Ser Lell Ser Ile Ser Asp Ser Ser Ile Arg Wall 105 11 O

Glin Gly Arg Trp Wall Arg Lys Ser Phe Phe Lell Glin Gly Ser 115 12 O 125

Phe Asp Wall Ser Wall Gly Ile Ser Ile Ser Wall Asn Luell Luell Luell 13 O 135 14 O

Gly Ser Glu Ser Ser Gly Arg Pro Thir Wall Thir Ala Ser Ser Ser 145 150 155 160

Ser Asp Ile Ala Asp Wall Glu Wall Asp Met Ser Gly Asp Luell Gly Trp 1.65 17s

Lell Luell Asn Luell Phe His Asn Glin Ile Glu Ser Phe Glin Wall 18O 185 19 O

Lell Glu Ser Arg Ile Glu Met Ile Glin Lys Ser Wall Ser Ser Asp 195

Lell Glin Pro Lell Glin Thir Luell Pro Wall Thir Thir Glu Ile Asp Ser 21 O 215

Phe Ala Asp Ile Asp Tyr Ser Luell Wall Glu Ala Pro Arg Ala Thir Ala 225 23 O 235 24 O

Glin Met Luell Glu Wall Met Phe Gly Glu Ile Phe His Arg Asn His 245 250 255

Arg Ser Pro Wall Thir Lell Lell Ala Ala Wall Met Ser Lell Pro Glu Glu 26 O 265 27 O

His Asn Lys Met Wall Phe Ala Ile Ser Asp Wall Phe Asn Thir 27s 285

Ala Ser Luell Wall Tyr His Glu Glu Gly Tyr Luell Asn Phe Ser Ile Thir 29 O 295 3 OO

Asp Asp Met Ile Pro Pro Asp Ser Asn Ile Arg Lell Thir Thir Ser 3. OS 310 315

Phe Arg Pro Phe Wall Pro Arg Luell Ala Arg Luell Tyr Pro Asn Met Asn 3.25 330 335

Lell Glu Luell Glin Gly Ser Wall Pro Ser Ala Pro Lell Lell Asn Phe Ser 34 O 345 35. O

Pro Gly Asn Luell Ser Wall Asp Pro Met Glu Ile Asp Ala Phe Wall 355 360 365

Lell Luell Pro Ser Ser Ser Lys Glu Pro Wall Phe Arg Lell Ser Wall Ala 37 O 375

Thir Asn Wall Ser Ala Thir Lell Thir Phe Asn Thir Ser Ile Thir Gly 385 390 395 4 OO

Phe Luell Pro Gly Lys Wall Wall Glu Luell Glu Ser Lys Wall 4 OS 415

Gly Luell Phe Asn Ala Glu Lell Luell Glu Ala Luell Lell Asn Tyr Ile 425 43 O

Lell Asn Thir Phe Tyr Pro Phe Asn Asp Lell Ala Glu Gly Phe 435 44 O 445

Pro Luell Pro Luell Lell Arg Wall Glin Luell Tyr Asp Lell Gly Luell Glin 450 45.5 460

Ile His Asp Phe Lell Phe Luell Gly Ala ASn Wall Glin Met Arg 465 470 47s 48O US 8,535,891 B2 131 132 - Continued

Wall

<210s, SEQ ID NO 24 &211s LENGTH: 322 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 24 Met Ala Arg Cys Phe Ser Lieu Val Lieu. Leu Lieu. Thir Ser Ile Trp Thr 1. 5 1O 15 Thir Arg Lieu. Lieu Val Glin Gly Ser Lieu. Arg Ala Glu Glu Lieu. Ser Ile 2O 25 3O Glin Val Ser Cys Arg Ile Met Gly Ile Thir Lieu Val Ser Lys Lys Ala 35 4 O 45 Asn Glin Glin Lieu. Asn. Phe Thr Glu Ala Lys Glu Ala Cys Arg Lieu. Lieu SO 55 6 O Gly Lieu. Ser Lieu Ala Gly Lys Asp Glin Val Glu Thir Ala Lieu Lys Ala 65 70 7s 8O Ser Phe Glu Thr Cys Ser Tyr Gly Trp Val Gly Asp Gly Phe Val Val 85 90 95 Ile Ser Arg Ile Ser Pro ASn Pro Llys Cys Gly Lys Asn Gly Val Gly 1OO 105 11 O Val Lieu. Ile Trp Llys Val Pro Val Ser Arg Glin Phe Ala Ala Tyr Cys 115 12 O 125 Tyr Asn Ser Ser Asp Thir Trp Thr Asn Ser Cys Ile Pro Glu Ile Ile 13 O 135 14 O Thir Thr Lys Asp Pro Ile Phe Asn Thr Glin Thr Ala Thr Glin Thir Thr 145 150 155 160 Glu Phe Ile Val Ser Asp Ser Thr Tyr Ser Val Ala Ser Pro Tyr Ser 1.65 17O 17s

