USOO9482672B2

(12) United States Patent (10) Patent No.: US 9.482,672 B2 Gong et al. (45) Date of Patent: Nov. 1, 2016

(54) METHODS FOR DIAGNOSING IRRITABLE 2002fO197661 A1 12/2002 Niles et al. BOWEL, SYNDROME 2003,022903.0 A1 12/2003 Theoharides 2008.OO57590 A1 3/2008 Urdea et al. (71) Applicant: NESTEC S.A., Vevey (CH) 2008/0085524 A1 4/2008 Lois (72) Inventors: Hua Gong, San Diego, CA (US); Shui FOREIGN PATENT DOCUMENTS Long Wang, San Diego, CA (US); JP 2000-065820 A 3, 2000 Sharat Singh, Rancho Santa Fe, CA JP 2003-277378. A 10, 2003 (US) JP 2007-506100 A 3, 2007 JP 2007-1864.57 A 7/2007 JP 2008-521817. A 6, 2008 (73) Assignee: Nestec S.A., Vevey (CH) JP 2008-530537 A 8, 2008 JP 2009-52.1690 A 6, 2009 (*) Notice: Subject to any disclaimer, the term of this WO O3.011812 A1 2, 2003 patent is extended or adjusted under 35 WO 2005/O29091 A2 3, 2005 U.S.C. 154(b) by 0 days. WO 2007/076411 A1 7/2005 WO 2006/06877O A1 6, 2006 WO 2006/084.299 A2 8, 2006 (21) Appl. No.: 14/222,531 WO 2008/O14414 A2 1, 2008 WO 2008/022177 A2 2, 2008 (22) Filed: Mar. 21, 2014 WO 2009,045291 A2 4/2009

(65) Prior Publication Data OTHER PUBLICATIONS US 2014/O1997.09 A1 Jul. 17, 2014 Jones et al. The Lancet, vol. 320, Issue 8308, pp. 1115-1172, Nov. 20, 1982, Abstract Only.* Related U.S. Application Data Thevarajah et al. (Practical Gastroenterology, May 2005, p. 62-74).* J.D. Wood (Gut, 2006, vol. 55, pp. 445-447, Commentaries).* (60) Division of application No. 13/352.225, filed on Jan. Gittlen et al. (Gut, 1990, vol. 31, pp. 96-99).* 17, 2012, now Pat. No. 8,709,733, which is a division Barbara, G. et al. “Activated Mast Cells in Proximity to Colonic of application No. 12/862,707, filed on Aug. 24, 2010, Nerves Correlate with Abdominal Pain in Irritable Bowel Syn now Pat. No. 8,114,616, which is a continuation of drome.” American Gastroenterological Association, 2004, vol. 126, pp. 693-702. application No. PCT/US2010/039866, filed on Jun. Clarke, G. et al., “Irritable bowel syndrome: towards biomarker 24, 2010. identification,” Trends in Molecular Medicine, Elsevier Current Trends, GB, vol. 15, No. 10, Oct. 1, 2009, pp. 478-479. (60) Provisional application No. 61/220,525, filed on Jun. Clarke, G. et al., “W2034 Polyunsaturated Fatty Acids Contribute to 25, 2009, provisional application No. 61/252,094, the Inflammatory Phenotype in Irritable Bowel Syndrome.” Gas filed on Oct. 15, 2009. troenterology, Elsevier, Philadelphia, PA. vol. 136, No. 5, May 1, 2009, pp. A-777. (51) Int. Cl. Gittlen, S.D. et al., “Raised histamine concentrations in chronic GOIN 33/53 (2006.01) cholestatic liver disease,” Gut, 1990, vol. 31, pp. 96-99. GOIN 3L/00 (2006.01) Guilarte, M. et al., “Diarrhoea-predominant IBS patients show mast cell activation and hyperplasia in the jejunum.” Gut, 56(2), 203-209, GOIN 33/573 (2006.01) 2007. GOIN 33/68 (2006.01) Lembo, A.J. et al., “Use of serum biomarkers in a diagnostic test for GOIN 33/74. (2006.01) irritable bowel syndrome.” Alimentary Pharmacology & Therapeu (52) U.S. Cl. tics, vol. 29, No. 8, Apr. 1, 2009, pp. 834-842. Patnaik, Mrinal et al., “Systemic mastocytosis—A concise clinical CPC ...... G0IN 33/573 (2013.01); G0IN 33/6893 and laboratory review.” Archives of Pathology & Laboratory Medi (2013.01); G0IN 33/74 (2013.01); G0IN cine, 131(5), 784-791, 2007. 2800/065 (2013.01); G0IN 2800/56 (2013.01); Samineni, Sridhar et al., “Optimization, Comparison, and Applica Y10S 436/809 (2013.01); Y10T 436/147777 tion of Colorimetric vs. Chemiluminescence Based Indirect Sand (2015.01) wich ELISA for Measurement of Human IL-23,” Journal of (58) Field of Classification Search Immunoassay & Immunochemistry, 2006, vol. 27, pp. 183-193. CPC. A61K 38/00; A61K 39/00; G01N 33/582: (Continued) G01N 33/6893; G01N 33/54366; C07K 5700; C07K 14/705; C07K 16/18 Primary Examiner — Lisa Cook See application file for complete search history. (74) Attorney, Agent, or Firm — Kilpatrick Townsend & Stockton LLP (56) References Cited (57) ABSTRACT The invention provides an ELISA assay for the determina U.S. PATENT DOCUMENTS tion of serum mast cell B-tryptase levels using rabbit anti 5,744,319 A 4, 1998 Niles et al. tryptase as the capture antibody and alkaline phosphatase 6,867,197 B1 3/2005 Davis et al. conjugated G3 as the detecting antibody. Luminescent Sub 8, 114,616 B2 * 2/2012 Gong ...... GO1N 33,573 strate CPSD was used to enhance the assay sensitivity. Also 427/287 provided are methods for aiding in the diagnosis of irritable 8,624,002 B2 * 1/2014 Gu ...... CO7K 16,26 bowel syndrome by detecting the serum level of B-tryptase, 530,387.1 8,709,733 B2 * 4/2014 Gong ...... GO1N 33,573 histamine and/or prostaglandin E. 435/287.9 21 Claims, 13 Drawing Sheets US 9,482.672 B2 Page 2

(56) References Cited Wang. Sherman et al., “Evaluation on the diagnostic utility of serum tryptase level in irritable bowel syndrome.” Am. J. Gastroenterol OTHER PUBLICATIONS ogy, 104(Suppl 3); S498, 2009. Taira, M. et al., "Serum B12 tryptase level as a marker of allergic Wood, J.D., "Histamine, mast cells, and the enteric nervous system airway inflammation in asthma,” Journal of Asthma, Asthma Pub- in the irritable bowel syndrome, enteritis, and food allergies.” Gut, lications Society, Ossining, New York. 39(4), 315-322, 2002. Thevarajah, Sharmela et al., “Gastrointestinal Tract and Irritable 2006, vol. 55, pp. 445-447. Bowel Syndrome.” Practical Gastroenterology, May 2005, pp. 62-74. * cited by examiner U.S. Patent Nov. 1, 2016 Sheet 1 of 13 US 9,482.672 B2 Optimize the Coating Ab

Dose Response Curve of Human Tryptase with Different Amount of Coating Antibody 11000 is 4 ug/ml 10000 A 2 ug/ml 9000 V 1 lug/ml 8000 0 0.5 g/ml 7000 6000 0.03371 5000 0.07571 4000

Log Tryptase uglml

FIG. 1 U.S. Patent Nov. 1, 2016 Sheet 2 of 13 US 9,482.672 B2

Optimize the Detecting Ab

Dose Response Curve of Tryptase with Different Dilutions of Detecting Ab AP-G3

20000

15000

0.05956 0.1236

Log Tryptase uglml

FIG. 2 U.S. Patent Nov. 1, 2016 Sheet 3 of 13 US 9,482.672 B2

Development of Sensitive Human Tryptase ELISA for the Detection of Tryptase in Human Serum

Standard Curve of Tryptase ELISA 10000

7500

STD 5000 EC50 2500 0.06455

-5 -4 -3 -2 -1 O 1 Log Tryptase ng/ml)

FIG. 3 U.S. Patent Nov. 1, 2016 Sheet 4 of 13 US 9,482.672 B2

Human Tryptase Concentration in Serum from Gil Control (71), IBS-C (61), and IBS-D (203) Patients. Values are expressed as mean (SEM). *p = 0.0019 for IBS-D; Mann Whitney U test.

FIG. 4 U.S. Patent Nov. 1, 2016 Sheet 5 of 13 US 9,482.672 B2

Tryptase Log Value Distribution

1 3. 2) 25 Q4 6 (38 & 1 & 8 8 BSD BSA

FIG. 5 U.S. Patent Nov. 1, 2016 Sheet 6 of 13 US 9,482.672 B2

Density Analysis for Tryptase Data O6BSO3 COhorts

c BS) --no BSC MMM ESA, on EATY e

e

FIG. 6 U.S. Patent Nov. 1, 2016 Sheet 7 of 13 US 9,482.672 B2

Human tryptase concentration in serum from healthy control (n=156), IBS-D (n=209), IBS-C (n=119), and IBS-A (n=57) Values are expressed as mean (SEM), p<0.05 for IBS-D, Mann Whitney U test

8& &&

FIG. 7 U.S. Patent Nov. 1, 2016 Sheet 8 of 13 US 9,482.672 B2

Standard Curve of Human Tryptase 10000

8000

6000

4000

2000

O N N N N N N Q SS S. Q Log Tryptase lug/ml

FIG. 8

U.S. Patent Nov. 1, 2016 Sheet 10 of 13 US 9,482.672 B2

FIG. 10 U.S. Patent Nov. 1, 2016 Sheet 11 of 13 US 9,482.672 B2

009 009), 0001, ?uffida)

O O O o 009), o O u/fd eulueSH U.S. Patent Nov. 1, 2016 Sheet 12 of 13 US 9,482.672 B2