Thir Ile Pro Ala Pro Thir Thir Thr Pro Pro Ala Pro Ala Ser Thir Ser 18O 185 19 O Ile Pro Arg Arg Llys Llys Lieu. Ile Cys Val Thr Glu Val Phe Met Glu 195 2OO 2O5 Thir Ser Thr Met Ser Thr Glu Thr Glu Pro Phe Val Glu Asn Lys Ala 21 O 215 22O Ala Phe Lys Asn. Glu Ala Ala Gly Phe Gly Gly Val Pro Thr Ala Lieu 225 23 O 235 24 O Lieu Val Lieu Ala Lieu Lleu Phe Phe Gly Ala Ala Ala Gly Lieu. Gly Phe 245 250 255 Cys Tyr Val Lys Arg Tyr Val Lys Ala Phe Pro Phe Thr Asn Lys Asn 26 O 265 27 O Glin Gln Lys Glu Met Ile Glu Thir Lys Val Val Lys Glu Glu Lys Ala 27s 28O 285 Asn Asp Ser Asn Pro Asn. Glu Glu Ser Lys Llys Thr Asp Lys Asn Pro 29 O 295 3 OO Glu Glu Ser Lys Ser Pro Ser Lys Thir Thr Val Arg Cys Lieu. Glu Ala 3. OS 310 315 32O

Glu Wall

<210s, SEQ ID NO 25 &211s LENGTH: 471 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 25 US 8,535,891 B2 133 134 - Continued

Met Glu Asn Luell His Ile Ile Thir Luell Gly Glin Wall Ile His Lys 15

Arg Glu Glu Lys Lys Glin Cys Arg Arg Luell Gly 25 3O

His Arg Wall Luell Gly Lell Ile Lys Pro Luell Glu Met Lell Glin Asp Glin 35 4 O 45

Gly Lys Arg Ser Wall Pro Ser Glu Luell Thir Thir Ala Met Asn Arg SO 55 6 O

Phe Ala Ala Lell Glu Glu Ala Asn Gly Glu Ile Glu Phe Ser 65 70

Asn Arg Ser Asn Ile Arg Phe Luell Thir Ala Ser Glin Asp Lys Ile 85 90 95

Lell Phe Asp Wall Asn Arg Luell Ser Asp Wall Trp Lys Glu Luell 105 11 O

Ser Luell Luell Luell Glin Wall Glu Glin Arg Met Pro Wall Ser Pro Ile Ser 115 12 O 125

Glin Gly Ala Ser Trp Ala Glin Glu Asp Glin Glin Asp Ala Asp Glu Asp 13 O 135 14 O

Arg Arg Ala Phe Glin Met Lell Arg Arg Asp ASn Glu Ile Glu Ala 145 150 155 160

Ser Luell Arg Arg Lell Glu Ile Asn Met Lys Glu Ile Glu Thir Luell 1.65 17s

Arg Glin Luell Pro Pro Met Glin Glu Ile Pro Glin Glu Glin 18O 185 19 O

Ile Glu Ile Glu Glin Luell Ser Gly Ser Pro Trp Ile Luell 195

Lell Arg Glu Asn Glu Wall Ser Thir Luell Tyr Lys Gly Glu His Arg 21 O 215 22O

Ala Pro Wall Ala Ile Lys Wall Phe Luell Glin Ala Gly Ser Ile 225 23 O 235 24 O

Ala Ile Wall Arg Glin Thir Phe Asn Glu Ile Thir Met Lys Lys 245 250 255

Phe Glu Ser Pro Asn Ile Lell Arg Ile Phe Gly Ile Ile Asp Glu 26 O 265 27 O

Thir Wall Thir Pro Pro Glin Phe Ser Ile Wall Met Glu Tyr Glu Luell 27s 28O 285

Gly Thir Luell Arg Glu Lell Lell Asp Arg Glu Lys Asp Lell Thir Luell Gly 29 O 295 3 OO

Lys Arg Met Wall Lell Wall Lell Gly Ala Ala Arg Gly Lell Arg Luell 3. OS 310 315

His His Ser Glu Ala Pro Glu Luell His Gly Lys Ile Arg Ser Ser Asn 3.25 330 335

Phe Luell Wall Thir Glin Gly Tyr Glin Wall Luell Ala Gly Phe Glu Luell 34 O 345 35. O

Arg Thir Glin Thir Ser Met Ser Luell Gly Thir Thir Arg Glu Thir 355 360 365

Asp Arg Wall Ser Thir Ala Luell Ser Pro Glin Glu Luell Glu Asp 37 O 375

Wall Phe Glin Tyr Asp Wall Ser Glu Ile Ser Phe Gly Ile 385 390 395 4 OO

Wall Luell Glu Ile Ala Thir Gly Asp Ile Pro Phe Glin Gly Cys Asn 4 OS 415

Ser Glu Ile Arg Lell Wall Ala Wall Lys Arg Glin Glin Glu Pro 42O 425 43 O US 8,535,891 B2 135 136 - Continued