U.S. Patent Nov. 1, 2016 Sheet 13 of 13 US 9,482.672 B2

1. 200

FIG. 13 US 9,482,672 B2 1. 2 METHODS FOR DIAGNOSING RRTABLE are considered important contributors to symptom patho BOWEL, SYNDROME genesis (Quigley, Scand. J. Gastroenterol. 38(Suppl. 237): 1-8 (2003); Mayer et al., Gastroenterol., 122:2032-2048 CROSS-REFERENCES TO RELATED (2002)), this condition is now generally viewed as a disorder APPLICATIONS of the brain-gut axis. Recently, roles for enteric infection and intestinal inflammation have also been proposed. Studies This application is a divisional application of U.S. patent have documented the onset of IBS following bacteriologi application Ser. No. 13/352.225, filed Jan. 17, 2013, now cally confirmed gastroenteritis, while others have provided U.S. Pat. No. 8,709,733 B2, which application is a divisional evidence of low-grade mucosal inflammation (Spiller et al., of U.S. patent application Ser. No. 12/862,707, filed Aug. 10 Gut, 47:804-811 (2000); Dunlop et al., Gastroenterol. 125: 24, 2010, now U.S. Pat. No. 8,114,616 B2, which applica 1651-1659 (2003); Cumberland et al., Epidemiol. Infect. tion is a continuation of PCT/US2010/039866, which appli 130:453-460 (2003)) and immune activation (Gwee et al., cation claims priority to U.S. Provisional Application No. Gut, 52:523-526 (2003); Pimentel et al., Am. J. Gastroen 61/220,525, filed Jun. 25, 2009 and U.S. Provisional Appli terol. 95:3503-3506 (2000)) in IBS. The enteric flora has cation No. 61/252,094, filed Oct. 15, 2009, the disclosures of 15 also been implicated, and a recent study demonstrated the which are herein incorporated by reference in their entirety efficacy of the probiotic organism Bifidobacterium in treat for all purposes. ing the disorder through modulation of immune activity (O'Mahony et al., Gastroenterol., 128:541-551 (2005)). BACKGROUND OF THE INVENTION The hypothalamic-pituitary-adrenal axis (HPA) is the core endocrine stress system in humans (De Wied et al. Front. Irritable bowel syndrome (IBS) is the most common of all Neuroendocrinol., 14:251-302 (1993)) and provides an gastrointestinal disorders, affecting 10-20% of the general important link between the brain and the gut immune population and accounting for more than 50% of all patients system. Activation of the axis takes place in response to both with digestive complaints. However, studies suggest that physical and psychological stressors (Dinan, Br. J. Psychia only about 10% to 50% of those afflicted with IBS actually 25 try, 164:365-371 (1994)), both of which have been impli seek medical attention. Patients with IBS present with cated in the pathophysiology of IBS (Cumberland et al., disparate symptoms such as, for example, abdominal pain Epidemiol. Infect., 130:453-460 (2003)). Patients with IBS predominantly related to defecation, diarrhea, constipation have been reported as having an increased rate of sexual and or alternating diarrhea and constipation, abdominal disten physical abuse in childhood together with higher rates of tion, gas, and excessive mucus in the stool. More than 40% 30 stressful life events in adulthood (Gaynes et al., Baillieres of IBS patients have symptoms so severe that they have to Clin. Gastroenterol. 13:437-452 (1999)). Such psychosocial take time off from work, curtail their social life, avoid sexual trauma or poor cognitive coping strategy profoundly affects intercourse, cancel appointments, stop traveling, take medi symptom severity, daily functioning, and health outcome. cation, and even stay confined to their house for fear of Although the etiology of IBS is not fully characterized, embarrassment. The estimated health care cost of IBS in the 35 the medical community has developed a consensus defini United States is S8 billion per year (Talley et al., Gastro tion and criteria, known as the Rome II criteria, to aid in the enterol., 109:1736-1741 (1995)). diagnosis of IBS based upon patient history. The Rome II The precise pathophysiology of IBS is not well under criteria requires three months of continuous or recurrent stood. Nevertheless, there is a heightened sensitivity to abdominal pain or discomfort over a one-year period that is visceral pain perception, known as peripheral sensitization. 40 relieved by defecation and/or associated with a change in This sensitization involves a reduction in the threshold and stool frequency or consistency as well as two or more of the an increase in the gain of the transduction processes of following: altered stool frequency, altered stool form, altered primary afferent neurons, attributable to a variety of media stool passage, passage of mucus, or bloating and abdominal tors including monoamines (e.g., catecholamines and distention. The absence of any structural or biochemical indoleamines), Substance P, and a variety of cytokines and 45 disorders that could be causing the symptoms is also a prostanoids such as E-type prostaglandins (see, e.g., Mayer necessary condition. As a result, the Rome II criteria can be et al., Gastroenterol., 107:271-293 (1994)). Also implicated used only when there is a Substantial patient history and is in the etiopathology of IBS is intestinal motor dysfunction, reliable only when there is no abnormal intestinal anatomy which leads to abnormal handling of intraluminal contents or metabolic process that would otherwise explain the and/or gas (see, e.g., Kellow et al., Gastroenterol., 92: 1885 50 symptoms. Similarly, the Rome III criteria recently devel 1893 (1987); Levitt et al., Ann. Int. Med., 124:422-424 oped by the medical community can be used only when there (1996)). Psychological factors may also contribute to IBS is presentation of a specific set of symptoms, a detailed symptoms appearing in conjunction with, if not triggered by, patient history, and a physical examination. disturbances including depression and anxiety (see, e.g., It is well documented that diagnosing a patient as having Drossman et al., Gastroenterol. Int, 8:47-90 (1995)). 55 IBS can be challenging due to the similarity in Symptoms The causes of IBS are not well understood. The walls of between IBS and other diseases or disorders. In fact, because the intestines are lined with layers of muscle that contract the symptoms of IBS are similar or identical to the symp and relax as they move food from the stomach through the toms of So many other intestinal illnesses, it can take years intestinal tract to the rectum. Normally, these muscles con before a correct diagnosis is made. For example, patients tract and relax in a coordinated rhythm. In IBS patients, 60 who have inflammatory bowel disease (IBD), but who these contractions are typically stronger and last longer than exhibit mild signs and symptoms Such as bloating, diarrhea, normal. As a result, food is forced through the intestines constipation, and abdominal pain, may be difficult to dis more quickly in some cases causing gas, bloating, and tinguish from patients with IBS. As a result, the similarity in diarrhea. In other cases, the opposite occurs: food passage symptoms between IBS and IBD renders rapid and accurate slows and stools become hard and dry causing constipation. 65 diagnosis difficult. The difficulty in differentially diagnosing The precise pathophysiology of IBS remains to be eluci IBS and IBD hampers early and effective treatment of these dated. While gut dysmotility and altered visceral perception diseases. Unfortunately, rapid and accurate diagnostic meth US 9,482,672 B2 3 4 ods for definitively distinguishing IBS from other intestinal wherein a higher level of B-tryptase in the second sample diseases or disorders presenting with similar symptoms are relative to the first sample is indicative of the progression of currently not available. The present invention satisfies this IBS in the subject and a lower level of B-tryptase in the need and provides related advantages as well. second sample relative to the first sample is indicative of the regression of IBS in the subject. BRIEF SUMMARY OF THE INVENTION In another aspect, the present invention provides a method for monitoring the progression or regression of irritable The present invention provides methods, assays, systems, bowel syndrome (IBS) in a subject, the method comprising: and code for accurately classifying whether a sample from (a) contacting a first blood or serum sample taken from the an individual is associated with irritable bowel syndrome 10 Subject at a first time with a histamine binding moiety under (IBS). As a non-limiting example, the present invention is conditions suitable to transform histamine present in the useful for classifying a sample from an individual as an IBS sample into a complex comprising histamine and the hista sample using a statistical algorithm and/or empirical data. mine binding moiety; (b) determining the level of the Similarly, the present invention also provides methods, complex, thereby determining the level of histamine present assays, systems, and code for aiding in the diagnosis of 15 in the first sample; (c) contacting a second blood or serum irritable bowel syndrome (IBS) in a subject. The present sample taken from the Subject at a second time with a invention is also useful for ruling out one or more diseases histamine binding moiety under conditions Suitable to trans or disorders that present with IBS-like symptoms and ruling form histamine present in the sample into a complex com in IBS using a combination of statistical algorithms and/or prising histamine and the histamine binding moiety; (d) empirical data. Thus, the present invention provides an determining the level of the complex, thereby determining accurate diagnostic prediction of IBS and prognostic infor the level of histamine present in the second sample; and (e) mation useful for guiding treatment decisions. comparing the level of histamine present in the first sample In one aspect, the present invention provides a method for to the level of histamine present in the second sample, aiding in the diagnosis of irritable bowel syndrome (IBS) in wherein a higher level of histamine in the second sample a Subject, the method comprising: (a) contacting a blood or 25 relative to the first sample is indicative of the progression of serum sample from the Subject with a B-tryptase binding IBS in the subject and a lower level of histamine in the moiety under conditions Suitable to transform B-tryptase second sample relative to the first sample is indicative of the present in the sample into a complex comprising B-tryptase regression of IBS in the subject. and the B-tryptase binding moiety; and (b) determining the In yet another aspect, the present invention provides a level of the complex, thereby determining the level of 30 method for monitoring the progression or regression of B-tryptase present in the sample. irritable bowel syndrome (IBS) in a subject, the method In another aspect, the present invention provides a method comprising: (a) contacting a first blood or serum sample for aiding in the diagnosis of IBS in a subject, the method taken from the Subject at a first time with a prostaglandin E2 comprising: (a) contacting a blood or serum sample from the (PGE2) binding moiety under conditions suitable to trans Subject with a histamine binding moiety under conditions 35 form PGE2 present in the sample into a complex comprising Suitable to transform histamine present in the sample into a PGE2 and the PGE2 binding moiety; (b) determining the complex comprising histamine and the histamine binding level of the complex, thereby determining the level of PGE2 moiety; and (b) determining the level of the complex, present in the first sample; (c) contacting a second blood or thereby determining the level of histamine present in the serum sample taken from the Subject at a second time with sample. 40 a PGE2 binding moiety under conditions suitable to trans In yet another aspect, the present invention provides a form PGE2 present in the sample into a complex comprising method for aiding in the diagnosis of IBS in a subject, the PGE2 and the PGE2 binding moiety; (d) determining the method comprising: (a) contacting a blood or serum sample level of the complex, thereby determining the level of PGE2 from the subject with a prostaglandin E2 (PGE2) binding present in the second sample; and (e) comparing the level of moiety under conditions suitable to transform PGE2 present 45 PGE2 present in the first sample to the level of PGE2 present in the sample into a complex comprising PGE2 and the in the second sample, wherein a higher level of PGE2 in the PGE2 binding moiety; and (b) determining the level of the second sample relative to the first sample is indicative of the complex, thereby determining the level of PGE2 present in progression of IBS in the subject and a lower level of PGE2 the sample. in the second sample relative to the first sample is indicative In one aspect, the present invention provides a method for 50 of the regression of IBS in the subject. monitoring the progression or regression of irritable bowel In one aspect, the present invention provides a computer syndrome (IBS) in a Subject, the method comprising: (a) readable medium comprising code for controlling one or contacting a first blood or serum sample taken from the more processors to classify whether a serum or blood sample Subject at a first time with a B-tryptase binding moiety under from an subject is associated with irritable bowel syndrome conditions Suitable to transform B-tryptase present in the 55 (IBS), the code comprising instructions to apply a statistical sample into a complex comprising B-tryptase and the process to a data set comprising a diagnostic marker profile B-tryptase binding moiety; (b) determining the level of the to produce a statistically derived decision classifying the complex, thereby determining the level off-tryptase present sample as an IBS sample or non-IBS sample based upon the in the first sample; (c) contacting a second blood or serum diagnostic marker profile, wherein the diagnostic marker sample taken from the Subject at a second time with a 60 profile indicates the level of at least one diagnostic marker B-tryptase binding moiety under conditions suitable to trans selected from the group consisting of B-tryptase, histamine, form B-tryptase present in the sample into a complex com prostaglandin E2 (PGE2), and a combination thereof. prising B-tryptase and the B-tryptase binding moiety; (d) In another aspect, the present invention provides a system determining the level of the complex, thereby determining for classifying whether a serum or blood sample from a the level of B-tryptase present in the second sample; and (e) 65 subject is associated with irritable bowel syndrome (IBS), comparing the level of B-tryptase present in the first sample the system comprising: (a) a data acquisition module con to the level of B-tryptase present in the second sample, figured to produce a data set comprising a diagnostic marker US 9,482,672 B2 5 6 profile, wherein the diagnostic marker profile indicates the TABLE 1-continued presence or level of at least one diagnostic marker selected from the group consisting of B-tryptase, histamine, prosta Exemplary diagnostic markers suitable glandin E2 (PGE2), and a combination thereof; (b) a data for use in IBS classification. processing module configured to process the data set by 5 Family Biomarker applying a statistical process to the data set to produce a MMP MMP-9 statistically derived decision classifying the sample as an TIMP TIMP-1 IBS sample or non-IBS sample based upon the diagnostic Alpha-globulin Alpha-2-macroglobulin (Ca2-MG) marker profile; and (c) a display module configured to Haptoglobin precursor alpha-2 (HpC.2) 10 Orosomucoid display the statistically derived decision. Actin-severing protein Gelsolin In yet another aspect, the present invention provides a S100 protein Calgranulin AS100A8/MRP-8 Fibrinopeptide Fibrinopeptide A (FIBA) method for the detection of B-tryptase in a blood or serum Serine protease Tryptase sample, the method comprising the steps of: (a) coating a Prostaglandin Prostaglandin E2 (PGE2) Solid phase Surface with a first anti-B-tryptase capture anti 15 Others Lactoferrin body; (b) contacting the solid phase surface with a blood or Anti-tissue transglutaminase (tTG) antibody serum sample under conditions suitable to transform Calcitonin gene-related peptide (CGRP) B-tryptase present in the sample into a complex comprising Histamine B-tryptase and the anti-B-tryptase capture antibody; (c) contacting the B-tryptase and the anti-B-tryptase complex with a second detecting antibody conjugated to alkaline BRIEF DESCRIPTION OF THE DRAWINGS phosphatase under conditions suitable to form a ternary FIG. 1 illustrates a dose response curve in one embodi complex; and (d) contacting the ternary complex with a ment of the present invention. CPSD luminescent substrate. FIG. 2 illustrates a dose response curve in one embodi In one aspect, the present invention provides a method for 25 ment of the present invention. classifying whether a sample from an individual is associ FIG. 3 illustrates a standard curve in one embodiment of ated with IBS, the method comprising: (a) determining a the present invention. Rabbit anti-human tryptase was diagnostic marker profile by detecting the presence or level coated onto an immunoassay plate and blocked with assay of at least one diagnostic marker in the sample; and (b) buffer (5% BSA in PBS). Different concentrations of human classifying the sample as an IBS sample or non-IBS sample 30 tryptase (Standard curve) or human serum samples (diluted using an algorithm based upon the diagnostic marker profile. in assay buffer 1:5 and 1:10) were added to the wells and In preferred embodiments of the various methods and incubated for 2 hours at RT. The plate was washed and assays of the present invention, the presence or level of 1, 2, alkaline phosphatase (AP) labeled McAb against tryptase 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, (G3) were added and incubated for 2 hours at RT. The plate 21, 22, 23, 24, 25, or more of the biomarkers shown in Table 35 was washed and AP substrate (CSPD) was added to each 1 is detected to generate a diagnostic marker profile that is well. A luminescence plate reader was used to detect the useful for predicting IBS. In certain instances, the biomark luminescent light. Serum tryptase concentration was calcu ers described herein are analyzed using an immunoassay lated using the standard curve. (Tryptase detection range Such as an enzyme-linked immunosorbent assay (ELISA) or 0.019-5000 ng/ml. EC50=65 ng/ml: Recovery 81.5% with an immunohistochemical assay. 40 20 ng/ml of tryptase spiked in normal pooled serum). See Example 16. TABLE 1. FIG. 4 illustrates a bar graph with tryptase concentrations in serum of GI control, IBS-C and IBS-D. Exemplary diagnostic markers suitable for use in IBS classification. FIG. 5 illustrates a bar graph with tryptase log value 45 distribution. Family Biomarker FIG. 6 illustrates a density analysis for tryptase data. Cytokine CXCL8 IL-8 FIG. 7 illustrates a bar graph with tryptase concentrations IL-1B in serum of GI control, IBS-C, IBS-D and IBS-A. TNF-related weak inducer of apoptosis (TWEAK) FIG. 8 illustrates the dose response curve of human Leptin 50 tryptase in one embodiment of the present invention. Osteoprotegerin (OPG) FIGS. 9A-C illustrate the optimization of the tryptase ELISA. FIG. 9A: Comparison of tryptase standard curve CXCL1 GRO1 GROC with different amount of capture antibody. FIG. 9B: Opti CXCL7:NAP-2 mization of detecting antibody dilutions. FIG.9C: Compari Growth Factor Epidermal growth factor (EGF) 55 son of tryptase standard curves in the presence of different Vascular endothelial growth factor (VEGF) Pigment epithelium-derived factor (PEDF) amounts of normal human serum (NHS). Brain-derived neurotrophic factor (BDNF) FIG. 10 illustrates tryptase levels in serum from healthy Schwannoma-derived growth factor (SDGF). controls (n=139) and from subjects with IBS (n=378). amphiregulin FIG. 11 illustrates the increased serum level of histamine Anti-neutrophil Anti-neutrophil cytoplasmic antibody (ANCA) antibody Perinuclear anti-neutrophil cytoplasmic antibody 60 and PGE in IBS patients versus healthy controls. (pANCA) FIG. 12 illustrates one embodiment of a molecular path ASCA ASCA-IgA way derived from the IBS markers identified and disclosed ASCA-IgG herein. Antimicrobial Anti-Outer membrane protein C (OmpC) antibody antibody Anti-Cbir-1 flagellin antibody FIG. 13 illustrates a disease classification system (DCS) Lipocalin Neutrophil gelatinase-associated lipocalin 65 according to one embodiment of the present invention. (NGAL) U.S. Patent Publication Nos. 2008/0085524, filed Aug. 14, 2007 and published Apr. 10, 2008; 2008/0166719, filed US 9,482,672 B2 7 8 Aug. 20, 2007, published Jul. 10, 2008, are herein incorpo certain diagnostic markers (e.g., B-tryptase, prostaglandin rated by reference in there entirety for all purposes. E, histamine, cytokines, growth factors, anti-neutrophil antibodies, anti-Saccharomyces cerevisiae antibodies, anti DETAILED DESCRIPTION OF THE microbial antibodies, lactoferrin, etc.), alone or in combi INVENTION nation with identifying the presence or severity of IBS related symptoms based upon the individual’s response to I. Introduction one or more questions (e.g., “Are you currently experiencing any symptoms?”). FIG. 12 shows a non-limiting example of Diagnosing a patient as having irritable bowel syndrome a molecular pathway derived from the IBS markers identi (IBS) can be challenging due to the similarity in symptoms 10 fied and disclosed herein. In some aspects, the present between IBS and other diseases or disorders. For example, invention uses statistical algorithms to aid in the classifica patients who have inflammatory bowel disease (IBD), but tion of a sample as an IBS sample or non-IBS sample. In who exhibit mild signs and symptoms such as bloating, other aspects, the present invention uses statistical algo diarrhea, constipation, and abdominal pain can be difficult to rithms for ruling out other intestinal disorders (e.g., IBD), distinguish from patients with IBS. As a result, the similarity 15 and then classifying the non-IBD sample to aid in the in symptoms between IBS and IBD renders rapid and classification of IBS. accurate diagnosis difficult and hampers early and effective treatment of the disease. II. Definitions Although the pathology of irritable bowel syndrome (IBS) is not completely understood, many studies have led to the As used herein, the following terms have the meanings hypothesis that IBS is a disorder caused by the dysregulation ascribed to them unless specified otherwise. of the brain-gut axis (for review, see, Ohman and Simrén, The term “classifying includes “to associate' or “to Nat Rev. Gastroenterol Hepatol. 2010 March; 7(3):163–73). categorize a sample with a disease state. In certain One observation that supports this theory is the repeated instances, “classifying is based on statistical evidence, finding that an increased number of mast cells can be found 25 empirical evidence, or both. In certain embodiments, the in the intestinal mucosa of patients diagnosed with IBS methods and systems of classifying use a so-called training (Guilarte, M. et al. Gut 56, 203-209 (2007); Walker, M. M. set of samples having known disease states. Once estab et al., Pharmacol. Ther: 29, 765-773 (2009); Akbar, A. et al. lished, the training data set serves as a basis, model, or Gut 57,923-929 (2008); Barbara, G. et al, Gastroenterology template against which the features of an unknown sample 126, 693-702 (2004); Barbara, G. et al. Gastroenterology 30 are compared, in order to classify the unknown disease state 132, 26-37 (2007); Cremon, C. et al. Am. J. Gastroenterol. of the sample. In certain instances, classifying the sample is 104, 392–400 (2009); and O'Sullivan, M. et al, Neurogas akin to diagnosing the disease state of the sample. In certain troenterol. Motil., 12, 449-457 (2000)). Similarly, some other instances, classifying the sample is akin to differenti studies have also found that levels of mediators released ating the disease state of the sample from another disease from these cells, including histamine and serine proteases 35 State. (e.g., tryptase), are found in the colonic mucosa of IBS The term “irritable bowel syndrome' or “IBS” includes a patients (Buhner et al., Gastroenterology 2009 October; group of functional bowel disorders characterized by one or 137(4); Barbara et al., Gastroenterology, Volume: 122, more symptoms including, but not limited to, abdominal Number: 4 Suppl. 1, Page: A-276, April 2002). However, pain, abdominal discomfort, change in bowel pattern, loose this finding remains controversial, as other studies have 40 or more frequent bowel movements, diarrhea, and consti reported that no such correlation exists (Guilarte et al., Gut pation, typically in the absence of any apparent structural 2007 February: 56(2):203-9). abnormality. There are at least three forms of IBS, depend Regardless of the existence of increased levels of mast ing on which symptom predominates: (1) diarrhea-predomi cell mediators in the colonic mucosa of IBS patients, such a nant (IBS-D); (2) constipation-predominant (IBS-C); and correlation has a limited diagnostic value since colonic 45 (3) IBS with alternating stool pattern (IBS-A). IBS can also biopsies are invasive procedures. While researchers have occur in the form of a mixture of symptoms (IBS-M). There looked for similar patterns in the blood/serum of IBS are also various clinical Subtypes of IBS. Such as post patients, fluids well Suited for non-invasive diagnostics, no infectious IBS (IBS-PI). correlation has been reported to date (Lessof et al., Ann The term “transforming the sample' includes a physical Allergy. 1983 August; 51(2 Pt 2):249-50; Guilarte, M. etal, 50 and/or chemical change of the sample to extract a marker or Gut 56, 203-209 (2007): Ohman and Simrén, Nat Rev to change or modify a marker as defined herein. An extrac Gastroenterol Hepatol. 2010 March; 7(3):163–73). tion, a manipulation, a chemical precipitation, an ELISA, a Advantageously, the present invention provides, among complexation, an immuno-extraction, a physical or chemical other aspects, non-invasive methods and assays for the modification of the sample or marker to measure a level or diagnosis and classification of irritable bowel syndrome. In 55 concentration of a marker all constitute a transformation. As certain embodiments, these methods and assays are related long as the sample or marker is not identical before and after to the detection of the presence or concentration level of the transformation step, the change or modification is a various IBS biomarkers in the blood and/or serum of Sub transformation. jects Suspected of, or previously diagnosed as having IBS. In The term 'sample includes any biological specimen preferred embodiments, the methods comprise the detection 60 obtained from an individual. Suitable samples for use in the off-tryptase, and/or histamine, and/or prostaglandin E in present invention include, without limitation, whole blood, blood and serum samples from a subject. Additional markers plasma, serum, saliva, urine, stool (i.e., feces), tears, and any can also be used. other bodily fluid, or a tissue sample (i.e., biopsy) Such as a The present invention is based, in part, upon the Surpris Small intestine or colon sample, and cellular extracts thereof ing discovery that the accuracy of classifying a biological 65 (e.g., red blood cellular extract). In a preferred embodiment, sample from an individual as an IBS sample can be Sub the sample is a blood, plasma, or serum sample. In a more stantially improved by detecting the presence or level of preferred embodiment, the sample is a serum sample. The US 9,482,672 B2 10 use of samples such as serum, saliva, and urine is well wise, a “symptom profile' can include a set of data that known in the art (see, e.g., Hashida et al., J. Clin. Lab. Anal., represents the presence, severity, frequency, and/or duration 11:267-86 (1997)). One skilled in the art will appreciate that of one or more symptoms associated with IBS and/or IBD. samples such as serum samples can be diluted prior to the In some embodiments, a panel for measuring one or more analysis of marker levels. of the diagnostic markers and/or diagnostic profiles The term “biomarker” or “marker includes any diagnos described above can be constructed and used for classifying tic marker Such as a biochemical marker, serological marker, the sample as an IBS sample or non-IBS sample. One skilled genetic marker, or other clinical or echographic character in the art will appreciate that the presence or level of a istic that can be used to classify a sample from an individual plurality of diagnostic markers can be determined simulta as an IBS sample or to rule out one or more diseases or 10 neously or sequentially, using, for example, an aliquot or disorders associated with IBS-like symptoms in a sample dilution of the individuals sample. In certain instances, the from an individual. The term “biomarker” or “marker” also level of a particular diagnostic marker in the individuals encompasses any classification marker Such as a biochemi sample is considered to be elevated when it is at least about cal marker, serological marker, genetic marker, or other 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, clinical or echographic characteristic that can be used to 15 175%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, classify IBS into one of its various forms or clinical sub 600%. 