Lell Gly Glu Asp Cys Pro Ser Glu Lieu. Arg Glu Ile Ile Asp Glu. Cys 435 44 O 445

Arg Ala His Asp Pro Ser Val Arg Pro Ser Val Asp Glu Ile Lieu Lys 450 45.5 460

Lys Luell Ser Thr Phe Ser Lys 465 470

<210s, SEQ ID NO 26 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 26

Met Ala Thir Ala Gly Gly Gly Ser Gly Ala Asp Pro Gly Ser Arg Gly 1. 5 15

Lell Luell Arg Luell Lell Ser Phe Wall Luell Luell Ala Gly Luell Arg 25 3O

Gly Asn Ser Wall Glu Arg Ile Ile Pro Lell Asn Thir Ala 35 4 O 45

Pro Cys Wall Arg Lell Lell Asn Ala Thir His Glin Ile Gly Glin Ser SO 55 6 O

Ser Ile Ser Gly Asp Thir Gly Wall Ile His Wall Wall Glu Glu Glu 65 70

Asp Luell Glin Trp Wall Lell Thir Asp Gly Pro ASn Pro Pro Met Wall 85 90 95

Lell Luell Glu Ser His Phe Thir Arg Asp Luell Met Glu Lys Luell 105 11 O

Gly Arg Thir Ser Arg Ile Ala Gly Luell Ala Wall Ser Lell Thir Pro 115 12 O 125

Ser Pro Ala Ser Gly Phe Ser Pro Ser Wall Glin Cys Pro Asn Gly 13 O 135 14 O

Phe Gly Wall Ser Asn Ser Gly Pro Glu Phe Ala His Arg 145 150 155 160

Glu Ile Glin Trp Asn Ser Lell Gly Asn Gly Luell Ala Glu Asp Phe 1.65 17O 17s

Ser Phe Pro Ile Phe Lell Lell Glu Asp Glu ASn Glu Thir Lys Wall Ile 18O 185 19 O

Glin Cys Glin Asp His Asn Luell Ser Glin Asn Gly Ser Ala Pro 195

Thir Phe Pro Luell Ala Met Glin Luell Phe Ser His Met His Ala Wall 21 O 215 22O

Ile Ser Thir Ala Thir Cys Met Arg Arg Ser Ser Ile Glin Ser Thir Phe 225 23 O 235 24 O

Ser Ile Asn Pro Glu Ile Wall Asp Pro Luell Ser Asp Asn Wall 245 250 255

Trp Ser Met Luell Pro Ile Asn Thir Thir Gly Thir Lell Lys Pro Asp 26 O 265 27 O

Asp Arg Wall Wall Wall Ala Ala Thir Arg Luell Asp Ser Arg Ser Phe Phe 28O 285

Trp Asn Wall Ala Pro Gly Ala Glu Ser Ala Wall Ala Ser Phe Wall Thir 29 O 295 3 OO

Glin Luell Ala Ala Ala Glu Ala Luell Glin Ala Pro Asp Wall Thir Thir 3. OS 310 315 32O

Lell Pro Arg Asn Wall Met Phe Wall Phe Phe Glin Gly Glu Thir Phe Asp 3.25 330 335 US 8,535,891 B2 137 138 - Continued

Ile Gly Ser Ser Arg Met Wall Tyr Asp Met Glu Lys Gly Lys Phe 34 O 345 35. O

Pro Wall Glin Luell Glu Asn Wall Asp Ser Phe Wall Glu Lell Gly Glin Wall 355 360 365

Ala Luell Arg Thir Ser Lell Glu Luell Trp Met His Thir Asp Pro Wall Ser 37 O 375

Glin Asn Glu Ser Wall Arg Asn Glin Wall Glu Asp Lell Luell Ala Thir 385 390 395 4 OO

Lell Glu Ser Gly Ala Gly Wall Pro Ala Wall Ile Lell Arg Arg Pro 4 OS 415

Asn Glin Ser Glin Pro Lell Pro Pro Ser Ser Luell Glin Arg Phe Luell Arg 425 43 O

Ala Arg Asn Ile Ser Gly Wall Wall Luell Ala Asp His Ser Gly Ala Phe 435 44 O 445

His Asn Tyr Glin Ser Ile Asp Thir Ala Glu Asn Ile Asn 450 45.5 460

Wall Ser Pro Glu Trp Lell Ser Pro Glu Glu Asp Lell Asn Phe Wall 465 470

Thir Asp Thir Ala Lys Ala Lell Ala Asp Wall Ala Thir Wall Luell Gly Arg 485 490 495

Ala Luell Glu Lell Ala Gly Gly Thir Asn Phe Ser Asp Thir Wall Glin SOO 505

Ala Asp Pro Glin Thir Wall Thir Arg Luell Luell Gly Phe Luell Ile 515 525

Ala Asn Asn Ser Trp Phe Glin Ser Ile Luell Arg Glin Asp Luell Arg Ser 53 O 535 54 O

Tyr Luell Gly Asp Gly Pro Lell Glin His Ile Ala Wall Ser Ser Pro 5.45 550 555 560

Thir Asn Thir Thir Tyr Wall Wall Glin Ala Luell Ala Asn Luell Thir Gly 565 st O sts

Thir Wall Wall Asn Lell Thir Arg Glu Glin Cys Glin Asp Pro Ser Wall 585 59 O

Pro Ser Glu Asn Lys Asp Lell Tyr Glu Tyr Ser Trp Wall Glin Gly Pro 595 605

Lell His Ser Asn Glu Thir Asp Arg Luell Pro Arg Cys Wall Arg Ser Thir 610 615

Ala Arg Luell Ala Arg Ala Lell Ser Pro Ala Phe Glu Lell Ser Glin Trp 625 630 635 64 O

Ser Ser Thir Glu Tyr Ser Thir Trp Thir Glu Ser Arg Trp Asp Ile 645 650 655

Arg Ala Arg Ile Phe Lell Ile Ala Ser Glu Lell Glu Luell Ile Thir 660 665 67 O

Lell Thir Wall Gly Phe Gly Ile Luell Ile Phe Ser Lell Ile Wall Thir Tyr 675 685

Ile Asn Ala Lys Ala Asp Wall Luell Phe Ile Ala Pro Arg Glu Pro 69 O. 695 7 OO

Gly Ala Wall Ser Tyr 7 Os

SEO ID NO 27 LENGTH: 469 TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 27 US 8,535,891 B2 139 140 - Continued

Met Ala Ala Ala Ser Pro Lell Arg Asp Cys Glin Ala Trp Lys Asp Ala 1O 15

Arg Luell Pro Luell Ser Thir Thir Ser Asn Glu Ala Lys Luell Phe Asp 25

Ala Thir Luell Thir Glin Wall Lys Trp Thir ASn Asp Lys Ser Luell Gly 35 4 O 45

Gly Ile Glu Gly Lell Ser Luell Ala Ala Asp Pro Thir Phe SO 55 6 O

Wall Met Gly His Ala Met Ala Thir Gly Luell Wall Lell Ile Gly Thir Gly 65 70

Ser Ser Wall Lell Asp Glu Luell Asp Luell Ala Wall Thir Met 85 90 95

Wall Glu Ile Ser Arg Thir Glin Pro Luell Thir Arg Arg Glu Glin Luell His 105 11 O

Wall Ser Ala Wall Glu Thir Phe Ala Asn Gly ASn Phe Pro Ala 115 12 O 125

Glu Luell Trp Glu Glin Ile Lell Glin Asp His Pro Thir Asp Met Luell Ala 13 O 135 14 O

Lell Phe Ser His Asp Ala Phe Luell Gly Glin Glu Glin 145 150 155 160

Met Arg Asp Ser Wall Ala Arg Ile Pro Phe Trp Thir Pro Asp Ile 1.65 17O 17s

Pro Luell Ser Ser Tyr Wall Gly Ile Ser Phe Gly Luell Met Glu 18O 185 19 O

Thir Asn Phe Asp Glin Ala Glu Luell Ala Glu Ala Luell Ser 195

Ile Asn Pro Thir Asp Ala Trp Ser Wall His Thir Wall Ala His Ile His 21 O 215 22O

Glu Met Ala Glu Ile Asp Gly Luell Glu Phe Met Glin His Ser 225 23 O 235 24 O

Glu Thir Phe Trp Lys Asp Ser Asp Met Luell Ala His Asn Tyr Trp 245 250 255

His Trp Ala Luell Tyr Lell Ile Glu Lys Gly Glu Glu Ala Ala Luell 26 O 265 27 O

Thir Ile Tyr Asp Thir His Ile Luell Pro Ser Luell Glin Ala Asn Asp Ala 27s 285

Met Luell Asp Wall Wall Asp Ser Ser Met Luell Tyr Arg Luell Glin Met 29 O 295 3 OO

Glu Gly Wall Ser Wall Gly Glin Arg Trp Glin Asp Wall Lell Pro Wall Ala 3. OS 310 315

Arg His Ser Arg Asp His Ile Luell Luell Phe Asn Asp Ala His Phe 3.25 330 335

Lell Met Ala Ser Lell Gly Ala His Asp Pro Glin Thir Thir Glin Glu Luell 34 O 345 35. O

Lell Thir Thir Luell Arg Asp Ala Ser Glu Ser Pro Gly Glu Asn Glin 355 360 365

His Luell Luell Ala Arg Asp Wall Gly Luell Pro Luell Cys Glin Ala Luell Wall 37 O 375

Glu Ala Glu Asp Gly Asn Pro Asp Arg Wall Luell Glu Lell Luell Luell Pro 385 390 395 4 OO

Ile Arg Arg Ile Wall Glin Luell Gly Gly Ser Asn Ala Glin Arg Asp 4 OS 415

Wall Phe Asn Glin Lell Lell Ile His Ala Ala Luell Asn Thir Ser Ser US 8,535,891 B2 141 142 - Continued

42O 425 43 O

Wal His Lys Asn Val Ala Arg Ser Lieu. Lieu Met Glu Arg Asp Ala Lieu 435 44 O 445 Lys Pro Asn. Ser Pro Lieu. Thr Glu Arg Lieu. Ile Arg Lys Ala Ala Thr 450 45.5 460

Wal His Lieu. Met Glin 465

<210s, SEQ ID NO 28 &211s LENGTH: 114 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 28

Met Ser Ala Lieu Ser Lieu. Lieu. Ile Luell Gly Luell Lell Thir Ala Wall Pro 1. 5 15

Pro Ala Ser Cys Glin Glin Gly Lieu Gly Asn Luell Glin Pro Trp Met Glin 25

Gly Luell Ile Ala Wall Ala Wall Phe Luell Wall Luell Wall Ala Ile Ala Phe 35 4 O 45