700%, 800%, 900%, or 1000% greater than the level types. Non-limiting examples of diagnostic markers Suitable of the same marker in a comparative sample (e.g., a normal, for use in the present invention are described below and GI control, IBD, and/or Celiac disease sample) or population include cytokines, growth factors, anti-neutrophil antibod of samples (e.g., greater than a median level of the same ies, anti-Saccharomyces cerevisiae antibodies, antimicrobial marker in a comparative population of normal, GI control, antibodies, anti-tissue transglutaminase (tTG) antibodies, IBD, and/or Celiac disease samples). In certain other lipocalins, matrix metalloproteinases (MMPs), tissue inhibi instances, the level of a particular diagnostic marker in the tor of metalloproteinases (TIMPs), alpha-globulins, actin individual’s sample is considered to be lowered when it is at severing proteins, S100 proteins, fibrinopeptides, calcitonin least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, gene-related peptide (CGRP), tachykinins, ghrelin, neuro 25 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or tensin, corticotropin-releasing hormone (CRH), serine pro 95% less than the level of the same marker in a comparative teases (e.g., tryptase, elastase), prostaglandin (e.g., PGE), sample (e.g., a normal, GI control, IBD, and/or Celiac histamine, C-reactive protein (CRP), lactoferrin, anti-lacto disease sample) or population of samples (e.g., less than a ferrin antibodies, calprotectin, hemoglobin, NOD2/ median level of the same marker in a comparative popula CARD15, serotonin reuptake transporter (SERT), trypto 30 tion of normal, GI control, IBD, and/or Celiac disease phan hydroxylase-1,5-hydroxytryptamine (5-HT), lactulose, samples). and the like. Examples of classification markers include, The term “individual,” “subject,” or “patient' typically without limitation, leptin, SERT, tryptophan hydroxylase-1, refers to humans, but also to other animals including, e.g., 5-HT, antrum mucosal protein 8, keratin-8, claudin-8, Zonu other primates, rodents, canines, felines, equines, ovines, lin, corticotropin releasing hormone receptor-1 (CRHR1). 35 porcines, and the like. corticotropin releasing hormone receptor-2 (CRHR2), As used herein, the term “substantially the same amino tryptase, histamine, prostaglandin E2 (PGE2) and the like. In acid sequence' includes an amino acid sequence that is Some embodiments, diagnostic markers can be used to similar, but not identical to, the naturally-occurring amino classify IBS into one of its various forms or clinical sub acid sequence. For example, an amino acid sequence that types. In other embodiments, classification markers can be 40 has substantially the same amino acid sequence as a natu used to classify a sample as an IBS sample or to rule out one rally-occurring peptide, polypeptide, or protein can have one or more diseases or disorders associated with IBS-like or more modifications such as amino acid additions, dele symptoms. One skilled in the art will know of additional tions, or Substitutions relative to the amino acid sequence of diagnostic and classification markers Suitable for use in the the naturally-occurring peptide, polypeptide, or protein, present invention. 45 provided that the modified sequence retains substantially at In certain instances, the presence or level of at least one least one biological activity of the naturally-occurring pep diagnostic marker is determined using an assay Such as a tide, polypeptide, or protein Such as immunoreactivity. hybridization assay or an amplification-based assay. Comparison for Substantial similarity between amino acid Examples of hybridization assays and amplification-based sequences is usually performed with sequences between assays Suitable for use in the methods of the present inven 50 about 6 and 100 residues, preferably between about 10 and tion are described above. In certain other instances, the 100 residues, and more preferably between about 25 and 35 presence or level of at least one diagnostic marker is residues. A particularly useful modification of a peptide, determined using an immunoassay or an immunohisto polypeptide, or protein of the present invention, or a frag chemical assay. Non-limiting examples of immunoassays ment thereof, is a modification that confers, for example, and immunohistochemical assays Suitable for use in the 55 increased Stability. Incorporation of one or more D-amino methods of the present invention are described herein. acids is a modification useful in increasing stability of a As used herein, the term “profile' includes any set of data polypeptide or polypeptide fragment. Similarly, deletion or that represents the distinctive features or characteristics substitution of lysine residues can increase stability by associated with a disease or disorder such as IBS or IBD. protecting the polypeptide or polypeptide fragment against The term encompasses a "diagnostic marker profile’ that 60 degradation. analyzes one or more diagnostic markers in a sample, a The term “monitoring the progression or regression of “symptom profile' that identifies one or more IBS-related IBS includes the use of the methods, systems, and code of clinical factors (i.e., symptoms) an individual is experienc the present invention to determine the disease state (e.g., ing or has experienced, and combinations thereof. For presence or severity of IBS) of an individual. In certain example, a "diagnostic marker profile' can include a set of 65 instances, the results of an algorithm (e.g., a learning sta data that represents the presence or level of one or more tistical classifier system) are compared to those results diagnostic markers associated with IBS and/or IBD. Like obtained for the same individual at an earlier time. In some US 9,482,672 B2 11 12 embodiments, the methods, systems, and code of the present 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, invention can be used to predict the progression of IBS, e.g., or more of the biomarkers shown in Table 1. In preferred by determining a likelihood for IBS to progress either embodiments, the methods provided herein rely on the rapidly or slowly in an individual based on an analysis of detection of at least one biomarker selected from B-tryptase, diagnostic markers and/or the identification or IBS-related 5 histamine, prostaglandin E. (PGE), and a combination symptoms. In other embodiments, the methods, systems, thereof. and code of the present invention can be used to predict the In certain embodiments, methods are provided for aiding regression of IBS, e.g., by determining a likelihood for IBS in the diagnosis of irritable bowel syndrome in a subject by to regress either rapidly or slowly in an individual based on determining the level of at least one biomarker selected from an analysis of diagnostic markers and/or the identification or 10 B-tryptase, histamine, prostaglandin E2 (PGE), and a com IBS-related symptoms. The term “monitoring drug efficacy in an individual bination thereof in conjunction with at least one biomarker receiving a drug useful for treating IBS includes the use of selected from the group consisting of Brain-Derived Neu the methods, systems, and code of the present invention to rotropic Factor (BDNF), Neutrophil Gelatinase-Associated determine the effectiveness of a therapeutic agent for treat 15 Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis ing IBS after it has been administered. In certain instances, (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), the results of an algorithm (e.g., a learning statistical clas Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro sifier system) are compared to those results obtained for the teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti same individual before initiation of use of the therapeutic body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti agent or at an earlier time in therapy. As used herein, a drug Human Neutrophil Cytoplasmic Antibody (ANCA), Anti useful for treating IBS is any compound or drug used to Human Tissue Transglutaminase IgA (tTG), and a improve the health of the individual and includes, without combination thereof. limitation, IBS drugs such as serotonergic agents, antide In yet other embodiments, methods are provided for pressants, chloride channel activators, chloride channel aiding in the diagnosis of irritable bowel syndrome in a blockers, guanylate cyclase agonists, antibiotics, opioids, 25 subject by determining the level of at least one biomarker neurokinin antagonists, antispasmodic or anticholinergic selected from B-tryptase, histamine, prostaglandin E agents, belladonna alkaloids, barbiturates, glucagon-like (PGE), and a combination thereof in conjunction with at peptide-1 (GLP-1) analogs, corticotropin releasing factor least one biomarker selected from the group consisting of a (CRF) antagonists, probiotics, free bases thereof, pharma cytokine (e.g., IL-8, IL-1 B, TWEAK, leptin, OPG, MIP-3|B, ceutically acceptable salts thereof, derivatives thereof, ana 30 GROC, CXCL4/PF-4, and/or CXCL7/NAP-2), growth fac logs thereof, and combinations thereof. tor (e.g., EGF, VEGF, PEDF, BDNF, and/or SDGF), anti The term “therapeutically effective amount or dose” neutrophilantibody (e.g., ANCA, p-ANCA, cANCA, NSNA, includes a dose of a drug that is capable of achieving a and/or SAPPA), ASCA (e.g., ASCA-IgA, ASCA-IgG, and/or therapeutic effect in a subject in need thereof. For example, ASCA-IgM), antimicrobial antibody (e.g., anti-OmpC anti a therapeutically effective amount of a drug useful for 35 body, anti-flagellin antibody, and/or anti-I2 antibody), lac treating IBS can be the amount that is capable of preventing toferrin, anti-tTG antibody, lipocalin (e.g., NGAL, NGAL/ or relieving one or more symptoms associated with IBS. The MMP-9 complex), MMP (e.g., MMP-9), TIMP (e.g., TIMP exact amount can be ascertainable by one skilled in the art 1), alpha-globulin (e.g., alpha-2-macroglobulin, using known techniques (see, e.g., Lieberman, Pharmaceu haptoglobin, and/or orosomucoid), actin-severing protein tical Dosage Forms, Vols. 1-3 (1992); Lloyd, The Art, 40 (e.g., gelsolin), S100 protein (e.g., calgranulin), fibrinopep Science and Technology of Pharmaceutical Compounding tide (e.g., FIBA), CGRP tachykinin (e.g., Substance P), (1999); Pickar, Dosage Calculations (1999); and Reming ghrelin, neurotensin, corticotropin-releasing hormone, and ton: The Science and Practice of Pharmacy, 20th Edition, combinations thereof. In yet other embodiments, the pres Gennaro, Ed., Lippincott, Williams & Wilkins (2003)). ence or level of other diagnostic markers such as, for 45 example, anti-lactoferrin antibody, L-selectin/CD62L, III. Description of the Embodiments elastase, C-reactive protein (CRP), calprotectin, anti-U1-70 kDa autoantibody, Zona occludens 1 (ZO-1), vasoactive The present invention provides methods, systems, and intestinal peptide (VIP), serum amyloid A, gastrin, and a code for aiding in the diagnosis of irritable bowel syndrome combination thereof may also be detected. in a Subject. Similarly, the present invention provides meth 50 In another aspect, the present invention provides a method ods, systems, and code for accurately classifying whether a for classifying whether a sample from an individual is sample from an individual is associated with irritable bowel associated with IBS, the method comprising: (a) determin syndrome (IBS). In some embodiments, the present inven ing a diagnostic marker profile by detecting the presence or tion relies on the use of a statistical algorithm (e.g., a level of at least one diagnostic marker in the sample; (b) learning statistical classifier system) and/or empirical data 55 classifying the sample as an IBD sample or non-IBD sample (e.g., the presence or level of an IBS marker). The present using a first statistical algorithm based upon the diagnostic invention is also useful for ruling out one or more diseases marker profile; and if the sample is classified as a non-IBD or disorders that present with IBS-like symptoms and ruling sample, (c) classifying the non-IBD Sample as an IBS in IBS using a combination of statistical algorithms and/or sample or non-IBS Sample using a second statistical algo empirical data. Accordingly, the present invention provides 60 rithm based upon the same diagnostic marker profile as an accurate diagnostic prediction of IBS and prognostic determined in step (a) or a different diagnostic marker information useful for guiding treatment decisions. profile. A. Methods for Aiding in the Diagnosis of Irritable Bowel The diagnostic markers used for ruling out IBD can be the Syndrome (IBS) same as the diagnostic markers used for ruling in IBS. In one aspect, the present invention provides methods for 65 Alternatively, the diagnostic markers used for ruling out IBD aiding in the diagnosis of irritable bowel syndrome in a can be different than the diagnostic markers used for ruling subject by determining the level of 1, 2, 3, 4, 5, 6, 7, 8, 9. in IBS. US 9,482,672 B2 13 14 In some embodiments, the method of first ruling out IBD Subject or an average level of B-tryptase present in blood or (i.e., classifying the sample as an IBD Sample or non-IBD serum samples from a cohort of healthy Subjects. In other sample) and then ruling in IBS (i.e., classifying the non-IBD embodiments, a control level is the level of B-tryptase sample as an IBS sample or non-IBS sample) comprises present in a blood or serum sample from a non-IBS subject determining a diagnostic marker profile optionally in com or an average level of B-tryptase present in blood or serum bination with a symptom profile, wherein the symptom samples from a cohort of non-IBS subjects. In another profile is determined by identifying the presence or severity embodiment, a control level is the level of B-tryptase present of at least one symptom in the individual; classifying the in a blood or serum sample from a diseased subject or an sample as an IBD Sample or non-IBD Sample using a first average level of B-tryptase present in blood or serum statistical algorithm based upon the diagnostic marker pro 10 samples from a cohort of diseased subjects. Non-limiting file and the symptom profile; and if the sample is classified examples of diseased subjects that are useful for determining as a non-IBD Sample, classifying the non-IBD sample as an a control level from include subjects with IBS, subjects with IBS sample or non-IBS Sample using a second statistical a non-IBS gastrointestinal disease, Subjects with inflamma algorithm based upon the same profiles as determined in step tory bowel disease (IBD), subjects with ulcerative colitis (a) or different profiles. One skilled in the art will appreciate 15 (UC), subjects with Crohn's disease (CD), subjects with that the diagnostic marker profile and the symptom profile celiac disease, Subjects with gastroesophageal reflux disease can be determined simultaneously or sequentially in any (GERD), subjects with cancer, subjects with a cancer of the order. gastrointestinal tract, Subjects with a cancer of the stomach, In additional embodiments, the methods of the present subjects with a cancer of the small or large bowel, and the invention further comprise ruling out intestinal inflamma like. tion. Non-limiting examples of intestinal inflammation In cases where the control level is a level of B-tryptase in include acute inflammation, diverticulitis, ileal pouch-anal a blood or serum sample of a healthy subject or healthy anastomosis, microscopic colitis, infectious diarrhea, and Subjects, an increased level off-tryptase present in a sample combinations thereof. In some instances, the intestinal from a subject, relative to the control level, is indicative of inflammation is ruled out based upon the presence or level 25 an increased likelihood of the subject having IBS. Con of C-reactive protein (CRP), lactoferrin, calprotectin, or versely, where the control is a healthy subject or subjects, a combinations thereof. similar or reduced level of B-tryptase present in a sample As described herein, the methods of the present invention from a subject, relative to the control level, is indicative of can further comprise sending the IBS classification results to an increased likelihood of the subject not having IBS. a clinician, e.g., a gastroenterologist or a general practitio 30 In cases where the control level is a level of B-tryptase in ner. The methods can also provide a diagnosis in the form of a blood or serum sample of a subject or subjects with IBS, a probability that the individual has IBS. For example, the a similar or increased level of B-tryptase present in a sample individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, from a subject, relative to the control level, is indicative of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, an increased likelihood of the subject having IBS. Con 80%, 85%, 90%, 95%, or greater probability of having IBS. 35 versely, where the control is a subject or subjects with IBS, In some instances, the methods of the present invention a reduced level of B-tryptase present in a sample from a further provide a prognosis of IBS in the individual. For subject, relative to the control level, is indicative of an example, the prognosis can be surgery, development of a increased likelihood of the subject not having IBS. category or clinical subtype of IBS, development of one or In certain aspects of the invention, methods are provided more symptoms, or recovery from the disease. 40 for aiding in the diagnosis of irritable bowel syndrome (IBS) 1. B-Tryptase in a subject comprising the detection off-tryptase in a blood In one aspect, a method for aiding in the diagnosis of or serum sample and at least one additional blood or serum irritable bowel syndrome (IBS) in a subject is provided, the biomarker selected from histamine and prostaglandin E method comprising: (a) contacting a blood or serum sample (PGE). In one embodiment, the method comprises detect from the Subject with a B-tryptase binding moiety under 45 ing or determining the level of 3-tryptase and histamine conditions Suitable to transform B-tryptase present in the from a blood or serum sample from a subject. In another sample into a complex comprising B-tryptase and the embodiment, the method comprises detecting or determin B-tryptase binding moiety; and (b) determining the level of ing the level of B-tryptase and PGE from a blood or serum the complex, thereby determining the level of B-tryptase sample from a subject. In a third embodiment, the method present in the sample. In one embodiment, the method 50 comprises detecting or determining the level of B-tryptase, further comprises: (c) comparing the level of B-tryptase histamine, and PGE from a blood or serum sample from a present in the sample to a control level, wherein a difference subject. Preferably, B-tryptase, histamine, and/or PGE are in the level of B-tryptase present in the sample relative to the detected from the same blood or serum sample, although in control level is indicative of an increased likelihood of the certain instances the biomarkers may be detected in different subject having IBS. 55 blood or serum samples taken from the same individual, for In a specific embodiment, the present invention provides example, at the same time or at different times. In certain an assay to aid in the diagnosis of IBS, the assay comprising: embodiments, the biomarkers may be detected in separate (a) contacting a sample having B-tryptase contained therein assays performed with different aliquots of a blood or serum under conditions Suitable to transform the B-tryptase into a sample from a subject. In other embodiments, the biomark complex comprising B-tryptase and a capture anti-tryptase 60 ers may be detected in a single multiplex detection assay, for antibody; (b) contacting the complex with an enzyme example, in a LumineXC XMAPC) assay. labeled indicator antibody to transform the complex into a In yet other aspects of the invention, methods are pro labeled complex; (c) contacting the labeled complex with a vided for aiding in the diagnosis of irritable bowel syndrome Substrate for the enzyme; and (d) detecting the presence or (IBS) in a subject comprising the detection of B-tryptase in level of B-tryptase in the sample. 65 a blood or serum sample and at least one additional bio In certain embodiments, a control level is the level of marker selected from Brain-Derived Neurotropic Factor B-tryptase present in a blood or serum sample from a healthy (BDNF), Neutrophil Gelatinase-Associated Lipocalin US 9,482,672 B2 15 16 (NGAL). TNF-related Weak Inducer of Apoptosis sample and Interleukin-1 Beta (IL-1B) from a biological (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), sample. In a preferred embodiment, Interleukin-1 Beta (IL Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro 1B) is detected in a blood or serum sample from the subject. teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti In another preferred embodiment, Interleukin-1 Beta (IL-1B) body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti is detected in a stool sample or a biopsy from the bowel of Human Neutrophil Cytoplasmic Antibody (ANCA), Anti the subject. In other embodiments, at least one additional Human Tissue Transglutaminase IgA (tTG), and a biomarker is also detected. combination thereof. In certain embodiments, at least 2 of In one embodiment, the method comprises detecting or the additional biomarkers may be detected. In other embodi determining the level of B-tryptase from a blood or serum ments, at least 3, 4, 5, 6, 7, 8, 9, or 10 of the additional 10 sample and Tissue Inhibitor of Metalloproteinase-1 (TIMP biomarkers may be detected. 1) from a biological sample. In a preferred embodiment, The sample used for detecting or determining the pres Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) is ence or level of at least one diagnostic marker is typically detected in a blood or serum sample from the subject. In whole blood, plasma, serum, saliva, urine, Stool (i.e., feces), another preferred embodiment, Tissue Inhibitor of Metallo tears, and any other bodily fluid, or a tissue sample (i.e., 15 proteinase-1 (TIMP-1) is detected in a stool sample or a biopsy) Such as a small intestine or colon sample. Preferably, biopsy from the bowel of the subject. In other embodiments, the sample is serum, whole blood, plasma, stool, urine, or a at least one additional biomarker is also detected. tissue biopsy. In certain instances, the methods of the present In one embodiment, the method comprises detecting or invention further comprise obtaining the sample from the determining the level of B-tryptase from a blood or serum individual prior to detecting or determining the presence or sample and Anti-Saccharomyces cerevisiae Antibody level of at least one diagnostic marker in the sample. In a (ASCA-IgA) from a biological sample. In a preferred preferred embodiment, the additional biomarker may be embodiment, Anti-Saccharomyces cerevisiae Antibody detected from a blood or serum sample. In other embodi (ASCA-IgA) is detected in a blood or serum sample from the ments, the additional biomarker may be detected from a subject. In another preferred embodiment, Anti-Saccharo stool sample or a biopsy from the bowel of the subject. 25 myces cerevisiae Antibody (ASCA-IgA) is detected in a In one embodiment, the method comprises detecting or stool sample or a biopsy from the bowel of the subject. In determining the level of B-tryptase from a blood or serum other embodiments, at least one additional biomarker is also sample and Brain-Derived Neurotropic Factor (BDNF) from detected. a biological sample. In a preferred embodiment, Brain In one embodiment, the method comprises detecting or Derived Neurotropic Factor (BDNF) is detected in a blood 30 determining the level of B-tryptase from a blood or serum or serum sample from the subject. In another preferred sample and Anti-CBir-1 Antibody (CBirl) from a biological embodiment, Brain-Derived Neurotropic Factor (BDNF) is sample. In a preferred embodiment, Anti-CBir-1 Antibody detected in a stool sample or a biopsy from the bowel of the (CBirl) is detected in a blood or serum sample from the subject. In other embodiments, at least one additional bio subject. In another preferred embodiment, Anti-CBir-1 Anti marker is also detected. 35 body (CBirl) is detected in a stool sample or a biopsy from In one embodiment, the method comprises detecting or the bowel of the subject. In other embodiments, at least one determining the level of B-tryptase from a blood or serum additional biomarker is also detected. sample and Neutrophil Gelatinase-Associated Lipocalin In one embodiment, the method comprises detecting or (NGAL) from a biological sample. In a preferred embodi determining the level of B-tryptase from a blood or serum ment, Neutrophil Gelatinase-Associated Lipocalin (NGAL) 40 sample and Anti-Human Neutrophil Cytoplasmic Antibody is detected in a blood or serum sample from the subject. In (ANCA) from a biological sample. In a preferred embodi another preferred embodiment, Neutrophil Gelatinase-As ment, Anti-Human Neutrophil Cytoplasmic Antibody sociated Lipocalin (NGAL) is detected in a stool sample or (ANCA) is detected in a blood or serum sample from the a biopsy from the bowel of the subject. In other embodi subject. In another preferred embodiment, Anti-Human ments, at least one additional biomarker is also detected. 45 Neutrophil Cytoplasmic Antibody (ANCA) is detected in a In one embodiment, the method comprises detecting or stool sample or a biopsy from the bowel of the subject. In determining the level of B-tryptase from a blood or serum other embodiments, at least one additional biomarker is also sample and TNF-related Weak Inducer of Apoptosis detected. (TWEAK) from a biological sample. In a preferred embodi In one embodiment, the method comprises detecting or ment, TNF-related Weak Inducer of Apoptosis (TWEAK) is 50 determining the level of B-tryptase from a blood or serum detected in a blood or serum sample from the subject. In sample and Anti-Human Tissue Transglutaminase IgA (tTG) another preferred embodiment, TNF-related Weak Inducer from a biological sample. In a preferred embodiment, Anti of Apoptosis (TWEAK) is detected in a stool sample or a Human Tissue Transglutaminase IgA (tTG) is detected in a biopsy from the bowel of the subject. In other embodiments, blood or serum sample from the subject. In another preferred at least one additional biomarker is also detected. 55 embodiment, Anti-Human Tissue Transglutaminase IgA In one embodiment, the method comprises detecting or (tTG) is detected in a stool sample or a biopsy from the determining the level of B-tryptase from a blood or serum bowel of the subject. In other embodiments, at least one sample and Growth-Related Oncogene Alpha (GRO-O.) additional biomarker is also detected. from a biological sample. In a preferred embodiment, In one embodiment, the method comprises detecting or Growth-Related Oncogene Alpha (GRO-O.) is detected in a 60 determining the level of B-tryptase from a blood or serum blood or serum sample from the subject. In another preferred sample and all of Brain-Derived Neurotropic Factor embodiment, Growth-Related Oncogene Alpha (GRO-O.) is (BDNF), Neutrophil Gelatinase-Associated Lipocalin detected in a stool sample or a biopsy from the bowel of the (NGAL). TNF-related Weak Inducer of Apoptosis subject. In other embodiments, at least one additional bio (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), marker is also detected. 