Ala Wall Asn His Phe Trp Cys Glin Glu Glu Pro Glu Pro Ala His Met SO 55 6 O

Ile Luell Thir Wall Gly Asn Lys Ala Asp Gly Wall Lell Wall Gly Thir Asp 65 70

Gly Arg Ser Ser Met Ala Ala Ser Phe Arg Ser Ser Glu His Glu 85 90 95

Asn Ala Glu Asn. Wall Pro Glu Glu Glu Gly Wall Arg Ser Thir 1OO 105 11 O

Pro Met

<210s, SEQ ID NO 29 &211s LENGTH: 212. TYPE : PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 29

Met Ser Ser Ser Pro Ser Arg Ser Ala Ala Asp Ile Ile Arg Glu 1. 5 15

Tyr Phe His Ser His Val Ser Gly Gly His Pro Glu Ala Thir Pro Luell 2O 25

Arg Wall Met Tyr Thir Asp Arg Pro Luell Ser Glin Thir Asp Pro Wall Thir 35 4 O 45

Lell Glin Tyr Cys Lieu. Thir Asp Asp Arg Glin Ala Phe Arg Pro Pro SO 55 6 O

Thir Arg Ala Glu Lieu Ala Arg His Arg Wall Wall Wall Thir Thir Thir Ser 65 70

Glin Ala Arg Glu Lieu. Arg Val Pro Wall Gly Phe Phe Ser His Ile Luell 85 90 95

Ile Asp Glu Ala Ala Gln Met Lieu. Glu Glu Ala Lell Thir Pro Luell 105 11 O

Ala Ala Ser His Gly Thr Arg Luell Wall Luell Ala Gly Asp His Met 115 12 O 125

Glin Wall Thir Pro Arg Lieu. Phe Ser Wall Ala Arg Ala Arg Ala Ala Glu 13 O 135 14 O

His Thir Luell Luell His Arg Lieu. Phe Luell Tyr Glin Glin Glu Thir His 145 150 155 160 US 8,535,891 B2 143 144 - Continued

Glu Wall Ala Arg Glin Ser Arg Luell Wall Phe His Glu Asn Tyr Arg 1.65 17O

Thir Asp Ala Ile Wall Ser Phe Ile Ser Arg His Phe Tyr Wall Ala 18O 185 19 O

Gly Asn Pro Ile His Ala Arg Gly Wall Pro Pro His Pro Arg His 195

Pro Luell Met Phe His Wall Ala Gly ASn Pro Asp Arg Asp Met 21 O 215 22O

Ser Met Ala Ser Trp Lell Asn Luell Ala Glu Ile Ala Glin Wall Wall Glu 225 23 O 235 24 O

Wall Glin Glu Ala Asn Thir Trp Pro Ser Trp Gly Gly Arg 245 250 255

Glu Glin Arg Cys Ile Wall Wall Ser His Gly Ala Glin Wall Ser Ala 26 O 265 27 O

Lell Arg Glin Glu Lell Arg Arg Arg Asp Luell Gly Glin Wall Ser Wall Gly 285

Ser Phe Glu Ile Lell Pro Gly Arg Glin Phe Arg Wall Wall Wall Luell Ser 29 O 295 3 OO

Thir Wall His Thir Cys Glin Ser Luell Luell Ser Pro Gly Ala Luell Ala Pro 3. OS 310 315

Glu Phe Phe Thir Asp Ala Arg Wall Luell Asn Thir Wall Lell Thir Arg Ala 3.25 330 335

Glin Ser Glin Luell Wall Wall Wall Gly Asp Ala Wall Ala Lell Cys Ser Phe 34 O 345 35. O

Gly Ala Cys Gly Lys Lell Trp Glu Ser Phe Ile Arg Glu Wall 355 360 365

Arg His Ser Wall Cys Pro Glu Gly Luell Ser Met Glu Glin Wall Glu 37 O 375

Gly Wall Ala Glin Arg Arg Arg Trp Pro Pro Arg Gly Thir Glin Ala 385 390 395

Ala Ala Gly Asn Trp Glu Ala Ala Pro Glu Pro Wall Gly Asp Luell 4 OS 415

Glu Glu Glin Ala Ala Wall Wall Thir Ala Met Wall Ala Glu Pro 425 43 O

Asp Glu Ala Luell Ser Pro Ala Ser Arg Asp Ile Thir Ala Thir Thir 435 44 O 445

Glin Thir Glu Ala Ala Ala Ala Pro Gly Asp Ala Wall Glu Asp 450 45.5 460

Wall Wall Pro Gly Ala Cys Ala Ala Ala Ala Ala Ala Ala Gly Wall 465 470

Glu Ser Thir Glu Ala Glu Asp Ala Ala Asp Phe Trp Pro Trp Asp 485 490 495

Gly Glu Luell Asn Ala Asp Asp Ala Luell Arg Glu Lell Luell Asp Glu SOO

Ser Glin Lys Wall Met Wall Thir Wall Glu Asp Gly Lell Luell Asp Thir 515 525

Wall Ala Arg Pro Glu Ser Lell Glin Ala Arg Lell Glu Asn Luell 53 O 535 54 O

Pro Pro Ala Ala Lell Arg Luell Luell Arg Ala Glu Pro Glu Arg Tyr 5.45 550 555 560

Arg His Ser Phe Wall Pro Glu Thir Phe Glu Arg Ala Ser Ala Ile 565 st O sts

Pro Luell Asp Asp Ala Ser Ser Gly Pro Ile Glin Wall Arg Gly Arg Luell 58O 585 59 O US 8,535,891 B2 145 146 - Continued