65 Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro In one embodiment, the method comprises detecting or teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti determining the level of B-tryptase from a blood or serum body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti US 9,482,672 B2 17 18 Human Neutrophil Cytoplasmic Antibody (ANCA), and fying the presence or severity of at least one symptom in the Anti-Human Tissue Transglutaminase IgA (tTG) from a Subject; and (e) diagnosing the Subject as having IBS or not blood or serum sample, a stool sample, or a biopsy from the having IBS using an algorithm based upon the level of an bowel of the subject. IBS biomarker and the system profile. In a preferred In certain embodiments, the presence or level of at least embodiment, the method for aiding in the diagnosis of one diagnostic marker is determined using an assay Such as irritable bowel syndrome (IBS) in a subject comprises a hybridization assay or an amplification-based assay. determining a diagnostic marker profile, for example for Examples of hybridization assays suitable for use in the B-tryptase or a combination thereof as described herein, methods of the present invention include, but are not limited optionally in combination with a symptom profile, wherein to, Northern blotting, dot blotting, RNase protection, and a 10 the symptom profile is determined by identifying the pres combination thereof. A non-limiting example of an ampli ence or severity of at least one symptom in the individual; fication-based assay suitable for use in the methods of the and classifying the sample as an IBS sample or non-IBS present invention includes a reverse transcriptase-poly sample using an algorithm based upon the diagnostic marker merase chain reaction (RT-PCR). profile and the symptom profile. In certain other embodiments, the presence or level of at 15 The symptom profile is typically determined by identify least one diagnostic marker is determined using an immu ing the presence or severity of at least one symptom selected noassay or an immunohistochemical assay. A non-limiting from the group consisting of chest pain, chest discomfort, example of an immunoassay Suitable for use in the methods heartburn, uncomfortable fullness after having a regular of the present invention includes an enzyme-linked immu sized meal, inability to finish a regular-sized meal, abdomi nosorbent assay (ELISA). Examples of immunohistochemi nal pain, abdominal discomfort, constipation, diarrhea, cal assays Suitable for use in the methods of the present bloating, abdominal distension, negative thoughts or feel invention include, but are not limited to, immunofluores ings associated with having pain or discomfort, and combi cence assays Such as direct fluorescent antibody assays, nations thereof. indirect fluorescent antibody (IFA) assays, anticomplement In preferred embodiments, the presence or severity of 1. immunofluorescence assays, and avidin-biotin immunofluo 25 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the symptoms described rescence assays. Other types of immunohistochemical herein is identified to generate a symptom profile that is assays include immunoperoxidase assays. useful for predicting IBS. In certain instances, a question In a preferred embodiment, the presence or level of naire or other form of written, verbal, or telephone survey is B-tryptase is determined using a sandwich enzyme-linked used to produce the symptom profile. The questionnaire or immunosorbent assay (ELISA). Any suitable antibody pair 30 Survey typically comprises a standardized set of questions may be used for the capture and detecting antibodies in a and answers for the purpose of gathering information from sandwich ELISA. One of skill in the art will know and respondents regarding their current and/or recent IBS-re appreciate how to select an appropriate antibody pair for the lated symptoms. assay. Generally, two antibodies are selected that bind to the In some embodiments, the symptom profile is produced target of interest, e.g., B-tryptase, at different epitopes such 35 by compiling and/or analyzing all or a Subset of the answers that the binding of the first (capture) antibody does not to the questions set forth in the questionnaire or Survey. In interfere with the second (detecting) antibody. In certain other embodiments, the symptom profile is produced based embodiments, the detecting antibody will be conjugated to upon the individual’s response to the following question: an enzyme, for example, alkaline phophatase, to aid in the “Are you currently experiencing any symptoms?’ The detection of the complex. In other embodiments, a second 40 symptom profile generated in accordance with either of ary antibody conjugated to an enzyme (e.g., alkaline these embodiments can be used in combination with a phophatase), which binds to the detecting antibody, may be diagnostic marker profile in the algorithmic-based methods used in the assay. Generally, the complex will then be described herein to improve the accuracy of predicting IBS. detected by the use of a luminescent Substrate, for example, In some embodiments, the methods provided herein fur Ultra LITETM (NAG Research Laboratories); SensoLyte(R) 45 ther include providing a probability that a subject has IBS. (AnaSpec); SuperSignal ELISA Femto Maximum Sensitiv In certain embodiments, the method may comprise provid ity Substrate (Thermo Scientific); SuperSignal ELISA Pico ing a probability that the is subject highly unlikely, unlikely, Chemiluminescent Substrate (Thermo Scientific); CPSD likely, or highly likely has IBS. In related embodiments, (disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'- methods are provided that further include providing a prob chloro)tricyclo[3.3.1.13.7 decan-4-yl)phenyl phosphate: 50 ability that a sample classified as an IBS sample or a Tropix, Inc). In a preferred embodiment, the B-tryptase non-IBS sample is from a subject with IBS. In certain sandwich ELISA comprises the use of an alkaline phos embodiments, the method may comprise providing a prob phatase conjugated anti-tryptase antibody as the detecting ability that the is sample is from a subject that highly antibody and a CPSD containing luminescent substrate to unlikely, unlikely, likely, or highly likely has IBS. enhance the assay sensitivity. The CPSD substrate can be 55 In other embodiments, the methods provided herein fur found in chemiluminescent detection systems, such as the ther include classifying a diagnosis of IBS as IBS-consti ELISA-LightTM System (Applied Biosystems). In a particu pation (IBS-C), IBS-diarrhea (IBS-D), IBS-mixed (IBS-M), larly preferred embodiment, the detection antibody used in IBS-alternating (IBS-A), or post-infectious IBS (IBS-PI). In the sandwich ELISA is the anti-tryptase antibody G3 (sc related embodiments, methods are provided herein to further 33676; Santa Cruz, Biotechnology, Inc.; Santa Cruz, Calif.). 60 classify a sample as an IBS-constipation (IBS-C), IBS In some embodiments, the methods provided herein fur diarrhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating ther include the step of determining a symptom profile for (IBS-A), or post-infectious IBS (IBS-PI) sample. the subject, wherein the symptom profile is determined by In one embodiment, the present invention provides an identifying the presence or severity of at least one symptom assay to aid in the differentiation of IBS-D and IBS-A from in the subject. In a preferred embodiment, the method 65 IBS-C, the assay comprising: (a) contacting a sample having comprises: (d) determining a symptom profile for the Sub B-tryptase contained therein under conditions suitable to ject, wherein the symptom profile is determined by identi transform the B-tryptase into a complex comprising US 9,482,672 B2 19 20 B-tryptase and a capture anti-tryptase antibody; (b) contact examples of diseased subjects that are useful for determining ing the complex with an enzyme labeled indicator antibody a control level from include subjects with IBS, subjects with to transform the complex into a labeled complex; (c) con a non-IBS gastrointestinal disease, Subjects with inflamma tacting the labeled complex with a substrate for the enzyme; tory bowel disease (IBD), subjects with ulcerative colitis and (d) detecting the presence or level of B-tryptase in the (UC), subjects with Crohn's disease (CD), subjects with sample. celiac disease, Subjects with gastroesophageal reflux disease In some instances, the assay to aid in the differentiation of (GERD), subjects with cancer, subjects with a cancer of the IBS-D and IBS-A from IBS-C further comprises detecting gastrointestinal tract, Subjects with a cancer of the stomach, the presence or level of a prostaglandin (e.g., PGE); and/or subjects with a cancer of the small or large bowel, and the histamine in the sample. 10 like. In yet other embodiments, methods provided herein fur In cases where the control level is a level of histamine in ther include diagnosing a Subject not having IBS as having a blood or serum sample of a healthy subject or healthy IBD, as not having IBD, as having celiac disease, as not Subjects, an increased level of histamine present in a sample having celiac disease, as being a healthy Subject, or as not from a subject, relative to the control level, is indicative of having a gastrointestinal disease. In a related embodiment, 15 an increased likelihood of the subject having IBS. Con methods are provided herein that further classify a non-IBS versely, where the control is a healthy subject or subjects, a sample as an IBD Sample, a non-IBD sample, a healthy similar or reduced level of histamine present in a sample sample, a non-gastrointestinal disease sample, a celiac from a subject, relative to the control level, is indicative of sample, and the like. an increased likelihood of the subject not having IBS. In one embodiment, the methods provided herein com In cases where the control level is a level of histamine in prise the use of an algorithm based upon the level of an IBS a blood or serum sample of a subject or subjects with IBS, biomarker. In certain embodiments, the algorithm is further a similar or increased level of histamine present in a sample based upon the system profile. In a preferred embodiment, from a subject, relative to the control level, is indicative of the algorithm comprises a statistical algorithm, for example an increased likelihood of the subject having IBS. Con a learning statistical classifier system. In a more preferred 25 versely, where the control is a subject or subjects with IBS, embodiment, the algorithm comprises a combination of at a reduced level of histamine present in a sample from a least two learning statistical classifier systems. In a most subject, relative to the control level, is indicative of an preferred embodiment, the combination of at least two increased likelihood of the subject not having IBS. learning statistical classifier systems comprises a random In certain aspects of the invention, methods are provided forest classifier and a neural network classifier. 30 for aiding in the diagnosis of irritable bowel syndrome (IBS) 2. Histamine in a subject comprising the detection of histamine in a blood In one specific aspect, a method for aiding in the diagnosis or serum sample and at least one additional blood or serum of irritable bowel syndrome (IBS) in a subject is provided, biomarker selected from prostaglandin E. (PGE) and the method comprising: (a) contacting a blood or serum B-tryptase. In one embodiment, the method comprises sample from the Subject with a histamine binding moiety 35 detecting or determining the level of histamine and prosta under conditions suitable to transform histamine present in glandin E. (PGE) from a blood or serum sample from a the sample into a complex comprising histamine and the Subject. In another embodiment, the method comprises histamine binding moiety; and (b) determining the level of detecting or determining the level of histamine and the complex, thereby determining the level of histamine B-tryptase from a blood or serum sample from a Subject. In present in the sample. In one embodiment, the method 40 a third embodiment, the method comprises detecting or further comprises: (c) comparing the level of histamine determining the level of histamine, prostaglandin E. (PGE), present in the sample to a control level, wherein a difference and B-tryptase from a blood or serum sample from a Subject. in the level of histamine present in the sample relative to the Preferably, histamine, prostaglandin E. (PGE), and/or control level is indicative of an increased likelihood of the B-tryptase are detected from the same blood or serum subject having IBS. 45 sample, although in certain instances the biomarkers may be In a specific embodiment, the present invention provides detected in different blood or serum samples taken from the an assay to aid in the diagnosis of IBS, the assay comprising: same individual, for example, at the same time or at different (a) contacting a sample having histamine contained therein times. In certain embodiments, the biomarkers may be under conditions suitable to transform the histamine into a detected in separate assays performed with different aliquots complex comprising histamine and a capture anti-tryptase 50 of a blood or serum sample from a subject. In other antibody; (b) contacting the complex with an enzyme embodiments, the biomarkers may be detected in a single labeled indicator antibody to transform the complex into a multiplex detection assay, for example, in a LumineXC) labeled complex; (c) contacting the labeled complex with a XMAPC) assay. Substrate for the enzyme; and (d) detecting the presence or In yet other aspects of the invention, methods are pro level of histamine in the sample. 55 vided for aiding in the diagnosis of irritable bowel syndrome In certain embodiments, a control level is the level of (IBS) in a subject comprising the detection of histamine in histamine present in a blood or serum sample from a healthy a blood or serum sample and at least one additional bio Subject or an average level of histamine present in blood or marker selected from Brain-Derived Neurotropic Factor serum samples from a cohort of healthy Subjects. In other (BDNF), Neutrophil Gelatinase-Associated Lipocalin embodiments, a control level is the level of histamine 60 (NGAL). TNF-related Weak Inducer of Apoptosis present in a blood or serum sample from a non-IBS subject (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), or an average level of histamine present in blood or serum Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro samples from a cohort of non-IBS subjects. In another teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti embodiment, a control level is the level of histamine present body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti in a blood or serum sample from a diseased subject or an 65 Human Neutrophil Cytoplasmic Antibody (ANCA), Anti average level of histamine present in blood or serum Human Tissue Transglutaminase IgA (tTG), and a samples from a cohort of diseased subjects. Non-limiting combination thereof. In certain embodiments, at least 2 of US 9,482,672 B2 21 22 the additional biomarkers may be detected. In other embodi In one embodiment, the method comprises detecting or ments, at least 3, 4, 5, 6, 7, 8, 9, or 10 of the additional determining the level of histamine from a blood or serum biomarkers may be detected. sample and Tissue Inhibitor of Metalloproteinase-1 (TIMP The sample used for detecting or determining the pres 1) from a biological sample. In a preferred embodiment, ence or level of at least one diagnostic marker is typically Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) is whole blood, plasma, serum, saliva, urine, Stool (i.e., feces), detected in a blood or serum sample from the subject. In tears, and any other bodily fluid, or a tissue sample (i.e., another preferred embodiment, Tissue Inhibitor of Metallo biopsy) Such as a small intestine or colon sample. Preferably, proteinase-1 (TIMP-1) is detected in a stool sample or a the sample is serum, whole blood, plasma, stool, urine, or a biopsy from the bowel of the subject. In other embodiments, 10 at least one additional biomarker is also detected. tissue biopsy. In certain instances, the methods of the present In one embodiment, the method comprises detecting or invention further comprise obtaining the sample from the determining the level of histamine from a blood or serum individual prior to detecting or determining the presence or sample and Anti-Saccharomyces cerevisiae Antibody level of at least one diagnostic marker in the sample. In a (ASCA-IgA) from a biological sample. In a preferred preferred embodiment, the additional biomarker may be 15 embodiment, Anti-Saccharomyces cerevisiae Antibody detected from a blood or serum sample. In other embodi (ASCA-IgA) is detected in a blood or serum sample from the ments, the additional biomarker may be detected from a subject. In another preferred embodiment, Anti-Saccharo stool sample or a biopsy from the bowel of the subject. myces cerevisiae Antibody (ASCA-IgA) is detected in a In one embodiment, the method comprises detecting or stool sample or a biopsy from the bowel of the subject. In determining the level of histamine from a blood or serum other embodiments, at least one additional biomarker is also sample and Brain-Derived Neurotropic Factor (BDNF) from detected. a biological sample. In a preferred embodiment, Brain In one embodiment, the method comprises detecting or Derived Neurotropic Factor (BDNF) is detected in a blood determining the level of histamine from a blood or serum or serum sample from the subject. In another preferred sample and Anti-CBir-1 Antibody (CBirl) from a biological embodiment, Brain-Derived Neurotropic Factor (BDNF) is 25 sample. In a preferred embodiment, Anti-CBir-1 Antibody detected in a stool sample or a biopsy from the bowel of the (CBirl) is detected in a blood or serum sample from the subject. In other embodiments, at least one additional bio subject. In another preferred embodiment, Anti-CBir-1 Anti marker is also detected. body (CBirl) is detected in a stool sample or a biopsy from In one embodiment, the method comprises detecting or the bowel of the subject. In other embodiments, at least one determining the level of histamine from a blood or serum 30 additional biomarker is also detected. sample and Neutrophil Gelatinase-Associated Lipocalin In one embodiment, the method comprises detecting or (NGAL) from a biological sample. In a preferred embodi determining the level of histamine from a blood or serum ment, Neutrophil Gelatinase-Associated Lipocalin (NGAL) sample and Anti-Human Neutrophil Cytoplasmic Antibody is detected in a blood or serum sample from the subject. In (ANCA) from a biological sample. In a preferred embodi another preferred embodiment, Neutrophil Gelatinase-As 35 ment, Anti-Human Neutrophil Cytoplasmic Antibody sociated Lipocalin (NGAL) is detected in a stool sample or (ANCA) is detected in a blood or serum sample from the a biopsy from the bowel of the subject. In other embodi subject. In another preferred embodiment, Anti-Human ments, at least one additional biomarker is also detected. Neutrophil Cytoplasmic Antibody (ANCA) is detected in a In one embodiment, the method comprises detecting or stool sample or a biopsy from the bowel of the subject. In determining the level of histamine from a blood or serum 40 other embodiments, at least one additional biomarker is also sample and TNF-related Weak Inducer of Apoptosis detected. (TWEAK) from a biological sample. In a preferred embodi In one embodiment, the method comprises detecting or ment, TNF-related Weak Inducer of Apoptosis (TWEAK) is determining the level of histamine from a blood or serum detected in a blood or serum sample from the subject. In sample and Anti-Human Tissue Transglutaminase IgA (tTG) another preferred embodiment, TNF-related Weak Inducer 45 from a biological sample. In a preferred embodiment, Anti of Apoptosis (TWEAK) is detected in a stool sample or a Human Tissue Transglutaminase IgA (tTG) is detected in a biopsy from the bowel of the subject. In other embodiments, blood or serum sample from the subject. In another preferred at least one additional biomarker is also detected. embodiment, Anti-Human Tissue Transglutaminase IgA In one embodiment, the method comprises detecting or (tTG) is detected in a stool sample or a biopsy from the determining the level of histamine from a blood or serum 50 bowel of the subject. In other embodiments, at least one sample and Growth-Related Oncogene Alpha (GRO-O.) additional biomarker is also detected. from a biological sample. In a preferred embodiment, In one embodiment, the method comprises detecting or Growth-Related Oncogene Alpha (GRO-O.) is detected in a determining the level of histamine from a blood or serum blood or serum sample from the subject. In another preferred sample and all of Brain-Derived Neurotropic Factor embodiment, Growth-Related Oncogene Alpha (GRO-O.) is 55 (BDNF), Neutrophil Gelatinase-Associated Lipocalin detected in a stool sample or a biopsy from the bowel of the (NGAL). TNF-related Weak Inducer of Apoptosis subject. In other embodiments, at least one additional bio (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), marker is also detected. Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro In one embodiment, the method comprises detecting or teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti determining the level of histamine from a blood or serum 60 body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti sample and Interleukin-1 Beta (IL-1B) from a biological Human Neutrophil Cytoplasmic Antibody (ANCA), and sample. In a preferred embodiment, Interleukin-1 Beta (IL Anti-Human Tissue Transglutaminase IgA (tTG) from a 1B) is detected in a blood or serum sample from the subject. blood or serum sample, a stool sample, or a biopsy from the In another preferred embodiment, Interleukin-1 Beta (IL-1B) bowel of the subject. is detected in a stool sample or a biopsy from the bowel of 65 In certain embodiments, the presence or level of at least the subject. In other embodiments, at least one additional one diagnostic marker is determined using an assay Such as biomarker is also detected. a hybridization assay or an amplification-based assay. US 9,482,672 B2 23 24 Examples of hybridization assays suitable for use in the these embodiments can be used in combination with a methods of the present invention include, but are not limited diagnostic marker profile in the algorithmic-based methods to, Northern blotting, dot blotting, RNase protection, and a described herein to improve the accuracy of predicting IBS. combination thereof. A non-limiting example of an ampli In some embodiments, the methods provided herein fur fication-based assay suitable for use in the methods of the ther include providing a probability that a subject has IBS. present invention includes a reverse transcriptase-poly In certain embodiments, the method may comprise provid merase chain reaction (RT-PCR). ing a probability that the Subject is highly unlikely, unlikely, In certain other embodiments, the presence or level of at likely, or highly likely has IBS. In related embodiments, least one diagnostic marker is determined using an immu methods are provided that further include providing a prob noassay or an immunohistochemical assay. A non-limiting 10 ability that a sample classified as an IBS sample or a example of an immunoassay Suitable for use in the methods non-IBS sample is from a subject with IBS. In certain of the present invention includes an enzyme-linked immu embodiments, the method may comprise providing a prob nosorbent assay (ELISA). Examples of immunohistochemi ability that the sample is from a subject that highly unlikely, cal assays Suitable for use in the methods of the present unlikely, likely, or highly likely has IBS. invention include, but are not limited to, immunofluores 15 In other embodiments, the methods provided herein fur cence assays Such as direct fluorescent antibody assays, ther include classifying a diagnosis of IBS as IBS-consti indirect fluorescent antibody (IFA) assays, anticomplement pation (IBS-C), IBS-diarrhea (IBS-D), IBS-mixed (IBS-M), immunofluorescence assays, and avidin-biotin immunofluo IBS-alternating (IBS-A), or post-infectious IBS (IBS-PI). In rescence assays. Other types of immunohistochemical related embodiments, methods are provided herein to further assays include immunoperoxidase assays. classify a sample as an IBS-constipation (IBS-C), IBS In some embodiments, the methods provided herein fur diarrhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating ther include the step of determining a symptom profile for (IBS-A), or post-infectious IBS (IBS-PI) sample. the subject, wherein the symptom profile is determined by In one embodiment, the present invention provides an identifying the presence or severity of at least one symptom assay to aid in the differentiation of IBS-D and IBS-A from in the subject. In a preferred embodiment, the method 25 IBS-C, the assay comprising: (a) contacting a sample having comprises: (d) determining a symptom profile for the Sub histamine contained therein under conditions suitable to ject, wherein the symptom profile is determined by identi transform the histamine into a complex comprising hista fying the presence or severity of at least one symptom in the mine and a capture anti-histamine antibody; (b) contacting Subject; and (e) diagnosing the Subject as having IBS or not the complex with an enzyme labeled indicator antibody to having IBS using an algorithm based upon the level of an 30 transform the complex into a labeled complex; (c) contact IBS biomarker and the system profile. In a preferred ing the labeled complex with a substrate for the enzyme; and embodiment, the method for aiding in the diagnosis of (d) detecting the presence or level of histamine in the irritable bowel syndrome (IBS) in a subject comprises sample. determining a diagnostic marker profile, for example for In some instances, the assay to aid in the differentiation of histamine or a combination thereof as described herein, 35 IBS-D and IBS-A from IBS-C further comprises detecting optionally in combination with a symptom profile, wherein the presence or level of B-tryptase; and/or prostaglandin E the symptom profile is determined by identifying the pres (PGE) in the sample. ence or severity of at least one symptom in the individual; In yet other embodiments, methods provided herein fur and classifying the sample as an IBS sample or non-IBS ther include diagnosing a subject not having IBS as having sample using an algorithm based upon the diagnostic marker 40 IBD, as not having IBD, as having celiac disease, as not profile and the symptom profile. having celiac disease, as being a healthy Subject, or as not The symptom profile is typically determined by identify having a gastrointestinal disease. In a related embodiment, ing the presence or severity of at least one symptom selected methods are provided herein that further classify a non-IBS from the group consisting of chest pain, chest discomfort, sample as an IBD Sample, a non-IBD sample, a healthy heartburn, uncomfortable fullness after having a regular 45 sample, a non-gastrointestinal disease sample, a celiac sized meal, inability to finish a regular-sized meal, abdomi sample, and the like. nal pain, abdominal discomfort, constipation, diarrhea, In one embodiment, the methods provided herein com bloating, abdominal distension, negative thoughts or feel prise the use of an algorithm based upon the level of an IBS ings associated with having pain or discomfort, and combi biomarker. In certain embodiments, the algorithm is further nations thereof. 