Asp Gly Met Ala Phe Ala Gly Asp Glu Wall Lell Wall Glin Luell Luell 595 605

Ser Gly Asp Ala Pro Glu Gly Arg Luell Arg Gly Arg Wall Luell Gly 610 615

Wall Luell Arg Arg His Glu Luell Ala Phe Wall Arg Met Asp 625 630 635 64 O

Thir Trp Asp Pro Arg Ile Met Wall Pro Ile ASn Gly Ser Wall Thir 645 650 655

Ile Phe Wall Ala Glu Lell Asp Pro Ser Glin Wall Pro Ile Ser 660 665 67 O

Lell Arg Lys Gly Arg Lell Glin Arg Wall Gly Luell Glu Arg Luell Thir Ala 675 685

Glu Ala Arg His Ser Arg Lell Phe Trp Wall Glin Ile Wall Luell Trp Arg 69 O. 695 7 OO

Glin Gly Phe Tyr Pro Lell Gly Ile Wall Arg Glu Wall Luell Pro Glu 7 Os

Ala Ser Thir Trp Glu Glin Gly Luell Arg Ile Luell Gly Lell Glu Tyr Ser 72 73 O 73

Lell Arg Wall Pro Pro Ser Asp Glin Ala Thir Ile Thir Wall Luell Glin 740 74. 7 O

His Thir Glu Lell Gly Arg Wall Ala Gly Arg Arg Glu Asp 760 765

Arg Ala Phe Luell Thir Phe Thir Wall Asp Pro Glin Gly Ala Asn Luell 770 775

Asp Asp Ala Luell Ser Wall Arg Asp Luell Gly Pro Arg Glu Wall Ala 79 O 79.

Wall His Ile Thir Asp Wall Ala Ser Phe Wall Pro Arg Asp Gly Wall Luell 805 810 815

Asp Wall Glu Ala Arg Arg Glin Gly Ala Ala Phe Ala Pro Gly Arg 825 83 O

Glu Pro Wall Pro Met Lell Pro Ala Ser Luell Cys Glin Asp Wall Luell Ser 835 84 O 845

Lell Luell Pro Gly Arg Asp Arg Luell Ala Ile Ser Lell Phe Luell Thir Met 850 855 860

Glu Ala Ser Gly Glin Lell Ser Luell Arg Phe Ala Pro Ser Wall 865

Wall Glin Ser Asp Arg Glin Lell Ser Glu Glu Ala Glu Glu Wall Ile 885 890 895

Arg Glin His Pro Gly Ala Gly Arg Glu Luell Pro Ala Arg Luell Asp Ser 9 OO 905 91 O

Wall Asp Ala Wall Wall Ala Ala Phe Ser Arg Luell Luell Arg 915 92 O 925

Arg His Arg Luell Arg Ser Asp Phe Tyr Glu Glin Pro Asp Glu Asp 93 O 935 94 O

Gly Thir Luell Gly Phe Arg Ala Ala His Ile Met Wall Glu Tyr Met 945 950 955 96.O

Ile Glin Phe Asn Arg Lell Wall Ala Glu Phe Luell Wall Gly Ser Glu 965 97.

Thir Arg Thir Wall Thir Pro Lell Arg Trp Glin Pro Ala Pro Arg Ser Glin 985 99 O

Glin Luell Lys Ala Lell Cys Glu Lys His Gly Asp Arg Val Pro Luel Ser 995 1OOO

Lell His Lieu. Gly His His Lieu. His Gly Gly Gly Gly Ser Pro Pro US 8,535,891 B2 147 148 - Continued