50 based upon the system profile. In a preferred embodiment, In preferred embodiments, the presence or severity of 1. the algorithm comprises a statistical algorithm, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the symptoms described a learning statistical classifier system. In a more preferred herein is identified to generate a symptom profile that is embodiment, the algorithm comprises a combination of at useful for predicting IBS. In certain instances, a question least two learning statistical classifier systems. In a most naire or other form of written, verbal, or telephone survey is 55 preferred embodiment, the combination of at least two used to produce the symptom profile. The questionnaire or learning statistical classifier systems comprises a random Survey typically comprises a standardized set of questions forest classifier and a neural network classifier. and answers for the purpose of gathering information from 3. Prostaglandin E. (PGE) respondents regarding their current and/or recent IBS-re In one specific aspect, a method for aiding in the diagnosis lated symptoms. 60 of irritable bowel syndrome (IBS) in a subject is provided, In some embodiments, the symptom profile is produced the method comprising: (a) contacting a blood or serum by compiling and/or analyzing all or a Subset of the answers sample from the Subject with a prostaglandin E. (PGE2) to the questions set forth in the questionnaire or Survey. In binding moiety under conditions Suitable to transform pros other embodiments, the symptom profile is produced based taglandin E. (PGE2) present in the sample into a complex upon the individual’s response to the following question: 65 comprising prostaglandin E2 (PGE2) and the prostaglandin “Are you currently experiencing any symptoms?’ The E. (PGE) binding moiety; and (b) determining the level of symptom profile generated in accordance with either of the complex, thereby determining the level of prostaglandin US 9,482,672 B2 25 26 E. (PGE) present in the sample. In one embodiment, the (PGE) and histamine from a blood or serum sample from a method further comprises: (c) comparing the level of pros Subject. In another embodiment, the method comprises taglandin E2 (PGE2) present in the sample to a control level. detecting or determining the level of prostaglandin E wherein a difference in the level of prostaglandin E. (PGE) (PGE) and B-tryptase from a blood or serum sample from present in the sample relative to the control level is indica a Subject. In a third embodiment, the method comprises tive of an increased likelihood of the subject having IBS. detecting or determining the level of prostaglandin E In a specific embodiment, the present invention provides (PGE), histamine, and B-tryptase from a blood or serum an assay to aid in the diagnosis of IBS, the assay comprising: sample from a subject. Preferably, prostaglandin E. (PGE), (a) contacting a sample having prostaglandin E. (PGE) histamine, and/or B-tryptase are detected from the same contained therein under conditions suitable to transform the 10 blood or serum sample, although in certain instances the prostaglandin E2 (PGE2) into a complex comprising prosta biomarkers may be detected in different blood or serum glandin E2 (PGE2) and a capture anti-tryptase antibody; (b) samples taken from the same individual, for example, at the contacting the complex with an enzyme labeled indicator same time or at different times. In certain embodiments, the antibody to transform the complex into a labeled complex: biomarkers may be detected in separate assays performed (c) contacting the labeled complex with a substrate for the 15 with different aliquots of a blood or serum sample from a enzyme; and (d) detecting the presence or level of prosta subject. In other embodiments, the biomarkers may be glandin E. (PGE) in the sample. detected in a single multiplex detection assay, for example, In certain embodiments, a control level is the level of in a LuminexC, XMAPC) assay. prostaglandin E. (PGE2) present in a blood or serum sample In yet other aspects of the invention, methods are pro from a healthy Subject or an average level of prostaglandin vided for aiding in the diagnosis of irritable bowel syndrome E. (PGE) present in blood or serum samples from a cohort (IBS) in a Subject comprising the detection of prostaglandin of healthy subjects. In other embodiments, a control level is E2 (PGE2) in a blood or serum sample and at least one the level of prostaglandin E. (PGE) present in a blood or additional biomarker selected from Brain-Derived Neuro serum sample from a non-IBS Subject or an average level of tropic Factor (BDNF), Neutrophil Gelatinase-Associated prostaglandin E2 (PGE) present in blood or serum samples 25 Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis from a cohort of non-IBS subjects. In another embodiment, (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), a control level is the level of prostaglandin E. (PGE) present Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro in a blood or serum sample from a diseased subject or an teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti average level of prostaglandin E. (PGE) present in blood or body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti serum samples from a cohort of diseased subjects. Non 30 Human Neutrophil Cytoplasmic Antibody (ANCA), Anti limiting examples of diseased subjects that are useful for Human Tissue Transglutaminase IgA (tTG), and a determining a control level from include subjects with IBS, combination thereof. In certain embodiments, at least 2 of Subjects with a non-IBS gastrointestinal disease, Subjects the additional biomarkers may be detected. In other embodi with inflammatory bowel disease (IBD), subjects with ulcer ments, at least 3, 4, 5, 6, 7, 8, 9, or 10 of the additional ative colitis (UC), subjects with Crohn's disease (CD), 35 biomarkers may be detected. Subjects with celiac disease, Subjects with gastroesophageal The sample used for detecting or determining the pres reflux disease (GERD), subjects with cancer, subjects with ence or level of at least one diagnostic marker is typically a cancer of the gastrointestinal tract, Subjects with a cancer whole blood, plasma, serum, saliva, urine, Stool (i.e., feces), of the stomach, Subjects with a cancer of the Small or large tears, and any other bodily fluid, or a tissue sample (i.e., bowel, and the like. 40 biopsy) Such as a small intestine or colon sample. Preferably, In cases where the control level is a level of prostaglandin the sample is serum, whole blood, plasma, stool, urine, or a E. (PGE) in a blood or serum sample of a healthy subject tissue biopsy. In certain instances, the methods of the present or healthy Subjects, an increased level of prostaglandin E invention further comprise obtaining the sample from the (PGE2) present in a sample from a subject, relative to the individual prior to detecting or determining the presence or control level, is indicative of an increased likelihood of the 45 level of at least one diagnostic marker in the sample. In a subject having IBS. Conversely, where the control is a preferred embodiment, the additional biomarker may be healthy subject or subjects, a similar or reduced level of detected from a blood or serum sample. In other embodi prostaglandin E. (PGE) present in a sample from a subject, ments, the additional biomarker may be detected from a relative to the control level, is indicative of an increased stool sample or a biopsy from the bowel of the subject. likelihood of the subject not having IBS. 50 In one embodiment, the method comprises detecting or In cases where the control level is a level of prostaglandin determining the level of prostaglandin E. (PGE) from a E. (PGE) in a blood or serum sample of a subject or blood or serum sample and Brain-Derived Neurotropic subjects with IBS, a similar or increased level of prosta Factor (BDNF) from a biological sample. In a preferred glandin E. (PGE2) present in a sample from a subject, embodiment, Brain-Derived Neurotropic Factor (BDNF) is relative to the control level, is indicative of an increased 55 detected in a blood or serum sample from the subject. In likelihood of the subject having IBS. Conversely, where the another preferred embodiment, Brain-Derived Neurotropic control is a subject or subjects with IBS, a reduced level of Factor (BDNF) is detected in a stool sample or a biopsy from prostaglandin E2 (PGE2) present in a sample from a subject, the bowel of the subject. In other embodiments, at least one relative to the control level, is indicative of an increased additional biomarker is also detected. likelihood of the subject not having IBS. 60 In one embodiment, the method comprises detecting or In certain aspects of the invention, methods are provided determining the level of prostaglandin E. (PGE) from a for aiding in the diagnosis of irritable bowel syndrome (IBS) blood or serum sample and Neutrophil Gelatinase-Associ in a subject comprising the detection of prostaglandin E ated Lipocalin (NGAL) from a biological sample. In a (PGE) in a blood or serum sample and at least one addi preferred embodiment, Neutrophil Gelatinase-Associated tional blood or serum biomarker selected from histamine 65 Lipocalin (NGAL) is detected in a blood or serum sample and 3-tryptase. In one embodiment, the method comprises from the subject. In another preferred embodiment, Neutro detecting or determining the level of prostaglandin E phil Gelatinase-Associated Lipocalin (NGAL) is detected in US 9,482,672 B2 27 28 a stool sample or a biopsy from the bowel of the subject. In plasmic Antibody (ANCA) from a biological sample. In a other embodiments, at least one additional biomarker is also preferred embodiment, Anti-Human Neutrophil Cytoplas detected. mic Antibody (ANCA) is detected in a blood or serum In one embodiment, the method comprises detecting or sample from the subject. In another preferred embodiment, determining the level of prostaglandin E. (PGE) from a Anti-Human Neutrophil Cytoplasmic Antibody (ANCA) is blood or serum sample and TNF-related Weak Inducer of detected in a stool sample or a biopsy from the bowel of the Apoptosis (TWEAK) from a biological sample. In a pre subject. In other embodiments, at least one additional bio ferred embodiment, TNF-related Weak Inducer of Apoptosis marker is also detected. (TWEAK) is detected in a blood or serum sample from the In one embodiment, the method comprises detecting or subject. In another preferred embodiment, TNF-related 10 determining the level of prostaglandin E. (PGE) from a Weak Inducer of Apoptosis (TWEAK) is detected in a stool blood or serum sample and Anti-Human Tissue Transgluta sample or a biopsy from the bowel of the subject. In other minase IgA (tTG) from a biological sample. In a preferred embodiments, at least one additional biomarker is also embodiment, Anti-Human Tissue Transglutaminase IgA detected. (tTG) is detected in a blood or serum sample from the In one embodiment, the method comprises detecting or 15 subject. In another preferred embodiment, Anti-Human Tis determining the level of prostaglandin E. (PGE) from a Sue Transglutaminase IgA (tTG) is detected in a stool sample blood or serum sample and Growth-Related Oncogene or a biopsy from the bowel of the subject. In other embodi Alpha (GRO-O.) from a biological sample. In a preferred ments, at least one additional biomarker is also detected. embodiment, Growth-Related Oncogene Alpha (GRO-O.) is In one embodiment, the method comprises detecting or detected in a blood or serum sample from the subject. In determining the level of prostaglandin E. (PGE) from a another preferred embodiment, Growth-Related Oncogene blood or serum sample and all of Brain-Derived Neurotropic Alpha (GRO-O.) is detected in a stool sample or a biopsy Factor (BDNF), Neutrophil Gelatinase-Associated Lipoca from the bowel of the subject. In other embodiments, at least lin (NGAL). TNF-related Weak Inducer of Apoptosis one additional biomarker is also detected. (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), In one embodiment, the method comprises detecting or 25 Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro determining the level of prostaglandin E. (PGE) from a teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti blood or serum sample and Interleukin-1 Beta (IL-1B) from body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti a biological sample. In a preferred embodiment, Interleu Human Neutrophil Cytoplasmic Antibody (ANCA), and kin-1 Beta (IL-1B) is detected in a blood or serum sample Anti-Human Tissue Transglutaminase IgA (tTG) from a from the subject. In another preferred embodiment, Inter 30 blood or serum sample, a stool sample, or a biopsy from the leukin-1 Beta (IL-1B) is detected in a stool sample or a bowel of the subject. biopsy from the bowel of the subject. In other embodiments, In certain embodiments, the presence or level of at least at least one additional biomarker is also detected. one diagnostic marker is determined using an assay Such as In one embodiment, the method comprises detecting or a hybridization assay or an amplification-based assay. determining the level of prostaglandin E. (PGE) from a 35 Examples of hybridization assays suitable for use in the blood or serum sample and Tissue Inhibitor of Metallopro methods of the present invention include, but are not limited teinase-1 (TIMP-1) from a biological sample. In a preferred to, Northern blotting, dot blotting, RNase protection, and a embodiment, Tissue Inhibitor of Metalloproteinase-1 combination thereof. A non-limiting example of an ampli (TIMP-1) is detected in a blood or serum sample from the fication-based assay suitable for use in the methods of the subject. In another preferred embodiment, Tissue Inhibitor 40 present invention includes a reverse transcriptase-poly of Metalloproteinase-1 (TIMP-1) is detected in a stool merase chain reaction (RT-PCR). sample or a biopsy from the bowel of the subject. In other In certain other embodiments, the presence or level of at embodiments, at least one additional biomarker is also least one diagnostic marker is determined using an immu detected. noassay or an immunohistochemical assay. A non-limiting In one embodiment, the method comprises detecting or 45 example of an immunoassay Suitable for use in the methods determining the level of prostaglandin E. (PGE) from a of the present invention includes an enzyme-linked immu blood or serum sample and Anti-Saccharomyces cerevisiae nosorbent assay (ELISA). Examples of immunohistochemi Antibody (ASCA-IgA) from a biological sample. In a pre cal assays Suitable for use in the methods of the present ferred embodiment, Anti-Saccharomyces cerevisiae Anti invention include, but are not limited to, immunofluores body (ASCA-IgA) is detected in a blood or serum sample 50 cence assays Such as direct fluorescent antibody assays, from the subject. In another preferred embodiment, Anti indirect fluorescent antibody (IFA) assays, anticomplement Saccharomyces cerevisiae Antibody (ASCA-IgA) is immunofluorescence assays, and avidin-biotin immunofluo detected in a stool sample or a biopsy from the bowel of the rescence assays. Other types of immunohistochemical subject. In other embodiments, at least one additional bio assays include immunoperoxidase assays. marker is also detected. 55 In some embodiments, the methods provided herein fur In one embodiment, the method comprises detecting or ther include the step of determining a symptom profile for determining the level of prostaglandin E. (PGE) from a the subject, wherein the symptom profile is determined by blood or serum sample and Anti-CBir-1 Antibody (CBirl) identifying the presence or severity of at least one symptom from a biological sample. In a preferred embodiment, Anti in the subject. In a preferred embodiment, the method CBir-1 Antibody (CBirl) is detected in a blood or serum 60 comprises: (d) determining a symptom profile for the Sub sample from the subject. In another preferred embodiment, ject, wherein the symptom profile is determined by identi Anti-CBir-1 Antibody (CBirl) is detected in a stool sample fying the presence or severity of at least one symptom in the or a biopsy from the bowel of the subject. In other embodi Subject; and (e) diagnosing the Subject as having IBS or not ments, at least one additional biomarker is also detected. having IBS using an algorithm based upon the level of an In one embodiment, the method comprises detecting or 65 IBS biomarker and the system profile. In a preferred determining the level of prostaglandin E. (PGE) from a embodiment, the method for aiding in the diagnosis of blood or serum sample and Anti-Human Neutrophil Cyto irritable bowel syndrome (IBS) in a subject comprises US 9,482,672 B2 29 30 determining a diagnostic marker profile, for example for ing the labeled complex with a substrate for the enzyme; and prostaglandin E. (PGE) or a combination thereof as (d) detecting the presence or level of prostaglandin E described herein, optionally in combination with a symptom (PGE) in the sample. profile, wherein the symptom profile is determined by iden In some instances, the assay to aid in the differentiation of tifying the presence or severity of at least one symptom in IBS-D and IBS-A from IBS-C further comprises detecting the individual; and classifying the sample as an IBS sample the presence or level of B-tryptase; and/or histamine in the or non-IBS sample using an algorithm based upon the sample. diagnostic marker profile and the symptom profile. In yet other embodiments, methods provided herein fur The symptom profile is typically determined by identify ther include diagnosing a subject not having IBS as having 10 IBD, as not having IBD, as having celiac disease, as not ing the presence or severity of at least one symptom selected having celiac disease, as being a healthy Subject, or as not from the group consisting of chest pain, chest discomfort, having a gastrointestinal disease. In a related embodiment, heartburn, uncomfortable fullness after having a regular methods are provided herein that further classify a non-IBS sized meal, inability to finish a regular-sized meal, abdomi sample as an IBD Sample, a non-IBD sample, a healthy nal pain, abdominal discomfort, constipation, diarrhea, 15 sample, a non-gastrointestinal disease sample, a celiac bloating, abdominal distension, negative thoughts or feel sample, and the like. ings associated with having pain or discomfort, and combi In one embodiment, the methods provided herein com nations thereof. prise the use of an algorithm based upon the level of an IBS In preferred embodiments, the presence or severity of 1. biomarker. In certain embodiments, the algorithm is further 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the symptoms described based upon the system profile. In a preferred embodiment, herein is identified to generate a symptom profile that is the algorithm comprises a statistical algorithm, for example useful for predicting IBS. In certain instances, a question a learning statistical classifier system. In a more preferred naire or other form of written, verbal, or telephone survey is embodiment, the algorithm comprises a combination of at used to produce the symptom profile. The questionnaire or least two learning statistical classifier systems. In a most Survey typically comprises a standardized set of questions 25 preferred embodiment, the combination of at least two and answers for the purpose of gathering information from learning statistical classifier systems comprises a random respondents regarding their current and/or recent IBS-re forest classifier and a neural network classifier. lated symptoms. 4. Symptom Profiles In some embodiments, the symptom profile is produced In some embodiments, the method for aiding in the 30 diagnosis of IBS comprises determining a diagnostic marker by compiling and/or analyzing all or a Subset of the answers profile optionally in combination with a symptom profile, to the questions set forth in the questionnaire or survey. In wherein the symptom profile is determined by identifying other embodiments, the symptom profile is produced based the presence or severity of at least one symptom in the upon the individual’s response to the following question: individual; and classifying the sample as an IBS sample or “Are you currently experiencing any symptoms?’ The 35 non-IBS Sample using an algorithm based upon the diag symptom profile generated in accordance with either of nostic marker profile and the symptom profile. One skilled these embodiments can be used in combination with a in the art will appreciate that the diagnostic marker profile diagnostic marker profile in the algorithmic-based methods and the symptom profile can be determined simultaneously described herein to improve the accuracy of predicting IBS. or sequentially in any order. In some embodiments, the methods provided herein fur 40 The symptom profile is typically determined by identify ther include providing a probability that a subject has IBS. ing the presence or severity of at least one symptom selected In certain embodiments, the method may comprise provid from the group consisting of chest pain, chest discomfort, ing a probability that the is subject highly unlikely, unlikely, heartburn, uncomfortable fullness after having a regular likely, or highly likely has IBS. In related embodiments, sized meal, inability to finish a regular-sized meal, abdomi methods are provided that further include providing a prob 45 nal pain, abdominal discomfort, constipation, diarrhea, ability that a sample classified as an IBS sample or a bloating, abdominal distension, negative thoughts or feel non-IBS sample is from a subject with IBS. In certain ings associated with having pain or discomfort, and combi embodiments, the method may comprise providing a prob nations thereof. ability that the is sample is from a subject that highly In preferred embodiments, the presence or severity of 1. unlikely, unlikely, likely, or highly likely has IBS. 50 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of the symptoms described In other embodiments, the methods provided herein fur herein is identified to generate a symptom profile that is ther include classifying a diagnosis of IBS as IBS-consti useful for predicting IBS. In certain instances, a question pation (IBS-C), IBS-diarrhea (IBS-D), IBS-mixed (IBS-M), naire or other form of written, verbal, or telephone survey is IBS-alternating (IBS-A), or post-infectious IBS (IBS-PI). In used to produce the symptom profile. The questionnaire or related embodiments, methods are provided herein to further 55 Survey typically comprises a standardized set of questions classify a sample as an IBS-constipation (IBS-C), IBS and answers for the purpose of gathering information from diarrhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating respondents regarding their current and/or recent IBS-re (IBS-A), or post-infectious IBS (IBS-PI) sample. lated symptoms. For instance. Example 13 provides exem In one embodiment, the present invention provides an plary questions that can be included in a questionnaire for assay to aid in the differentiation of IBS-D and IBS-A from 60 identifying the presence or severity of one or more IBS IBS-C, the assay comprising: (a) contacting a sample having related symptoms in the individual. prostaglandin E2 (PGE2) contained therein under conditions In certain embodiments, the symptom profile is produced suitable to transform the prostaglandin E. (PGE) into a by compiling and/or analyzing all or a Subset of the answers complex comprising prostaglandin E. (PGE) and a capture to the questions set forth in the questionnaire or Survey. In anti-prostaglandin E. (PGE2) antibody; (b) contacting the 65 certain other embodiments, the symptom profile is produced complex with an enzyme labeled indicator antibody to based upon the individual’s response to the following ques transform the complex into a labeled complex; (c) contact tion: “Are you currently experiencing any symptoms?’ The US 9,482,672 B2 31 32 symptom profile generated in accordance with either of In some instances, the data obtained from using the these embodiments can be used in combination with a learning statistical classifier system or systems can be pro diagnostic marker profile in the algorithmic-based methods cessed using a processing algorithm. Such a processing described herein to improve the accuracy of predicting IBS. algorithm can be selected, for example, from the group 5. Use of Statistical Algorithms consisting of a multilayer perceptron, backpropagation net In some embodiments, methods for aiding in the diagno work, and Levenberg-Marquardt algorithm. In other sis of irritable bowel syndrome (IBS) in a subject is based instances, a combination of Such processing algorithms can upon the diagnostic marker profile, alone or in combination be used, such as in a parallel or serial fashion. with a symptom profile, in conjunction with a statistical In certain other embodiments, the methods of the present algorithm. In certain instances, the statistical algorithm is a 10 invention further comprise sending the IBS classification learning statistical classifier system. The learning statistical results to a clinician, e.g., a gastroenterologist or a general classifier system can be selected from the group consisting practitioner. In another embodiment, the methods of the of a random forest (RF), classification and regression tree present invention provide a diagnosis in the form of a (C&RT), boosted tree, neural network (NN), support vector 15 probability that the individual has IBS. For example, the machine (SVM), general chi-squared automatic interaction individual can have about a 0%, 5%, 10%, 15%, 20%, 25%, detector model, interactive tree, multiadaptive regression 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, spline, machine learning classifier, and combinations 80%, 85%, 90%, 95%, or greater probability of having IBS. thereof. Preferably, the learning statistical classifier system In yet another embodiment, the methods of the present is a tree-based statistical algorithm (e.g., RF, C&RT, etc.) invention further provide a prognosis of IBS in the indi and/or a NN (e.g., artificial NN, etc.). Additional examples vidual. For example, the prognosis can be Surgery, devel of learning statistical classifier systems suitable for use in opment of a category or clinical subtype of IBS, develop the present invention are described in U.S. patent application ment of one or more symptoms, or recovery from the Ser. No. 1 1/368,285. In certain embodiments, the methods disease. comprise classifying a sample from the Subject as an IBS 25 B. Assays and Kits sample or non-IBS sample. In one aspect, the present invention provides an assay for In certain instances, the statistical algorithm is a single the detection of B-tryptase in a blood or serum sample, the learning statistical classifier system. Preferably, the single method comprising the steps of: (a) coating a Solid phase learning statistical classifier system comprises a tree-based Surface with a first anti-B-tryptase capture antibody; (b) statistical algorithm such as a RF or C&RT. As a non 30 contacting the Solid phase Surface with a blood or serum limiting example, a single learning statistical classifier sys sample under conditions suitable to transform B-tryptase tem can be used to classify the sample as an IBS sample or present in the sample into a complex comprising B-tryptase non-IBS sample based upon a prediction or probability value and the anti-B-tryptase capture antibody; (c) contacting the and the presence or level of at least one diagnostic marker B-tryptase and the anti-B-tryptase complex with a second (i.e., diagnostic marker profile), alone or in combination 35 detecting antibody under conditions suitable to form a with the presence or severity of at least one symptom (i.e., ternary complex; and (d) contacting the ternary complex symptom profile). The use of a single learning statistical with a luminescent or chemiluminescent Substrate. classifier system typically classifies the sample as an IBS In one embodiment, the detecting antibody is conjugated sample with a sensitivity, specificity, positive predictive to alkaline phosphatase. In other embodiments, the detecting value, negative predictive value, and/or overall accuracy of 40 