O15

Asp Arg Lell His Luell Luell Ala Ser Luell Trp Lys Glin Wall Glin O25 O3 O O35

Phe Ala Arg Thir Glin Glu Glin Met Wall Asp Lell Wall OSO

Thir Asp Asp Met His Phe Luell Ala Pro Ala Gly Arg Asp O65

Lell Ala Luell Glu Ser Ala Phe Gly Ala Arg

Gly Glin Glin Glin Gly His Ser Lell Wall Asp Trp

Trp Ala Thir Ser Ile Arg Arg Asp Wall Wall

Lell Arg Glin Ile Luell Ala Luell Gly His Gly Ser Ala

Ala Arg Asp Ile Gly Luell Glin Phe Ser Lell

Glin Ala Lell Ala Glin Glin Arg Arg Arg Ser Lell

His Ala Wall Glin Luell Ala Glin Pro Lell Lell Gly

Phe Wall Asp Wall Glu Gly Ser Arg Arg Lell Lell

Phe Ser Asn Arg Glu Luell Pro Asp Pro Pro Wall Pro

Ser Lell Glin Luell Glu His Pro His Ala Luell Ala Gly 2O5 21 O 215

Arg Pro Gly Lell Arg Luell Luell Trp Arg Arg Arg Wall Ser Ala 22O 225 23 O

Glin Ser Ser Pro Pro Luell Pro Luell Pro Gly Wall Pro Asp 24 O 245

Pro Thir Lell Ala Wall Glu Thir Ala Luell Trp Glin Lell Lell 255 26 O

Glu Luell Wall Glu Luell Glin Trp Pro Glu Ala Ala Ala Lell Ile 265 27 O 27s

Glin Glu Gly Glu Ala Ser Glin Arg Arg Glu Lell Wall Glin Wall 28O 285 29 O

Glin Ser His Gly Phe Luell Glu Wall Ala Arg Glu Lell 295 305

Gly Ser Gly Asp Thir Luell Glin Wall Glin Luell Gly Ser Lell Glin 310 315

His Phe Lell Wall Pro Ser Pro Glin Luell Trp Wall Ala Pro 3.25 33 O 335

Gly Phe Ser Lell Luell His Wall Glu Arg Gly Asp 34 O

Phe Ser Gly Arg Wall Tyr Ala Pro Arg Asp Arg Asp 355

Wall Glu Ala Cys Trp Glu Pro Phe Ala Lell Glu

Ser Thir Gly Ala Wall Glu Asn Asp Ser Wall Thir Lell Glin 395

His Luell Ser Wall Ser Trp Glu Ala Ser Arg Thir Pro Glin Gly Glin 4 OO 405 41 O US 8,535,891 B2 149 150 - Continued

Lell Gly Ala Phe Arg Luell Glu Ala Ala Phe Lell Glu Glu Asn 42O 425

Asp Ile Asn Phe Ser Lell Cys Ile Arg Lell 435 44 O

Glu Lell Pro Ala Pro Ala Ser Pro Arg Pro Gly Pro Ser 450 45.5

Ser Gly Pro Gly Luell Asn Wall Asp Pro Gly Thir Thir Trp 465 47 O

Wall His Gly Glin Thir Glin Asp Trp Asp Glin Glu Arg Arg Ala 485

Asp Glin Glu Ala Pro Arg Wall His Lell Phe Wall His His 495 SOO

Met Met Glu Wall Pro Glu Glu Wall Lell Pro Gly Thir 510 515

Lell Phe Thir Wall Glu Luell Luell Pro Glin Lell Pro Asp Lell Arg 52O 525 53 O

Glu Glu Ala Wall Arg Luell Glu Glu Ala Pro Lell Wall 535 545

Thir Ser Ile Ala Luell Gly Pro Wall Pro Glin Pro Luell Arg 550 560

Wall Pro Ser Arg Phe Luell Glu Arg Glin Thir yr ASn Ile Pro st O sts

Gly Arg His Luell Asn Pro Ser Glin Asn Wall Ala Wall Arg 585 590

Glu Lell Glu Pro Thir Wall Ile Glin Pro Pro Gly 605

Thir Thir Ile Wall Luell His Ile Wall Phe Trp Phe His 62O

Ser Asn Glin Glu Glin Glin Pro Gly Gly Pro Pro Arg Gly 625 635

Glu Lys Arg Lell Gly Gly Pro Ile Luell Gly Pro Ser 64 O 645 650

Asn Lys Ser Wall Asp Wall Luell Ala Gly Luell Lell Lell Arg Arg Met 655 660 665

Glu Lel Pro Luell Arg Tyr Ser Glu Glin Ala Glu Ala Ser 670 68O

Glu Phe Pro Wall Pro Arg Gly Ser Arg Lell Luell Arg 685 695

Ser Arg Glu Gly Arg Pro Asn Glin Ser Lell Ser Ile Thir 7Os

Lell His Arg Ile Arg Glin Ala Pro ASn Pro yr Ser Ser Glu 72 O 72

Ile Lys Ala Phe Asp Thir Luell Glin Arg Gly Glu Luell Phe Ser 73 O 73 74 O

Arg Glu Asp Lell Wall Trp yr Lys Wall Lell rp Glu Ala Arg 74. 7 O 7ss

Glu Lell Asp Arg Glu Wall Ile Lell Thir Ser 765 770

Ala Ser Ala Ser Luell Ile Luell Asp Wall Arg Glin Ile 78O 78s

Lell Asp Glu Ala Gly Met Ala Thir Glu Pro Glu Thir Lell Ile 79. 8OO

Pro Luell Wall Glin Phe Pro Glin Ala Glu Wall Wall Luell Lell Gly 805 810 815 US 8,535,891 B2 151 152 - Continued