antibody is not conjugated to an enzyme and the method at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, further comprises the steps of (i) contacting the ternary 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, complex with a third antibody conjugated to alkaline phos 93%, 94%, 95%, 96%, 97%, 98%, or 99%. As such, the phatase under conditions Suitable to form a quaternary classification of a sample as an IBS sample or non-IBS complex and (ii) contacting the quaternary complex with a sample is useful for aiding in the diagnosis of IBS in a 45 luminescent or chemiluminescent Substrate. Subject. Any suitable antibody pair may be used for the capture In certain other instances, the statistical algorithm is a and detecting antibodies in a sandwich ELISA. One of skill combination of at least two learning statistical classifier in the art will know and appreciate how to select an systems. Preferably, the combination of learning statistical appropriate antibody pair for the assay. Generally, two classifier systems comprises a RF and a NN, e.g., used in 50 antibodies are selected that bind to the target of interest, e.g., tandem or parallel. As a non-limiting example, a RF can first B-tryptase, at different epitopes such that the binding of the be used to generate a prediction or probability value based first (capture) antibody does not interfere with the second upon the diagnostic marker profile, alone or in combination (detecting) antibody. In certain embodiments, the detecting with a symptom profile, and a NN can then be used to antibody will be conjugated to an enzyme, for example, classify the sample as an IBS sample or non-IBS sample 55 alkaline phophatase, to aid in the detection of the complex. based upon the prediction or probability value and the same In other embodiments, a secondary antibody conjugated to or different diagnostic marker profile or combination of an enzyme (e.g., alkaline phophatase), which binds to the profiles. Advantageously, the hybrid RF/NN learning statis detecting antibody, may be used in the assay. tical classifier system of the present invention classifies the Generally, the complex will be detected by the use of a sample as an IBS sample with a sensitivity, specificity, 60 luminescent Substrate, for example, a luminescent Substrate positive predictive value, negative predictive value, and/or found in a kit such as Ultra LITETM (NAG Research overall accuracy of at least about 75%, 76%, 77%, 78%, Laboratories); SensoLyte R (AnaSpec); SuperSignal ELISA 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, Femto Maximum Sensitivity Substrate (Thermo Scientific); 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or SuperSignal ELISA Pico Chemiluminescent Substrate 99%. In a particularly preferred embodiment, the statistical 65 (Thermo Scientific); or CPSD (disodium 3-(4-methox algorithm is a random forest classifier or a combination of a yspiro 1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7 de random forest classifier and a neural network classifier. can-4-yl)phenyl phosphate; Tropix, Inc). US 9,482,672 B2 33 34 In a preferred embodiment, an assay for detecting the antibody; (b) contacting the complex with an enzyme presence or level of B-tryptase comprises a sandwich ELISA labeled indicator antibody to transform the complex into a that relies on the use of an alkaline phosphatase conjugated labeled complex; (c) contacting the labeled complex with a anti-tryptase antibody as the detecting antibody and a CPSD Substrate for the enzyme; and (d) detecting the presence or containing luminescent Substrate to enhance the assay sen level of B-tryptase in the sample. sitivity. The CPSD substrate can be found in chemilumines In one embodiment of an assay to aid in the differentiation cent detection systems, such as the ELISA-LightTM System of clinical subtypes of IBS, the assay aids in the differen (Applied Biosystems). In a particularly preferred embodi tiation of IBS-D and IBS-A from IBS-C. ment, the detection antibody used in the sandwich ELISA is In another embodiment of an assay to aid in the differ the anti-tryptase antibody G3 (sc-33676; Santa Cruz, Bio 10 entiation of clinical subtypes of IBS, the sample is human technology, Inc.; Santa Cruz, Calif.). S. In a preferred embodiment of the assay, the detection limit In another embodiment of an assay to aid in the differ of B-tryptase present in a blood or serum sample is less than entiation of clinical Subtypes of IBS, the assay is an enzyme about 500 pg/ml. In certain embodiments, the detection limit linked immunosorbent assay (ELISA). of B-tryptase present in a blood or serum sample is less than 15 In another embodiment of an assay to aid in the differ about 500 pg/ml, or less than about 400 pg/ml, 300 pg/ml, entiation of clinical subtypes of IBS, detecting the presence 250 pg/ml, 200 pg/ml, 150 pg/ml, 100 pg/ml, 75 pg/ml, 50 or level of B-tryptase in the sample comprises the use of a pg/ml. 40 pg/ml, 30 pg/ml, 25 pg/ml, 20 pg/ml, 15 pg/ml, or detection device. less than about 10 pg/ml. In a preferred embodiment, the In another embodiment of an assay to aid in the differ detection limit of B-tryptase present in a blood or serum entiation of clinical subtypes of IBS, the assay further sample is less than about 200 pg/ml. In a more preferred comprises detecting the presence or level of prostaglandin embodiment, the detection limit of B-tryptase present in a E2 (PGE) and/or histamine in the sample. blood or serum sample is less than about 100 pg/ml. In a In yet another aspect, the present invention provides an more preferred embodiment, the detection limit of assay to aid in the diagnosis of IBS, the assay comprising: B-tryptase present in a blood or serum sample is less than 25 (a) contacting a sample having B-tryptase, prostaglandin E, about 50 pg/ml. In a most preferred embodiment, the detec and/or histamine under conditions suitable to transform the tion limit of B-tryptase present in a blood or serum sample B-tryptase, prostaglandin E, and/or histamine into a com is less than about 25 pg/ml. plex comprising f-tryptase, prostaglandin E, and/or hista In another aspect, the present invention provides an assay mine and a capture anti-B-tryptase, anti-prostaglandin E, to aid in the diagnosis of IBS, the assay comprising: (a) 30 and/or anti-histamine antibody; (b) contacting the complex contacting a sample having B-tryptase under conditions with an enzyme-labeled indicator antibody to transform the suitable to transform the B-tryptase into a complex compris complex into a labeled complex; (c) contacting the labeled ing B-tryptase and a capture anti-tryptase antibody; (b) complex with a Substrate for the enzyme; and (d) detecting contacting the complex with an enzyme-labeled indicator the presence or level of B-tryptase, prostaglandin E, and/or antibody to transform the complex into a labeled complex: 35 histamine in the sample. (c) contacting the labeled complex with a substrate for the In certain embodiments, the assays provided herein may enzyme; and (d) detecting the presence or level off-tryptase further comprise detection of the presence or level of at least in the sample. one additional biomarker selected from the group consisting In one embodiment of an assay to aid in the diagnosis of of Brain-Derived Neurotropic Factor (BDNF), Neutrophil IBS, the sample is human serum. 40 Gelatinase-Associated Lipocalin (NGAL). TNF-related In another embodiment of an assay to aid in the diagnosis Weak Inducer of Apoptosis (TWEAK), Growth-Related of IBS, the sample is obtained from a subject suspected of Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-13), Tis having IBS. Sue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac In another embodiment of an assay to aid in the diagnosis charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 of IBS, a higher level of B-tryptase in the sample relative to 45 Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic healthy controls is indicative of an increased likelihood of Antibody (ANCA), Anti-Human Tissue Transglutaminase the subject having IBS. IgA (tTG), and a combination thereof. In another embodiment of an assay to aid in the diagnosis In yet other embodiments, the assays provided herein may of IBS, the assay is an enzyme-linked immunosorbent assay further comprise detection of the presence or level of at least (ELISA). 50 one additional biomarker selected from the group consisting In another embodiment of an assay to aid in the diagnosis of a cytokine (e.g., IL-8, IL-1B. TWEAK, leptin, OPG, of IBS, detecting the presence or level of B-tryptase in the MIP-3|B, GROC, CXCL4/PF-4, and/or CXCL7/NAP-2), sample comprises the use of a detection device. growth factor (e.g., EGF, VEGF, PEDF, BDNF, and/or In another embodiment of an assay to aid in the diagnosis SDGF), anti-neutrophil antibody (e.g., ANCA, p-ANCA, of IBS, the detection device comprises a luminescence plate 55 cANCA, NSNA, and/or SAPPA), ASCA (e.g., ASCA-IgA, reader. ASCA-IgG, and/or ASCA-IgM), antimicrobial antibody In another embodiment of an assay to aid in the diagnosis (e.g., anti-OmpC antibody, anti-flagellin antibody, and/or of IBS, the detection device comprises a spectrophotometer. anti-I2 antibody), lactoferrin, anti-tTG antibody, lipocalin In another embodiment of an assay to aid in the diagnosis (e.g., NGAL, NGAL/MMP-9 complex), MMP (e.g., MMP of IBS, the assay further comprises detecting the presence or 60 9), TIMP (e.g., TIMP-1), alpha-globulin (e.g., alpha-2- level of prostaglandin E2 (PGE) and/or histamine in the macroglobulin, haptoglobin, and/or orosomucoid), actin sample. severing protein (e.g., gelsolin), S100 protein (e.g., In another aspect, the present invention provides an assay calgranulin), fibrinopeptide (e.g., FIBA), CGRP, tachykinin to aid in the differentiation of clinical subtypes of IBS, the (e.g., Substance P), ghrelin, neurotensin, corticotropin-re assay comprising: (a) contacting a sample having B-tryptase 65 leasing hormone, and combinations thereof. In yet other under conditions Suitable to transform the B-tryptase into a embodiments, the presence or level of other diagnostic complex comprising B-tryptase and a capture anti-tryptase markers such as, for example, anti-lactoferrin antibody, US 9,482,672 B2 35 36 L-selectin/CD62L, elastase, C-reactive protein (CRP), cal detection antibody, the third antibody being conjugated to an protectin, anti-U1-70 kDa autoantibody, Zona occludens 1 enzyme, e.g., alkaline phophatase. (ZO-1), vasoactive intestinal peptide (VIP), serum amyloid In certain embodiments, the kit will also contain a lumi A, gastrin, and a combination thereof may also be detected. nescent or chemiluminescent Substrate, for example, a lumi In certain embodiments, the assays provided may com nescent substrate as found in Ultra LITETM (NAG Research prise the detection of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, Laboratories); SensoLyte R (AnaSpec); SuperSignal ELISA 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, Femto Maximum Sensitivity Substrate (Thermo Scientific); 28, 29, 30, or more of the biomarkers described herein. SuperSignal ELISA Pico Chemiluminescent Substrate In another aspect, kits are provided for aiding in the (Thermo Scientific); or CPSD (disodium 3-(4-methox diagnosis of IBS in a subject, or for classifying a sample as 10 yspiro 1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7 de an IBS sample or a non-IBS sample. In certain embodi can-4-yl)phenyl phosphate; Tropix, Inc). In a preferred ments, The kits provided herein contain a binding moiety for embodiment, the luminescent substrate is CPSD (disodium detecting the presence or level of an IBS biomarker selected 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo from ?-tryptase, prostaglandin E, and/or histamine. 3.3.1.13.7 decan-4-yl)phenyl phosphate. In other embodiments, the kit will also contain at least one 15 In certain embodiments, the kit for detecting the presence binding moiety for an additional biomarker selected from or level of B-tryptase is suitable for detecting B-tryptase the group consisting of Brain-Derived Neurotropic Factor present in a blood or serum sample at a concentration of less (BDNF), Neutrophil Gelatinase-Associated Lipocalin than about 500 pg/ml. In certain embodiments, the kit is (NGAL). TNF-related Weak Inducer of Apoptosis Suitable for detecting B-tryptase present in a blood or serum (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), sample at a concentration of less than about 500 pg/ml, or Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro less than about 400 pg/ml, 300 pg/ml, 250 pg/ml, 200 pg/ml, teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti 150 pg/ml, 100 pg/ml, 75 pg/ml, 50 pg/ml. 40 pg/ml, 30 body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti pg/ml, 25 pg/ml, 20 pg/ml, 15 pg/ml, or less than about 10 Human Neutrophil Cytoplasmic Antibody (ANCA), Anti pg/ml. In a preferred embodiment, the kit is suitable for Human Tissue Transglutaminase IgA (tTG), and a 25 detecting B-tryptase present in a blood or serum sample at a combination thereof. concentration of less than about 200 pg/ml. In a more In yet other embodiments, a kit will contain at least one preferred embodiment, the kit is suitable for detecting binding moiety for an additional biomarker selected from B-tryptase present in a blood or serum sample at a concen the group consisting of a cytokine (e.g., IL-8, IL-1B. tration of less than about 100 pg/ml. In a more preferred TWEAK, leptin, OPG, MIP-3 B, GROC, CXCL4/PF-4, and/ 30 embodiment, the kit is suitable for detecting B-tryptase or CXCL7/NAP-2), growth factor (e.g., EGF, VEGF, PEDF, present in a blood or serum sample at a concentration of less BDNF, and/or SDGF), anti-neutrophil antibody (e.g., than about 50 pg/ml. In a most preferred embodiment, the kit ANCA, p-ANCA, cANCA, NSNA, and/or SAPPA), ASCA is suitable for detecting 3-tryptase present in a blood or (e.g., ASCA-IgA, ASCA-IgG, and/or ASCA-IgM), antimi serum sample at a concentration of less than about 25 pg/ml. crobial antibody (e.g., anti-Ompo antibody, anti-flagellin 35 C. Methods for Monitoring and Assigning Therapy antibody, and/or anti-I2 antibody), lactoferrin, anti-tTG anti In some embodiments, the diagnosis of an individual as body, lipocalin (e.g., NGAL, NGAL/MMP-9 complex), having IBS is followed by administering to the individual a MMP (e.g., MMP-9), TIMP (e.g., TIMP-1), alpha-globulin therapeutically effective amount of a drug useful for treating (e.g., alpha-2-macroglobulin, haptoglobin, and/or orosomu one or more symptoms associated with IBS. Suitable IBS coid), actin-severing protein (e.g., gelsolin), S100 protein 40 drugs include, but are not limited to, serotonergic agents, (e.g., calgranulin), fibrinopeptide (e.g., FIBA), CGRP, antidepressants, chloride channel activators, chloride chan tachykinin (e.g., Substance P), ghrelin, neurotensin, corti nel blockers, guanylate cyclase agonists, antibiotics, opioid cotropin-releasing hormone, anti-lactoferrin antibody, L-se agonists, neurokinin antagonists, antispasmodic or anticho lectin/CD62L, elastase, C-reactive protein (CRP), calpro linergic agents, belladonna alkaloids, barbiturates, GLP-1 tectin, anti-U1-70 kDa autoantibody, Zona occludens 1 (ZO 45 analogs, CRF antagonists, probiotics, free bases thereof, 1), vasoactive intestinal peptide (VIP), serum amyloid A. pharmaceutically acceptable salts thereof, derivatives gastrin, and combinations thereof. thereof, analogs thereof, and combinations thereof. Other In certain embodiments, the kits provided may contain a IBS drugs include bulking agents, dopamine antagonists, binding moiety for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, carminatives, tranquilizers, dextofisopam, phenyloin, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 50 timolol, and diltiazem. Additionally, amino acids like glu 29, 30, or more of the biomarkers described herein. tamine and glutamic acid which regulate intestinal perme In a preferred embodiment, a kit is provided for detecting ability by affecting neuronal or glial cell signaling can be the presence or level of B-tryptase in a blood or serum administered to treat patients with IBS. sample. In one embodiment, the kit contains a first capture In other embodiments, the methods of the present inven antibody, which is optionally attached to a solid Surface, and 55 tion further comprise classifying an IBS sample or a diag a second detection antibody that binds to a different epitope nosis of IBS as an IBS-constipation (IBS-C), IBS-diarrhea on B-tryptase than the capture antibody. In certain embodi (IBS-D), IBS-mixed (IBS-M), IBS-alternating (IBS-A), or ments, the detection antibody will be conjugated to alkaline post-infectious IBS (IBS-PI) sample or diagnosis. In certain phosphatase. In other embodiments, the detection antibody instances, the classification of the IBS sample or diagnosis will not be conjugated to an enzyme, i.e., alkaline 60 into a category, form, or clinical subtype of IBS is based phophatase. In a preferred embodiment, the detection anti upon the presence or level of at least one, two, three, four, body is the anti-tryptase antibody G3 (sc-33676; Santa Cruz five, six, seven, eight, nine, ten, or more classification Biotechnology, Inc.; Santa Cruz, Calif.). In a more preferred markers provided herein. Preferably, at least one form of IBS embodiment, the anti-tryptase antibody G3 is further alka is distinguished from at least one other form of IBS based line phosphatase conjugated. In certain embodiments, 65 upon the presence or level of leptin, B-tryptase, prostaglan wherein the detection antibody is not enzyme-conjugated, din E, and/or histamine. In certain instances, the methods of the kit may further comprise a third antibody specific for the the present invention can be used to differentiate an IBS-C US 9,482,672 B2 37 38 sample from an IBS-A and/or IBS-D sample in an individual and determining the effectiveness of the drug using an previously identified as having IBS. In certain other algorithm based upon the diagnostic marker profile and the instances, the methods of the present invention can be used symptom profile. to classify a sample from an individual not previously In one embodiment, the present invention provides a diagnosed with IBS as an IBS-A sample, IBS-C sample, method for monitoring the progression or regression of IBS-D sample, or non-IBS sample. irritable bowel syndrome (IBS) in a subject, the method In certain embodiments, the methods further comprise comprising: (a) contacting a first blood or serum sample sending the results from the classification to a clinician. In taken from the subject at a first time with a 3-tryptase, certain other embodiments, the methods further provide a prostaglandin E, and/or histamine binding moiety under 10 conditions suitable to transform B-tryptase, prostaglandin diagnosis in the form of a probability that the individual has E, and/or histamine present in the sample into a complex IBS-A, IBS-C, IBS-D, IBS-M, or IBS-PI. The methods of comprising B-tryptase, prostaglandin E, and/or histamine the present invention can further comprise administering to and the B-tryptase, prostaglandin E, and/or histamine bind the individual a therapeutically effective amount of a drug ing moiety; (b) determining the level of the complex, useful for treating IBS-A, IBS-C, IBS-D, IBS-M, or IBS-PI. 15 thereby determining the level of B-tryptase, prostaglandin Suitable drugs include, but are not limited to, tegaserod E. and/or histamine present in the first sample; (c) contact (ZelnormTM), alosetron (LotroneXR), lubiprostone (Am ing a second blood or serum sample taken from the Subject itizatM), rifamixin (XifaxanTM), MD-1100, probiotics, and a at a second time with a B-tryptase, prostaglandin E, and/or combination thereof. histamine binding moiety under conditions Suitable to trans In instances where the sample is classified as an IBS-A or form B-tryptase, prostaglandin E, and/or histamine present IBS-C sample and/or the individual is diagnosed with IBS-A in the sample into a complex comprising B-tryptase, pros or IBS-C, a therapeutically effective dose of tegaserod or taglandin E, and/or histamine and the B-tryptase, prosta other 5-HT agonist (e.g., mosapride, renzapride, AG1-001, glandin E, and/or histamine binding moiety; (d) determin etc.) can be administered to the individual. In some ing the level of the complex, thereby determining the level instances, when the sample is classified as IBS-C and/or the 25 of B-tryptase, prostaglandin E, and/or histamine present in individual is diagnosed with IBS-C, a therapeutically effec the second sample; and (e) comparing the level of tive amount of lubiprostone or other chloride channel acti B-tryptase, prostaglandin E, and/or histamine present in the vator, rifamixin or other antibiotic capable of controlling first sample to the level off-tryptase, prostaglandin E, intestinal bacterial overgrowth, MD-1100 or other guanylate and/or histamine present in the second sample, wherein a cyclase agonist, asimadoline or other opioid agonist, or 30 higher level off-tryptase, prostaglandin E, and/or hista mine in the second sample relative to the first sample is talnetant or other neurokinin antagonist can be administered indicative of the progression of IBS in the subject and a to the individual. In other instances, when the sample is lower level off-tryptase, prostaglandin E, and/or histamine classified as IBS-D and/or the individual is diagnosed with in the second sample relative to the first sample is indicative IBS-D, a therapeutically effective amount of allosetron or 35 of the regression of IBS in the subject. other 5-HT antagonist (e.g., ramosetron, DDP-225, etc.). In certain embodiments of the methods of monitoring and crofelemer or other chloride channel blocker, talnetant or assigning therapy, the method further comprises detecting other neurokinin antagonist (e.g., saredutant, etc.), or an the presence of level of at least one additional biomarker antidepressant Such as a tricyclic antidepressant can be selected from the group consisting of Brain-Derived Neu administered to the individual. 40 rotropic Factor (BDNF), Neutrophil Gelatinase-Associated In yet another aspect, the present invention provides a Lipocalin (NGAL). TNF-related Weak Inducer of Apoptosis method for monitoring the progression or regression of IBS (TWEAK), Growth-Related Oncogene Alpha (GRO-O.), in an individual, the method comprising: (a) determining a Interleukin-1 Beta (IL-1B), Tissue Inhibitor of Metallopro diagnostic marker profile by detecting the presence or level teinase-1 (TIMP-1), Anti-Saccharomyces cerevisiae Anti of at least one diagnostic marker in the sample; and (b) 45 body (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), Anti determining the presence or severity of IBS in the individual Human Neutrophil Cytoplasmic Antibody (ANCA), Anti using an algorithm based upon the diagnostic marker profile. Human Tissue Transglutaminase IgA (tTG), and a In one embodiment, the method of monitoring the pro combination thereof. In certain embodiments, the method gression or regression of IBS comprises determining a comprises detecting the presence or level of at least two, diagnostic marker profile optionally in combination with a 50 three, four, five, six, seven, eight, nine, or all ten of the symptom profile, wherein the symptom profile is determined additional biomarkers provided above. by identifying the presence or severity of at least one In yet other embodiments, the method my further com symptom in the individual; and determining the presence or prises detecting the presence of level of at least one bio severity of IBS in the individual using an algorithm based marker selected from the group consisting of a cytokine upon the diagnostic marker profile and the symptom profile. 55 (e.g., IL-8, IL-1 B, TWEAK, leptin, OPG, MIP-3 B, GROC, In a related aspect, the present invention provides a CXCL4/PF-4, and/or CXCL7/NAP-2), growth factor (e.g., method for monitoring drug efficacy in an individual receiv EGF, VEGF, PEDF, BDNF, and/or SDGF), anti-neutrophil ing a drug useful for treating IBS, the method comprising: antibody (e.g., ANCA, p-ANCA, cANCA, NSNA, and/or (a) determining a diagnostic marker profile by detecting the SAPPA), ASCA (e.g., ASCA-IgA, ASCA-IgG, and/or presence or level of at least one diagnostic marker in the 60 ASCA-IgM), antimicrobial antibody (e.g., anti-OmpC anti sample; and (b) determining the effectiveness of the drug body, anti-flagellin antibody, and/or anti-I2 antibody), lac using an algorithm based upon the diagnostic marker profile. toferrin, anti-tTG antibody, lipocalin (e.g., NGAL, NGAL/ In one embodiment, the method of monitoring IBS drug MMP-9 complex), MMP (e.g., MMP-9), TIMP (e.g., TIMP efficacy comprises determining a diagnostic marker profile 1), alpha-globulin (e.g., alpha-2-macroglobulin, optionally in combination with a symptom profile, wherein 65 haptoglobin, and/or orosomucoid), actin-severing protein the symptom profile is determined by identifying the pres (e.g., gelsolin), S100 protein (e.g., calgranulin), fibrinopep ence or severity of at least one symptom in the individual; tide (e.g., FIBA), CGRP tachykinin (e.g., Substance P), US 9,482,672 B2 39 40 ghrelin, neurotensin, corticotropin-releasing hormone, and comprise determining the effectiveness of the IBS drug by combinations thereof. In yet other embodiments, the pres comparing the effectiveness of the IBS drug determined in ence or level of other diagnostic markers such as, for step (b) to the effectiveness of the IBS drug in the individual example, anti-lactoferrin antibody, L-selectin/CD62L, at an earlier time in therapy. In additional embodiments, the elastase, C-reactive protein (CRP), calprotectin, anti-U1-70 methods can further comprise sending the IBS monitoring kDa autoantibody, Zona occludens 1 (ZO-1), vasoactive results to a clinician, e.g., a gastroenterologist or a general intestinal peptide (VIP), serum amyloid A, gastrin, and a practitioner. combination thereof may also be detected. D. Computer Readable Medium and Systems for Classi In some embodiments, a panel for measuring one or more fying Samples of the diagnostic markers described above may be con 10 structed and used for determining the presence or severity of In one aspect, the present invention provides a computer IBS or for determining the effectiveness of an IBS drug. One readable medium comprising code for controlling one or skilled in the art will appreciate that the presence or level of more processors to classify whether a serum or blood sample a plurality of diagnostic markers can be determined simul from an subject is associated with irritable bowel syndrome taneously or sequentially, using, for example, an aliquot or 15 (IBS), the code comprising instructions to apply a statistical dilution of the individuals sample. As described above, the process to a data set comprising a diagnostic marker profile level of a particular diagnostic marker in the individuals to produce a statistically derived decision classifying the sample is generally considered to be elevated when it is at sample as an IBS sample or non-IBS sample based upon the least about 10%, 15%, 20%, 25%, 50%, 75%, 100%, 125%, diagnostic marker profile, wherein the diagnostic marker 150%, 175%, 200%, 250%, 300%, 350%, 400%, 450%, profile indicates the level of at least one diagnostic marker 500%. 600%, 700%, 800%, 900%, or 1000% greater than selected from the group consisting of B-tryptase, histamine, the level of the same marker in a comparative sample or prostaglandin E. (PGE2), and a combination thereof. population of Samples (e.g., greater than a median level). In other embodiments, the computer-readable medium for Similarly, the level of a particular diagnostic marker in the ruling in IBS comprises instructions to apply a statistical individual’s sample is typically considered to be lowered 25 process to a data set comprising a diagnostic marker profile when it is at least about 5%, 10%, 15%, 20%, 25%, 30%, optionally in combination with a symptom profile which 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, indicates the presence or severity of at least one symptom in 85%, 90%, or 95% less than the level of the same marker in the individual to produce a statistically derived decision a comparative sample or population of samples (e.g., less classifying the sample as an IBS sample or non-IBS sample than a median level). 