Asp His Lys Glin Lieu. Arg Pro Val Val Lys Asn. Glu Arg Lieu. Glin 82O 825 83 O

Asn Luell Gly Lieu. Asp Arg Ser Lieu. Phe Glu Arg Tyr His Glu Asp 835 84 O 845

Ala Met Lieu. Asp Thr Glin Tyr Arg Met His Glu Gly Ile Cys 850 855 86 O

Ala Phe Pro Ser Val Ala Phe Tyr Lys Ser Lys Lieu Lys Thr Trp 865 87 O 87s

Glin Lieu. Arg Arg Pro Pro Ser Val Lieu. Gly His Ala Gly Lys 885 890

Glu Ser Cys Pro Val Ile Phe Gly His Val Glin Gly His Glu Arg 895 9 OO 905

Ser Luell Lieu Val Ser Thir Asp Glu Gly Asn. Glu Asn. Ser Lys Ala 910 915 92 O

Asn Luell Glu Glu Val Ala Glu Val Val Arg Ile Thr Lys Glin Lieu. 925 93 O 935

Thir Luell Gly Arg Thr Val Glu Pro Glin Asp Ile Ala Val Lieu. Thr 94 O 945 950

Pro yr Asn Ala Glin Ala Ser Glu Ile Ser Lys Ala Lieu. Arg Arg 955 96.O 965

Glu Ile Ala Gly Val Ala Val Ser Ser Ile Thr Lys Ser Glin 97. 98 O

Gly Ser Glu Trp Arg Tyr Val Lieu Val Ser Thr Val Arg Thr Cys 985 990 995

Ala Lys Ser Asp Lieu. Asp Glin Arg Pro Thr Llys Ser Trp Lieu Lys 2OOO 2005 2010

Phe Lieu. Gly Phe Val Val Asp Pro Asn Glin Val Asn. Wall Ala 2015 2O2O 2O25

Wall Thir Arg Ala Glin Glu Gly Lieu. Cys Lieu. Ile Gly Asp His Lieu. 2O3O 2O35 2O4. O

Lell Luell Arg Cys Cys Pro Lieu. Trp Arg Ser Lieu. Lieu. Asp Phe Cys 2O45 2OSO 2O55

Glu Ala Gln Glin Thr Lieu Val Pro Ala Gly Glin Val Arg Val Cys 2O60 2O65 2. Of O

Arg Pro Thir Met Pro Ser 2O8 O

<210s, SEQ ID NO 3 O &211s LENGTH: 190 212. TYPE : PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 30 Met Ala Thr His His Thir Lieu. Trp Met Gly Lieu Ala Lieu. Lieu. Gly Val 1. 5 1O 15

Lell Gly Asp Lieu. Glin Ala Ala Pro Glu Ala Glin Val Ser Val Glin Pro 2O 25 3O

Asn Phe Glin Glin Asp Llys Phe Lieu. Gly Arg Trp Phe Ser Ala Gly Lieu. 35 4 O 45

Ala Ser Asn Ser Ser Trp Lieu. Arg Glu Lys Lys Ala Ala Lieu. Ser Met SO 55 6 O Cys Ser Val Val Ala Pro Ala Thr Asp Gly Gly Lieu. Asn Lieu. Thr 65 70 7s 8O

Ser Thir Phe Lieu. Arg Lys Asn Glin Cys Glu Thir Arg Thr Met Lieu. Lieu 85 90 95 US 8,535,891 B2 153 154 - Continued

Gln Pro Ala Gly Ser Lieu. Gly Ser Tyr Ser Tyr Arg Ser Pro His Trp 105 11 O

Gly Ser Thr Tyr Ser Val Ser Val Wall Glu Thir Asp Tyr Asp Glin 115 12 O 125

Ala Lieu. Lieu. Tyr Ser Glin Gly Ser Gly Pro Gly Glu Asp Phe Arg 13 O 135 14 O

Met Ala Thr Lieu. Tyr Ser Arg Thr Glin Thir Pro Arg Ala Glu Luell Lys 145 150 155 160

Glu Lys Phe Thr Ala Phe Cys Lys Ala Glin Gly Phe Thir Glu Asp Thir 1.65 17O 17s

Ile Val Phe Leu Pro Gln Thr Asp Lys Met Thir Glu Glin 18O 185 19 O

SEQ ID NO 31 LENGTH: 174 TYPE PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 31

Met Ala Ala Ala Met Val Pro Gly Arg Ser Glu Ser Trp Glu Arg Gly 1. 5 15

Glu Pro Gly Arg Pro Ala Lieu. Tyr Phe Gly Ser Ile Arg Gly Gly 25

Arg Glu Asp Arg Thr Lieu. Tyr Glu Arg Ile Wall Ser Arg Luell Arg Arg 35 4 O 45

Phe Gly Thr Val Lieu. Thr Glu. His Wall Ala Ala Ala Glu Luell Gly Ala SO 55 6 O

Arg Gly Glu Glu Ala Ala Gly Gly Asp Arg Luell Ile His Glu Glin Asp 65 70

Lieu. Glu Trp Lieu. Glin Glin Ala Asp Wall Wall Wall Ala Glu Wall Thir Glin 85 90 95

Pro Ser Leu Gly Val Gly Tyr Glu Luell Gly Arg Ala Wall Ala Phe Asn 105 11 O

Lys Arg Ile Lieu. Cys Lieu. Phe Arg Pro Glin Ser Gly Arg Wall Luell Ser 115 12 O 125

Ala Met Ile Arg Gly Ala Ala Asp Gly Ser Arg Phe Glin Wall Trp Asp 13 O 135 14 O

Tyr Glu Glu Gly Glu Val Glu Ala Luell Luell Asp Arg Phe Glu Ala 145 150 155 160

Asp Pro Pro Gly Glin Val Ala Ala Ser Pro Asp Pro Thir Thir 1.65 17O

SEQ ID NO 32 LENGTH: 114 TYPE PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 32

Met Thr Cys Llys Met Ser Gln Leu Glu Arg ASn Ile Glu Thir Ile Ile 1. 5 1O 15

Asn Thr Phe His Glin Tyr Ser Val Lys Luell Gly His Pro Asp Thir Luell 25 3O

Asn Glin Gly Glu Phe Lys Glu Lieu. Wall Arg Asp Lell Glin Asn Phe 35 4 O 45

Lieu Lys Lys Glu Asn Lys Asn. Glu Wall Ile Glu His Ile Met Glu SO 55 6 O