30 based upon the diagnostic marker profile and the symptom In certain embodiments, the method of monitoring the progression or regression of IBS comprises determining a profile. One skilled in the art will appreciate that the diagnostic marker profile optionally in combination with a statistical process can be applied to the diagnostic marker symptom profile, wherein the symptom profile is determined profile and the symptom profile simultaneously or sequen by identifying the presence or severity of at least one 35 tially in any order. symptom in the individual; and determining the presence or In a specific embodiment, the present invention provides severity of IBS in the individual using an algorithm based a computer-readable medium including code for controlling upon the diagnostic marker profile and the symptom profile. one or more processors to classify whether a sample from an In certain other embodiments, the method of monitoring IBS individual is associated with IBS, the code comprising drug efficacy comprises determining a diagnostic marker 40 instructions to apply a statistical process to a data set profile optionally in combination with a symptom profile, comprising a diagnostic marker profile to produce a statis wherein the symptom profile is determined by identifying tically derived decision classifying the sample as an IBS the presence or severity of at least one symptom in the sample or non-IBS sample based upon the diagnostic marker individual; and determining the effectiveness of the drug profile, wherein the diagnostic marker profile indicates the using an algorithm based upon the diagnostic marker profile 45 presence or level of at least one diagnostic marker in the and the symptom profile. One skilled in the art will appre sample. ciate that the diagnostic marker profile and the symptom In a related aspect, the present invention provides a profile can be determined simultaneously or sequentially in computer-readable medium including code for controlling any order. one or more processors to classify whether a sample from an In some embodiments, determining the presence or sever 50 individual is associated with IBS, the code comprising: (a) ity of IBS or the effectiveness of an IBS drug is based upon instructions to apply a first statistical process to a data set the diagnostic marker profile, alone or in combination with comprising a diagnostic marker profile to produce a statis a symptom profile, in conjunction with a statistical algo tically derived decision classifying the sample as an IBD rithm. In certain instances, the statistical algorithm is a sample or non-IBD Sample based upon the diagnostic learning statistical classifier system. The learning statistical 55 marker profile, wherein the diagnostic marker profile indi classifier system comprises any of the learning statistical cates the presence or level of at least one diagnostic marker classifier systems described herein. in the sample; and if the sample is classified as a non-IBD In certain embodiments, the methods of the present inven sample, (b) instructions to apply a second statistical process tion can further comprise comparing the presence or severity to the same or different data set to produce a second of IBS in the individual determined in step (b) to the 60 statistically derived decision classifying the non-IBD presence or severity of IBS in the individual at an earlier sample as an IBS sample or non-IBS sample. time. As a non-limiting example, the presence or severity of In one embodiment, the computer-readable medium for IBS determined for an individual receiving an IBS drug can ruling in IBS comprises instructions to apply a statistical be compared to the presence or severity of IBS determined process to a data set comprising a diagnostic marker profile for the same individual before initiation of use of the IBS 65 optionally in combination with a symptom profile which drug or at an earlier time in therapy. In certain other indicates the presence or severity of at least one symptom in embodiments, the methods of the present invention can the individual to produce a statistically derived decision US 9,482,672 B2 41 42 classifying the sample as an IBS sample or non-IBS sample In a related aspect, the present invention provides a based upon the diagnostic marker profile and the symptom system for classifying whether a sample from an individual profile. is associated with IBS, the system comprising: (a) a data In other embodiments, the computer-readable medium for acquisition module configured to produce a data set com first ruling out IBD and then ruling in IBS comprises prising a diagnostic marker profile, wherein the diagnostic instructions to apply a first statistical process to a data set marker profile indicates the presence or level of at least one comprising a diagnostic marker profile optionally in com diagnostic marker in the sample; (b) a data processing bination with a symptom profile which indicates the pres module configured to process the data set by applying a first ence or severity of at least one symptom in the individual to statistical process to the data set to produce a first statisti 10 cally derived decision classifying the sample as an IBD produce a statistically derived decision classifying the sample or non-IBD Sample based upon the diagnostic sample as an IBD sample or non-IBD sample based upon the marker profile; if the sample is classified as a non-IBD diagnostic marker profile and the symptom profile; and if the sample, a data processing module configured to apply a sample is classified as a non-IBD sample, instructions to second statistical process to the same or different data set to apply a second statistical process to the same or different 15 produce a second statistically derived decision classifying data set to produce a second statistically derived decision the non-IBD sample as an IBS sample or non-IBS sample: classifying the non-IBD sample as an IBS sample or non and (c) a display module configured to display the first IBS sample. One skilled in the art will appreciate that the and/or the second statistically derived decision. first and/or second statistical process can be applied to the In one embodiment, the system for first ruling out IBD diagnostic marker profile and the symptom profile simulta and then ruling in IBS comprises a data acquisition module neously or sequentially in any order. configured to produce a data set comprising a diagnostic In one embodiment, the first and second statistical pro marker profile optionally in combination with a symptom cesses are implemented in different processors. Alterna profile which indicates the presence or severity of at least tively, the first and second statistical processes are imple one symptom in the individual; a data processing module mented in a single processor. In another embodiment, the 25 configured to process the data set by applying a first statis first statistical process is a learning statistical classifier tical process to the data set to produce a first statistically system. Examples of learning statistical classifier systems derived decision classifying the sample as an IBD sample or suitable for use in the present invention are described above. non-IBD sample based upon the diagnostic marker profile In certain instances, the first and/or second statistical process and the symptom profile; if the sample is classified as a is a single learning statistical classifier system such as, for 30 non-IBD Sample, a data processing module configured to example, a RF or C&RT. In certain other instances, the first apply a second statistical process to the same or different and/or second statistical process is a combination of at least data set to produce a second statistically derived decision two learning statistical classifier systems. As a non-limiting classifying the non-IBD sample as an IBS sample or non example, the combination of learning statistical classifier IBS sample; and a display module configured to display the systems comprises a RF and a NN or SVM, e.g., used in 35 first and/or the second statistically derived decision. tandem. In some instances, the data obtained from using the In certain embodiments the diagnostic marker profile learning statistical classifier system or systems can be pro indicates the level of at least one additional diagnostic cessed using a processing algorithm. marker selected from the group consisting of Brain-Derived In another aspect, the present invention provides a system Neurotropic Factor (BDNF), Neutrophil Gelatinase-Associ for classifying whether a serum or blood sample from a 40 ated Lipocalin (NGAL). TNF-related Weak Inducer of subject is associated with irritable bowel syndrome (IBS), Apoptosis (TWEAK), Growth-Related Oncogene Alpha the system comprising: (a) a data acquisition module con (GRO-O.), Interleukin-1 Beta (IL-1B), Tissue Inhibitor of figured to produce a data set comprising a diagnostic marker Metalloproteinase-1 (TIMP-1), Anti-Saccharomyces cerevi profile, wherein the diagnostic marker profile indicates the siae Antibody (ASCA-IgA), Anti-CBir-1 Antibody (CBirl), presence or level of at least one diagnostic marker selected 45 Anti-Human Neutrophil Cytoplasmic Antibody (ANCA), from the group consisting of B-tryptase, histamine, prosta Anti-Human Tissue Transglutaminase IgA (tTG), and a glandin E. (PGE), and a combination thereof; (b) a data combination thereof. In certain embodiments, the method processing module configured to process the data set by comprises detecting the presence or level of at least two, applying a statistical process to the data set to produce a three, four, five, six, seven, eight, nine, or all ten of the statistically derived decision classifying the sample as an 50 additional biomarkers provided above. IBS sample or non-IBS sample based upon the diagnostic In yet other embodiments, the diagnostic marker profile marker profile; and (c) a display module configured to indicates the level of at least one additional diagnostic display the statistically derived decision. marker selected from the group consisting of a cytokine In certain embodiments, the system for classifying (e.g., IL-8, IL-1 B, TWEAK, leptin, OPG, MIP-3 B, GROC, whether a serum or blood sample is associated with IBS, 55 CXCL4/PF-4, and/or CXCL7/NAP-2), growth factor (e.g., aiding in the diagnosis of IBS, or ruling in IBS comprises a EGF, VEGF, PEDF, BDNF, and/or SDGF), anti-neutrophil data acquisition module configured to produce a data set antibody (e.g., ANCA, p-ANCA, cANCA, NSNA, and/or comprising a diagnostic marker profile optionally in com SAPPA), ASCA (e.g., ASCA-IgA, ASCA-IgG, and/or bination with a symptom profile which indicates the pres ASCA-IgM), antimicrobial antibody (e.g., anti-OmpC anti ence or severity of at least one symptom in the individual; 60 body, anti-flagellin antibody, and/or anti-I2 antibody), lac a data processing module configured to process the data set toferrin, anti-tTG antibody, lipocalin (e.g., NGAL, NGAL/ by applying a statistical process to the data set to produce a MMP-9 complex), MMP (e.g., MMP-9), TIMP (e.g., TIMP statistically derived decision classifying the sample as an 1), alpha-globulin (e.g., alpha-2-macroglobulin, IBS sample or non-IBS sample based upon the diagnostic haptoglobin, and/or orosomucoid), actin-severing protein marker profile and the symptom profile; and a display 65 (e.g., gelsolin), S100 protein (e.g., calgranulin), fibrinopep module configured to display the statistically derived deci tide (e.g., FIBA), CGRP tachykinin (e.g., Substance P), S1O. ghrelin, neurotensin, corticotropin-releasing hormone, and US 9,482,672 B2 43 44 combinations thereof. In yet other embodiments, the pres have elevated fecal lactoferrin and/or calprotectin levels in ence or level of other diagnostic markers such as, for addition to IBS-like symptoms. Lactoferrin is a glycoprotein example, anti-lactoferrin antibody, L-selectin/CD62L, secreted by mucosal membranes and is the major protein in elastase, C-reactive protein (CRP), calprotectin, anti-U1-70 the secondary granules of leukocytes. Leukocytes are com kDa autoantibody, Zona occludens 1 (ZO-1), vasoactive monly recruited to inflammatory sites where they are acti intestinal peptide (VIP), serum amyloid A, gastrin, and a vated, releasing granule content to the Surrounding area. combination thereof may also be detected. This process increases the concentration of lactoferrin in the stool. IV. Diseases and Disorders with IBS-Like Increased lactoferrin levels are observed in patients with Symptoms 10 ileal pouch-anal anastomosis (i.e., a pouch is created fol lowing complete resection of colon in severe cases of A variety of structural or metabolic diseases and disorders Crohn's disease) when compared to other non-inflammatory can cause signs or symptoms that are similar to IBS. As conditions of the pouch, like irritable pouch syndrome. non-limiting examples, patients with diseases and disorders Elevated levels of lactoferrin are also observed in patients such as inflammatory bowel disease (IBD), Celiac disease 15 with diverticulitis, a condition in which bulging pouches (CD), acute inflammation, diverticulitis, ileal pouch-anal (i.e., diverticula) in the digestive tract become inflamed anastomosis, microscopic colitis, chronic infectious diar and/or infected, causing severe abdominal pain, fever, nau rhea, lactase deficiency, cancer (e.g., colorectal cancer), a sea, and a marked change in bowel habits. Microscopic mechanical obstruction of the Small intestine or colon, an colitis is a chronic inflammatory disorder that is also asso enteric infection, ischemia, maldigestion, malabsorption, ciated with increased fecal lactoferrin levels. Microscopic endometriosis, and unidentified inflammatory disorders of colitis is characterized by persistent watery diarrhea (non the intestinal tract can present with abdominal discomfort bloody), abdominal pain usually associated with weight associated with mild to moderate pain and a change in the loss, a normal mucosa during colonoscopy and radiological consistency and/or frequency of stools that are similar to examination, and very specific histopathological changes. IBS. Additional IBS-like symptoms can include chronic 25 Microscopic colitis consists of two diseases, collagenous diarrhea or constipation or an alternating form of each, colitis and lymphocytic colitis. Collagenous colitis is of weight loss, abdominal distention or bloating, and mucus in unknown etiology and is found in patients with long-term the stool. watery diarrhea and a normal colonoscopy examination. Most IBD patients can be classified into one of two Both collagenous colitis and lymphocytic colitis are char distinct clinical Subtypes, Crohn's disease and ulcerative 30 acterized by increased lymphocytes in the lining of the colitis. Crohn's disease is an inflammatory disease affecting colon. Collagenous colitis is further characterized by a the lower part of the ileum and often involving the colon and thickening of the sub-epithelial collagen layer of the colon. other regions of the intestinal tract. Ulcerative colitis is Chronic infectious diarrhea is an illness that is also associ characterized by an inflammation localized mostly in the ated with increased fecal lactoferrin levels. Chronic infec mucosa and Submucosa of the large intestine. Patients Suf 35 tious diarrhea is usually caused by a bacterial, viral, or fering from these clinical subtypes of IBD typically have protozoan infection, with patients presenting with IBS-like IBS-like symptoms such as, for example, abdominal pain, symptoms such as diarrhea and abdominal pain. Increased chronic diarrhea, weight loss, and cramping. lactoferrin levels are also observed in patients with IBD. The clinical presentation of Celiac disease is also char In addition to determining CRP and/or lactoferrin and/or acterized by IBS-like symptoms such as abdominal discom 40 calprotectin levels, diseases and disorders associated with fort associated with chronic diarrhea, weight loss, and intestinal inflammation can also be ruled out by detecting the abdominal distension. Celiac disease is an immune-medi presence of blood in the stool. Such as fecal hemoglobin. ated disorder of the intestinal mucosa that is typically Intestinal bleeding that occurs without the patient’s knowl associated with villous atrophy, crypt hyperplasia, and/or edge is called occult or hidden bleeding. The presence of inflammation of the mucosal lining of the Small intestine. In 45 occult bleeding (e.g., fecal hemoglobin) is typically addition to the malabsorption of nutrients, individuals with observed in a stool sample from the patient. Other conditions Celiac disease are at risk for mineral deficiency, vitamin Such as ulcers (e.g., gastric, duodenal), cancer (e.g., stomach deficiency, osteoporosis, autoimmune diseases, and intesti cancer, colorectal cancer), and hemorrhoids can also present nal malignancies (e.g., lymphoma and carcinoma). It is with IBS-like symptoms including abdominal pain and a thought that exposure to proteins such as gluten (e.g., 50 change in the consistency and/or frequency of stools. glutenin and prolamine proteins which are present in wheat, In addition, fecal calprotectin levels can also be assessed. rye, barley, oats, millet, triticale, spelt, and kamut), in the Calprotectin is a calcium binding protein with antimicrobial appropriate genetic and environmental context, is respon activity derived predominantly from neutrophils and mono sible for causing Celiac disease. cytes. Calprotectin has been found to have clinical relevance Other diseases and disorders characterized by intestinal 55 in cystic fibrosis, rheumatoid arthritis, IBD, colorectal can inflammation that present with IBS-like symptoms include, cer, HIV, and other inflammatory diseases. Its level has been for example, acute inflammation, diverticulitis, ileal pouch measured in serum, plasma, oral, cerebrospinal and synovial anal anastomosis, microscopic colitis, and chronic infectious fluids, urine, and feces. Advantages of fecal calprotectin in diarrhea, as well as unidentified inflammatory disorders of GI disorders have been recognized: stable for 3-7 days at the intestinal tract. Patients experiencing episodes of acute 60 room temperature enabling sample shipping through regular inflammation typically have elevated C-reactive protein mail; correlated to fecal alpha 1-antitrypsin in patients with (CRP) levels in addition to IBS-like symptoms. CRP is Crohn's disease; and elevated in a great majority of patients produced by the liver during the acute phase of the inflam with gastrointestinal carcinomas and IBD. It was found that matory process and is usually released about 24 hours fecal calprotectin correlates well with endoscopic and his post-commencement of the inflammatory process. Patients 65 tological gradings of disease activity in ulcerative colitis, Suffering from diverticulitis, ileal pouch-anal anastomosis, and with fecal excretion of indium-111-labelled neutrophilic microscopic colitis, and chronic infectious diarrhea typically granulocytes, which is a standard of disease activity in IBD. US 9,482,672 B2 45 46 In view of the foregoing, it is clear that a wide array of comprises an anti-Ompo antibody, anti-flagellin antibody, diseases and disorders can cause IBS-like symptoms, anti-I2 antibody, and combinations thereof. thereby creating a substantial obstacle for definitively clas In certain instances, the lipocalin comprises one or more Sifying a sample as an IBS Sample. However, the present of the lipocalins described below. Preferably, the presence or invention overcomes this limitation by classifying a sample level of neutrophil gelatinase-associated lipocalin (NGAL) from an individual as an IBS sample using, for example, a and/or a complex of NGAL and a matrix metalloproteinase statistical algorithm, or by excluding (i.e., ruling out) those (e.g., NGAL/MMP-9 complex) is determined in the indi diseases and disorders that share a similar clinical presen viduals sample. In other instances, the matrix metallopro tation as IBS and identifying (i.e., ruling in) IBS in a sample teinase (MMP) comprises one or more of the MMPs using, for example, a combination of statistical algorithms. 10 described below. Preferably, the presence or level of MMP-9 is determined in the individual’s sample. In further V. Diagnostic Markers instances, the tissue inhibitor of metalloproteinase (TIMP) comprises one or more of the TIMPs described below. A variety of diagnostic markers are suitable for use in the Preferably, the presence or level of TIMP-1 is determined in methods, systems, and code of the present invention for 15 the individuals sample. In yet further instances, the alpha classifying a sample from an individual as an IBS sample or globulin comprises one or more of the alpha-globulins for ruling out one or more diseases or disorders associated described below. Preferably, the presence or level of alpha with IBS-like symptoms in a sample from an individual. 2-macroglobulin, haptoglobin, and/or orosomucoid is deter Examples of diagnostic markers include, without limitation, mined in the individuals sample. cytokines, growth factors, anti-neutrophil antibodies, anti In certain other instances, the actin-severing protein com Saccharomyces cerevisiae antibodies, antimicrobial anti prises one or more of the actin-severing proteins described bodies, anti-tissue transglutaminase (tTG) antibodies, below. Preferably, the presence or level of gelsolin is deter lipocalins, matrix metalloproteinases (MMPs), complexes of mined in the individual’s sample. In additional instances, the lipocalin and MMP, tissue inhibitor of metalloproteinases S100 protein comprises one or more of the S100 proteins (TIMPs), globulins (e.g., alpha-globulins), actin-severing 25 described below including, for example, calgranulin. In yet proteins, S100 proteins, fibrinopeptides, calcitonin gene other instances, the fibrinopeptide comprises one or more of related peptide (CGRP), tachykinins, ghrelin, neurotensin, the fibrinopeptides described below. Preferably, the presence corticotropin-releasing hormone (CRH), serine proteases or level of fibrinopeptide A (FIBA) is determined in the (e.g., tryptases such as B-tryptase, elastase, etc.), prostaglan individual’s sample. In further instances, the presence or din (e.g., PGE), histamine, C-reactive protein (CRP), lac 30 level of a tachykinin such as Substance P. neurokinin A, toferrin, anti-lactoferrin antibodies, calprotectin, hemoglo and/or neurokinin B is determined in the individuals bin, NOD2/CARD15, serotonin reuptake transporter sample. (SERT), tryptophan hydroxylase-1,5-hydroxytryptamine In some instances, the serine protease comprises one or (5-HT), lactulose, and combinations thereof. Additional more of the serine proteases described below. Preferably, the diagnostic markers for predicting IBS in accordance with the 35 presence or level of a serine protease Such as tryptase (e.g., present invention can be selected using the techniques B-tryptase) is determined in the individual’s sample. In other described in Example 14 from US Patent Publication No. instances, the prostaglandin comprises one or more of the 2008/0085524, filed Aug. 14, 2007, which is herein incor prostaglandins described below. Preferably, the presence or porated by reference in its entirety for all purposes. One level of a prostaglandin such as PGE is determined in the skilled in the art will also know of other diagnostic markers 40 individual’s sample. suitable for use in the present invention. The presence or level of other diagnostic markers such as, In particular embodiments, a diagnostic marker profile is for example, anti-lactoferrin antibody, L-selectin/CD62L, determined by detecting the presence or level of at least one, elastase, C-reactive protein (CRP), calprotectin, anti-U1-70 two, or all three of the following biomarkers: a serine kDa autoantibody, Zona occludens 1 (ZO-1), vasoactive protease (e.g., a tryptase Such as B-tryptase); a prostaglandin 45 intestinal peptide (VIP), serum amyloid A, and/or gastrin (e.g., PGE2); and/or histamine. can also be determined. In other embodiments, the presence or level of at least A. Serine Proteases two, three, four, five, six, seven, eight, nine, ten, or more The determination of the presence or level of at least one diagnostic markers are determined in the individuals serine protease in a sample is useful in the present invention. sample. In certain instances, the cytokine comprises one or 50 As used herein, the term “serine protease' includes any more of the cytokines described below. Preferably, the member of a family of proteases in which one of the amino presence or level of IL-8, IL-1B, TNF-related weak inducer acids at the active site is serine. Non-limiting examples of of apoptosis (TWEAK), leptin, osteoprotegerin (OPG), serine proteases include tryptase (e.g., C.-tryptase, MIP-3|B, GROC, CXCL4/PF-4, and/or CXCL7/NAP-2 is B-tryptase, Y-tryptase, and/or A-tryptase), elastase, chy determined in the individual’s sample. In certain other 55 motrypsin, trypsin, Subtilisin, and combinations thereof. instances, the growth factor comprises one or more of the Tryptase is an abundant specific neutral protease of human growth factors described below. Preferably, the presence or mast cells that can be measured in various biological fluids level of epidermal growth factor (EGF), vascular endothelial and can serve as a useful marker for mast cell activation. In growth factor (VEGF), pigment epithelium-derived factor fact, tryptase can be used as a marker for mast cell activation (PEDF), brain-derived neurotrophic factor (BDNF), and/or 60 in IBS. It is believed that tryptase, possibly by activation of amphiregulin (SDGF) is determined in the individuals protease-activated receptor-2, is a good candidate to explain sample. the pro-Secretory and pro-inflammatory effects of mast cells, In some instances, the anti-neutrophilantibody comprises however other mast cell mediators could also involved. In a ANCA, p-ANCA, cANCA, NSNA, SAPPA, and combina preferred embodiment, B-tryptase (TPBAB1) is detected in tions thereof. In other instances, the ASCA comprises 65 a blood or serum sample from a subject. 3-tryptase, also ASCA-IgA, ASCA-IgG, ASCA-IgM, and combinations commonly referred to as Tryptase beta-1 or Tryptase I, is thereof. In further instances, the antimicrobial antibody encoded by the tryptase alpha/beta 1 gene (TPSAB1; US 9,482,672 B2 47 48 NM 003294), which is translated to form a 275 amino acid D. Cytokines tryptase beta-1 precursor protein (NP 003285). The precur The determination of the presence or level of at least one Sor protein is then processed by the removal of a signal cytokine in a sample is particularly useful in the present peptide (amino acids 1-18) and activation peptide propeptide invention. As used herein, the term “cytokine' includes any (amino acids 19-30) resulting in the mature Tryptase beta-1 5 of a variety of polypeptides or proteins secreted by immune polypeptide (amino acids 31-275: UniProt: Q15661). cells that regulate a range of immune system functions and In certain instances, the presence or level of a particular encompasses Small cytokines such as chemokines. The term serine protease such as tryptase is detected at the level of “cytokine' also includes adipocytokines, which comprise a mRNA expression with an assay Such as, for example, a group of cytokines secreted by adipocytes that function, for hybridization assay or an amplification-based assay. In cer 10 example, in the regulation of body weight, hematopoiesis, tain other instances, the presence or level of a particular angiogenesis, wound healing, insulin resistance, the immune serine protease such as tryptase is detected at the level of response, and the inflammatory response. protein expression using, for example, an immunoassay In certain aspects, the presence or level of at least one (e.g., ELISA) or an immunohistochemical assay. Suitable cytokine including, but not limited to, TNF-C, TNF-related ELISA techniques for determining the presence or level of 15 weak inducer of apoptosis (TWEAK), osteoprotegerin tryptase in a serum sample are described herein. In a (OPG), IFN-O, IFN-?3. IFN-Y, IL-1C, IL-1 B, IL-1 receptor particularly preferred embodiment, the level off-tryptase is antagonist (IL-1 ra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 detected in a blood or serum sample using a sandwich receptor (sIL-6R), IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, ELISA assay in which the detecting antibody is an alkaline IL-15, IL-17, IL-23, and IL-27 is determined in a sample. In phosphatase conjugated anti-B-tryptase antibody, for certain other aspects, the presence or level of at least one example, commercial antibody G3. Aluminescent Substrate, chemokine such as, for example, CXCL1/GRO1/GROC. for example, CPSD (disodium 3-(4-methoxyspiro {1,2-di CXCL2/GRO2, CXCL3/GRO3, CXCL4/PF-4, CXCL5/ oxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7 decan-4-yl) ENA-78, CXCL6/GCP-2, CXCL7/NAP-2, CXCL9/MIG, phenyl phosphate), can then be used to enhance the sensi CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, tivity of the assay. 25 CXCL13/BCA-1, CXCL14/BRAK, CXCL15, CXCL16, B. Prostaglandins CXCL17/DMC, CCL1, CCL2/MCP-1, CCL3/MIP-1o, The determination of the presence or level of at least one CCL4/MIP-1B, CCL5/RANTES, CCL6/C10, CCL7/MCP prostaglandin in a sample is also useful in the present 3, CCL8/MCP-2, CCL9/CCL10, CCL11/Eotaxin, CCL12/ invention. As used herein, the term “prostaglandin' includes MCP-5, CCL13/MCP-4, CCL14/HCC-1, CCL15/MIP-5, any member of a group of lipid compounds that are derived 30 CCL16/LEC, CCL17/TARC, CCL18/MIP-4, CCL19/MIP enzymatically from fatty acids and have important functions 3 B, CCL20/MIP-3C, CCL21/SLC, CCL22/MDC, CCL23/ in the animal body. Every prostaglandin contains 20 carbon MPIF1, CCL24/Eotaxin-2, CCL25/TECK, CCL26/Eotaxin atoms, including a 5-carbon ring. Prostaglandins, together 3, CCL27/CTACK, CCL28/MEC, CL1, CL2, and CXCL1 with the thromboxanes and prostacyclins, form the pros is determined in a sample. In certain further aspects, the tanoid class offatty acid derivatives. The prostanoid class is 35 presence or level of at least one adipocytokine including, but a Subclass of the eicosanoids. Non-limiting examples of not limited to, leptin, adiponectin, resistin, active or total prostaglandins include prostaglandin I2 (PGI2), prostaglan plasminogen activator inhibitor-1 (PAI-1), visfatin, and reti din E2 (PGE), prostaglandin F2 (PGF2), and combina nol binding protein 4 (RBP4) is determined in a sample. tions thereof. In a preferred embodiment, prostaglandin E Preferably, the presence or level of IL-8, IL-1 B, TWEAK, (PGE) is detected in a blood or serum sample from a 40 leptin, OPG, MIP-3|B, GROC, CXCL4/PF-4, and/or Subject, for example, a Subject Suspected of having IBS. In CXCL7/NAP-2 is determined. certain embodiments, PGE may be detected by an ELISA or In certain instances, the presence or level of a particular chemiluminescent assay. Suitable ELISA kits for determin cytokine is detected at the level of mRNA expression with ing the presence or level of PGE in a serum sample are an assay Such as, for example, a hybridization assay or an available from, e.g., Cayman Chemical Co. (Ann Arbor, 45 amplification-based assay. In certain other instances, the Mich.). presence or level of a particular cytokine is detected at the C. Histamine level of protein expression using, for example, an immuno The determination of the presence or level of histamine in assay (e.g., ELISA) or an immunohistochemical assay. Suit a sample is also useful in the present invention. As used able ELISA kits for determining the presence or level of a herein, the term “histamine' includes a biogenic amine 50 cytokine such as IL-8, IL-1 B, MIP-3 B, GROC, CXCL4/PF involved in local immune responses as well as regulating 4, or CXCL7/NAP-2 in a serum, plasma, saliva, or urine physiological function in the gut and acting as a neurotrans sample are available from, e.g., R&D Systems, Inc. (Min mitter. Histamine triggers the inflammatory response. As neapolis, Minn.), Neogen Corp. (Lexington, Ky.), Alpco part of an immune response to foreign pathogens, histamine Diagnostics (Salem, N.H.). Assay Designs, Inc. (Ann Arbor, is produced by basophils and by mast cells found in nearby 55 Mich.), BD Biosciences Pharmingen (San Diego, Calif.), connective tissues. Histamine increases the permeability of Invitrogen (Camarillo, Calif.), Calbiochem (San Diego, the capillaries to white blood cells and other proteins, in Calif.), CHEMICON International, Inc. (Temecula, Calif.), order to allow them to engage foreign invaders in the Antigenix America Inc. (Huntington Station, N.Y.), QIA affected tissues. It is found in virtually all animal body cells. GEN Inc. (Valencia, Calif.), Bio-Rad Laboratories, Inc. In a preferred embodiment, histamine is detected in a blood 60 (Hercules, Calif.), and/or Bender MedSystems Inc. (Burl or serum sample from a subject, for example, a subject ingame, Calif.). Suspected of having IBS. In certain embodiments, histamine 1. TWEAK may be detected by an ELISA or chemiluminescent assay. TWEAK is a member of the TNF superfamily of struc Suitable ELISA kits for determining the presence or level of turally related cytokines. Full-length, membrane-anchored histamine in a serum sample are available from, e.g., Immu 65 TWEAK can be found on the surface of many cell types and notech (Czech Republic) and Cayman Chemical Co. (Ann a smaller, biologically active form, generated via proteolytic Arbor, Mich.). processing, has also been detected in the extracellular milieu US 9,482,672 B2 49 50 (see, e.g., Chicheportiche et al., J. Biol. Chem., 272:32401 class I cytokine Superfamily of receptors, which are char 32410 (1997)). TWEAK acts via binding to a TNF receptor acterized by extracellular motifs of four cysteine residues superfamily member named fibroblast growth factor-induc and a number of fibronectin type III domains (see, e.g., ible 14 (Fn 14; also known as tumor necrosis factor receptor Heim, Eur: J. Clin. Invest., 26:1-12 (1996)). The leptin superfamily member 12A or TNFRSF12A). TWEAK has 5 receptor is known to exist as a homodimer and is activated multiple biological activities, including stimulation of cell by conformational changes that occur following ligand growth and angiogenesis, induction of inflammatory cytok binding to the receptor (see, e.g., Devos et al., J. Biol. ines, and stimulation of apoptosis (see, e.g., Wiley et al., Chem., 272:18304-18310 (1997)). Six leptin receptor iso Cytokine Growth Factor Rev., 14:241-249 (2003)). In par forms, generated by alternate slicing, have been identified to ticular, TWEAK has been shown to induce the expression of 10 PGE2, MMP-1, IL-6, IL-8, RANTES, and IP-10 in fibro date (see, e.g., Wang et al., Nature, 393:684-688 (1998); Lee blasts and synoviocytes, and to upregulate ICAM-1, E-se et al., Nature, 379:632-635 (1996)). lectin, IL-8, and MCP-1 expression in endothelial cells (see, Suitable ELISA kits for determining the presence or level e.g., Campbell et al., Front. Biosci., 9:2273-2284 (2004)). It of leptin in a biological sample Such as a serum, plasma, has also been demonstrated that TWEAK binding to the 15 saliva, or urine sample are available from, e.g., R&D Sys Fn 14 receptor, or constitutive Fn 14 overexpression, acti tems, Inc. (Minneapolis, Minn.), B-Bridge International Vates the NF-KB signaling pathway, which plays an impor (Mountain View, Calif.), Neogen Corp. (Lexington, Ky.), tant role in immune and inflammatory processes, oncogen Assay Designs, Inc. (Ann Arbor, Mich.), Invitrogen (Cama esis, cancer therapy resistance, and tumorigenesis (see, e.g., rillo, Calif.), CHEMICON International, Inc. (Temecula, Winkles et al., Cancer Lett., 235:11-17 (2006); and Winkles Calif.), Antigenix America Inc. (Huntington Station, N.Y.), et al., Front. Biosci., 12:2761-2771 (2007)). One skilled in LINCOResearch, Inc. (St. Charles, Mo.), Diagnostic Sys the art will appreciate that TWEAK is also known as tumor tems Laboratories, Inc. (Webster, Tex.), Immuno-Biological necrosis factor ligand superfamily member 12 (TNFSF12), Laboratories, Inc. (Minneapolis, Minn.), and Cayman APO3 ligand (APO3L), CD255, DR3 ligand, FN14, and Chemical Co. (Ann Arbor, Mich.). UNQ181/PRO207. 25 E. Growth Factors Suitable ELISA kits for determining the presence or level The determination of the presence or level of one or more of TWEAK in a biological sample such as a serum, plasma, growth factors in a sample is also useful in the present saliva, or urine sample are available from, e.g., Antigenix invention. As used herein, the term “growth factor includes America Inc. (Huntington Station, N.Y.), Bender MedSys any of a variety of peptides, polypeptides, or proteins that tems Inc. (Burlingame, Calif.), Agdia Inc. (Elkhart, Ind.), 30 are capable of stimulating cellular proliferation and/or cel American Research Products Inc. (Belmont, Mass.), lular differentiation. Biomeda Corp. (Foster City, Calif.), BioVision, Inc. (Moun In certain aspects, the presence or level of at least one tain View, Calif.), and Kamiya Biomedical Co. (Seattle, growth factor including, but not limited to, epidermal Wash.). growth factor (EGF), heparin-binding epidermal growth 2. Osteoprotegerin (OPG) 35 factor (HB-EGF), vascular endothelial growth factor OPG is a 401-amino acid member of the TNF superfamily (VEGF), pigment epithelium-derived factor (PEDF; also of structurally related cytokines. OPG, which is homologous known as SERPINF1), amphiregulin (AREG; also known as to the receptor activator of NFkB (RANK), inhibits the schwannoma-derived growth factor (SDGF)), basic fibro differentiation of macrophages into osteoclasts and regulates blast growth factor (bfGF), hepatocyte growth factor the resorption of osteoclasts by acting as a soluble decoy 40 (HGF), transforming growth factor-O. (TGF-C.), transform receptor for RANK ligand (RANKL.; also known as OPG ing growth factor-B (TGF-B), bone morphogenetic proteins ligand (OPGL)). As a result, the OPG-RANK-RANKL (e.g., BMP1-BMP15), platelet-derived growth factor system plays a direct and essential role in the formation, (PDGF), nerve growth factor (NGF), f-nerve growth factor function, and survival of osteoclasts. The OPG-RANK (B-NGF), neurotrophic factors (e.g., brain-derived neuro RANKL system has also been shown to modulate cancer cell 45 trophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin migration, thus controlling the development of bone metas 4 (NT4), etc.), growth differentiation factor-9 (GDF-9), tases. One skilled in the art will appreciate that OPG is also granulocyte-colony stimulating factor (G-CSF), granulo known as osteoprotegrin and osteoclastogenesis inhibitory cyte-macrophage colony Stimulating factor (GM-CSF), factor (OCIF). myostatin (GDF-8), erythropoietin (EPO), and thrombopoi Suitable ELISA kits for determining the presence or level 50 etin (TPO) is determined in a sample. Preferably, the pres of OPG in a serum, plasma, saliva, or urine sample are ence or level of EGF, VEGF, PEDF, amphiregulin (SDGF), available from, e.g., Antigenix America Inc. (Huntington and/or BDNF is determined. Station, N.Y.), Immunodiagnostic Systems Ltd. (Boldon, In certain instances, the presence or level of a particular United Kingdom), and BioVendor, LLC (Candler, N.C.). growth factor is detected at the level of mRNA expression 3. Leptin 55 with an assay Such as, for example, a hybridization assay or Leptin, a member of the adipocytokine family of cytok an amplification-based assay. In certain other instances, the ines, is a 16-kD peptide hormone that plays a critical role in presence or level of a particular growth factor is detected at the regulation of body weight by inhibiting food intake and the level of protein expression using, for example, an stimulating energy expenditure. It is predominantly synthe immunoassay (e.g., ELISA) or an immunohistochemical sized by adipocytes and circulates in the plasma in amounts 60 assay. Suitable ELISA kits for determining the presence or proportional to body fat content (see, e.g., Maffei et al., Nat. level of a growth factor such as EGF, VEGF, PEDF, SDGF, Med., 1:1155-1161 (1995); Considine et al., Diabetes, or BDNF in a serum, plasma, saliva, or urine sample are 45:992-994 (1996)). Leptin displays a high degree of homol available from, e.g., Antigenix America Inc. (Huntington ogy among different species and it is also analogous in Station, N.Y.), Promega (Madison, Wis.), R&D Systems, structure to other cytokines (see, e.g., Madei et al., FEBS 65 Inc. (Minneapolis, Minn.), Invitrogen (Camarillo, Calif.), Lett., 373:13-18 (1995)). Leptin acts through the leptin CHEMICON International, Inc. (Temecula, Calif.), Neogen receptor, a single-transmembrane-domain receptor of the Corp. (Lexington, Ky.), PeproTech (Rocky Hill, N.J.), Alpco US 9,482,672 B2 51 52 Diagnostics (Salem, N.H.), Pierce Biotechnology, Inc. G. Matrix Metalloproteinases (Rockford, Ill.), and/or Abazyme (Needham, Mass.). The determination of the presence or level of at least one F. Lipocalins matrix metalloproteinase (MMP) in a sample is also useful in the present invention. As used herein, the term “matrix The determination of the presence or level of one or more metalloproteinase' or “MMP includes zinc-dependent lipocalins in a sample is also useful in the present invention. endopeptidases capable of degrading a variety of extracel As used herein, the term “lipocalin' includes any of a variety lular matrix proteins, cleaving cell Surface receptors, releas of small extracellular proteins that are characterized by ing apoptotic ligands, and/or regulating chemokines. MMPS several common molecular recognition properties: the abil are also thought to play a major role in cell behaviors such ity to bind a range of Small hydrophobic molecules; binding as cell proliferation, migration (adhesion/dispersion), differ to specific cell-surface receptors; and the formation of 10 entiation, angiogenesis, and host defense. complexes with soluble macromolecules (see, e.g., Flowers, In certain aspects, the presence or level of at least one at Biochem. J., 318:1-14 (1996)). The varied biological func least one MMP including, but not limited to, MMP-1 (inter tions of lipocalins are mediated by one or more of these stitial collagenase), MMP-2 (gelatinase-A), MMP-3 properties. The lipocalin protein family exhibits great func (stromelysin-1), MMP-7 (matrilysin), MMP-8 (neutrophil 15 collagenase), MMP-9 (gelatinase-B), MMP-10 (stromely tional diversity, with roles in retinol transport, invertebrate sin-2), MMP-11 (stromelysin-3), MMP-12 (macrophage cryptic coloration, olfaction and pheromone transport, and metalloelastase), MMP-13 (collagenase-3), MMP-14, prostaglandin synthesis. Lipocalins have also been impli MMP-15, MMP-16, MMP-17, MMP-18 (collagenase-4), cated in the regulation of cell homoeostasis and the modu MMP-19, MMP-20 (enamelysin), MMP-21, MMP-23, lation of the immune response, and, as carrier proteins, to act MMP-24, MMP-25, MMP-26 (matrilysin-2), MMP-27, and in the general clearance of endogenous and exogenous MMP-28 (epilysin) is determined in a sample. Preferably, compounds. Although lipocalins have great diversity at the the presence or level of MMP-9 is determined. sequence level, their three-dimensional structure is a unify In certain instances, the presence or level of a particular ing characteristic. Lipocalin crystal structures are highly MMP is detected at the level of mRNA expression with an conserved and comprise a single eight-stranded continu 25 assay Such as, for example, a hybridization assay or an ously hydrogen-bonded antiparallel beta-barrel, which amplification-based assay. In certain other instances, the encloses an internal ligand-binding site. presence or level of a particular MMP is detected at the level In certain aspects, the presence or level of at least one of protein expression using, for example, an immunoassay lipocalin including, but not limited to, neutrophil gelatinase (e.g., ELISA) or an immunohistochemical assay. Suitable associated lipocalin (NGAL; also known as human neutro 30 ELISA kits for determining the presence or level of an MMP phil lipocalin (HNL) or lipocalin-2), von Ebner's gland such as MMP-9 in a serum or plasma sample are available protein (VEGP; also known as lipocalin-1), retinol-binding from, e.g., Calbiochem (San Diego, Calif.), CHEMICON protein (RBP), purpurin (PURP), retinoic acid-binding pro International, Inc. (Temecula, Calif.), and R&D Systems, tein (RABP), C-globulin (A2U), major urinary protein Inc. (Minneapolis, Minn.). (MUP), bilin-binding protein (BBP), C-crustacyanin, preg 35 H. Tissue Inhibitor of Metalloproteinases nancy protein 14 (PP14), B-lactoglobulin (Big), C-micro The determination of the presence or level of at least one globulin (AIM), the gamma chain of C8 (C8), Apollipopro tissue inhibitor of metalloproteinase (TIMP) in a sample is tein D (Apod), lazarillo (LAZ), prostaglandin D2 synthase also useful in the present invention. As used herein, the term (PGDS), quiescence-specific protein (QSP), choroid plexus “tissue inhibitor of metalloproteinase” or “TIMP includes protein, odorant-binding protein (OBP), C-acid glycopro 40 proteins capable of inhibiting MMPs. tein (AGP), probasin (PBAS), aphrodisin, orosomucoid, and In certain aspects, the presence or level of at least one at progestagen-associated endometrial protein (PAEP) is deter least one TIMP including, but not limited to, TIMP-1, mined in a sample. In certain other aspects, the presence or TIMP-2, TIMP-3, and TIMP-4 is determined in a sample. level of at least one lipocalin complex including, for Preferably, the presence or level of TIMP-1 is determined. example, a complex of NGAL and a matrix metalloprotei 45 In certain instances, the presence or level of a particular nase (e.g., NGAL/MMP-9 complex) is determined. Prefer TIMP is detected at the level of mRNA expression with an ably, the presence or level of NGAL or a complex thereof assay Such as, for example, a hybridization assay or an with MMP-9 is determined. amplification-based assay. In certain other instances, the In certain instances, the presence or level of a particular presence or level of a particular TIMP is detected at the level lipocalin is detected at the level of mRNA expression with 50 of protein expression using, for example, an immunoassay an assay Such as, for example, a hybridization assay or an (e.g., ELISA) or an immunohistochemical assay. Suitable amplification-based assay. In certain other instances, the ELISA kits for determining the presence or level of a TIMP presence or level of a particular lipocalin is detected at the such as TIMP-1 in a serum or plasma sample are available level of protein expression using, for example, an immuno from, e.g., Alpco Diagnostics (Salem, N.H.), Calbiochem assay (e.g., ELISA) or an immunohistochemical assay. Suit 55 (San Diego, Calif.), Invitrogen (Camarillo, Calif.), CHEMI able ELISA kits for determining the presence or level of a CON International, Inc. (Temecula, Calif.), and R&D Sys lipocalin Such as NGAL in a serum, plasma, or urine sample tems, Inc. (Minneapolis, Minn.). are available from, e.g., AntibodyShop A/S (Gentofte, Den I. Globulins mark), LabClinics SA (Barcelona, Spain), Lucerna-Chem The determination of the presence or level of at least one AG (Luzern, Switzerland), R&D Systems, Inc. (Minneapo 60 globulin in a sample is also useful in the present invention. lis, Minn.), and Assay Designs, Inc. (Ann Arbor, Mich.). As used herein, the term “globulin' includes any member of Suitable ELISA kits for determining the presence or level of a heterogeneous series of families of serum proteins which the NGAL/MMP-9 complex are available from, e.g., R&D migrate less than albumin during serum electrophoresis. Systems, Inc. (Minneapolis, Minn.). Additional NGAL and Protein electrophoresis is typically used to categorize globu NGAL/MMP-9 complex ELISA techniques are described in, 65 lins into the following three categories: alpha-globulins (i.e., e.g., Kjeldsen et al., Blood, 83:799-807 (1994); and Kjeldsen alpha-1-globulins or alpha-2-globulins); beta-globulins; and et al., J. Immunol. Methods, 198:155-164 (1996). gamma-globulins. US 9,482,672 B2 53 54 Alpha-globulins comprise a group of globular proteins in phages, Langerhans cells, dendritic cells, and keratinocytes. plasma which are highly mobile in alkaline or electrically S100 proteins have been implicated in a variety of intrac charged solutions. They generally function to inhibit certain ellular and extracellular functions such as the regulation of blood protease and inhibitor activity. Examples of alpha protein phosphorylation, transcription factors, Ca' homeo globulins include, but are not limited to, alpha-2-macro Stasis, the dynamics of cytoskeleton constituents, enzyme globulin (C2-MG), haptoglobin (Hp), orosomucoid, alpha activities, cell growth and differentiation, and the inflam 1-antitrypsin, alpha-1-antichymotrypsin, alpha-2- matory response. antiplasmin, antithrombin, ceruloplasmin, heparin cofactor Calgranulin is an S100 protein that is expressed in mul II, retinol binding protein, and transcortin. Preferably, the tiple cell types, including renal epithelial cells and neutro presence or level of C2-MG, haptoglobin, and/or orosomu 10 phils, and are abundant in infiltrating monocytes and granu coid is determined. In certain instances, one or more hap locytes under conditions of chronic inflammation. Examples toglobin allotypes such as, for example, Hp precursor, Hp?3. of calgranulins include, without limitation, calgranulin A Hpo.1, and Hpo.2, are determined. (also known as S100A8 or MRP-8), calgranulin B (also In certain instances, the presence or level of a particular known as S100A9 or MRP-14), and calgranulin C (also globulin is detected at the level of mRNA expression with an 15 known as S100A12). assay Such as, for example, a hybridization assay or an In certain instances, the presence or level of a particular amplification-based assay. In certain other instances, the S100 protein is detected at the level of mRNA expression presence or level of a particular globulin is detected at the with an assay Such as, for example, a hybridization assay or level of protein expression using, for example, an immuno an amplification-based assay. In certain other instances, the assay (e.g., ELISA) or an immunohistochemical assay. Suit presence or level of a particular S100 protein is detected at able ELISA kits for determining the presence or level of a the level of protein expression using, for example, an globulin Such as C2-MG, haptoglobin, or orosomucoid in a immunoassay (e.g., ELISA) or an immunohistochemical serum, plasma, or urine sample are available from, e.g., assay. Suitable ELISA kits for determining the presence or GenWay Biotech, Inc. (San Diego, Calif.) and/or Immundi level of an S100 protein such as calgranulin A (S100A8) or agnostik AG (Bensheim, Germany) 25 calgranulin B (S100A9) in a serum, plasma, or urine sample J. Actin-Severing Proteins are available from, e.g., Peninsula Laboratories Inc. (San The determination of the presence or level of at least one Carlos, Calif.) and Hycult biotechnology b.v. (Uden, The actin-severing protein in a sample is also useful in the Netherlands). present invention. As used herein, the term “actin-severing Calprotectin, the complex of S100A8 and S100A9, is a protein’ includes any member of a family of proteins 30 calcium- and zinc-binding protein in the cytosol of neutro involved in actin remodeling and regulation of cell motility. phils, monocytes, and keratinocytes. Calprotectin is a major Non-limiting examples of actin-severing proteins include protein in neutrophilic granulocytes and macrophages and gelsolin (also known as brevin or actin-depolymerizing accounts for as much as 60% of the total protein in the factor), Villin, fragmin, and adseverin. For example, gelsolin cytosol fraction in these cells. It is therefore a Surrogate is a protein of leukocytes, platelets, and other cells which 35 marker of neutrophil turnover. Its concentration in stool severs actin filaments in the presence of Submicromolar correlates with the intensity of neutrophil infiltration of the calcium, thereby solating cytoplasmic actin gels. intestinal mucosa and with the severity of inflammation. In In certain instances, the presence or level of a particular Some instances, calprotectin can be measured with an actin-severing protein is detected at the level of mRNA ELISA using Small (50-100 mg) fecal samples (see, e.g., expression with an assay Such as, for example, a hybridiza 40 Johne et al., Scand J Gastroenterol., 36:291-296 (2001)). tion assay or an amplification-based assay. In certain other L. Tachykinins instances, the presence or level of a particular actin-severing The determination of the presence or level of at least one protein is detected at the level of protein expression using, tachykinin in a sample is also useful in the present invention. for example, an immunoassay (e.g., ELISA) or an immu As used herein, the term “tachykinin’ includes amidated nohistochemical assay. Suitable ELISA techniques for deter 45 neuropeptides that share the carboxy-terminal sequence mining the presence or level of an actin-severing protein Phe-X-Gly-Leu-Met-NH. Tachykinins typically bind to one Such as gelsolin in a plasma sample are described in, e.g., or more tachykinin receptors (e.g., TACR1, TACR2, and/or Smith et al., J. Lab. Clin. Med., 110:189-195 (1987); and TACR3). Hiyoshi et al., Biochem. Mol. Biol. Int, 32:755-762 (1994). In certain aspects, the presence or level of at least one K. S100 Proteins 50 tachykinin including, but not limited to, Substance P. neu The determination of the presence or level of at least one rokinin A, and neurokinin B is determined in a sample. S100 protein in a sample is also useful in the present Preferably, the presence or level of substance P is deter invention. As used herein, the term “S100 protein’ includes mined. Substance P is a peptide of 11 amino acids in length any member of a family of low molecular mass acidic that is released by nerve endings in both the central and proteins characterized by cell-type-specific expression and 55 peripheral nervous systems. Among the numerous biological the presence of 2 EF-hand calcium-binding domains. There sites innervated by Substance P-releasing neurons are the are at least 21 different types of S100 proteins in humans. skin, intestines, stomach, bladder, and cardiovascular sys The name is derived from the fact that S100 proteins are tem. 100% soluble in ammonium sulfate at neutral pH. Most In certain instances, the presence or level of a particular S100 proteins are homodimeric, consisting of two identical 60 tachykinin is detected at the level of mRNA expression with polypeptides held together by non-covalent bonds. Although an assay Such as, for example, a hybridization assay or an S100 proteins are structurally similar to calmodulin, they amplification-based assay. In certain other instances, the differ in that they are cell-specific, expressed in particular presence or level of a particular tachykinin is detected at the cells at different levels depending on environmental factors. level of protein expression using, for example, an immuno S-100 proteins are normally present in cells derived from the 65 assay (e.g., ELISA) or an immunohistochemical assay. Suit neural crest (e.g., Schwann cells, melanocytes, glial cells), able ELISA kits for determining the presence or level of a chondrocytes, adipocytes, myoepithelial cells, macro tachykinin Such as Substance P in a serum, plasma, Saliva, or US 9,482,672 B2 55 56 urine sample are available from, e.g., MD BioSciences Inc. corticotropin-releasing hormone receptors (e.g., CRHR1 (St. Paul, Minn.), Assay Designs, Inc. (Ann Arbor, Mich.), and/or CRHR2). CRH is expressed by the hypothalamus, R&D Systems, Inc. (Minneapolis, Minn.), Sigma-Aldrich spinal cord, stomach, spleen, duodenum, adrenal gland, and Corp. (St. Louis, Mo.), and Cayman Chemical Co. (Ann placenta. Arbor, Mich.). 5 In certain instances, the presence or level of CRH is M. Ghrelin detected at the level of mRNA expression with an assay such The determination of the presence or level of ghrelin in a as, for example, a hybridization assay or an amplification sample is also useful in the present invention. As used based assay. In certain other instances, the presence or level herein, the term “ghrelin' includes a peptide of 28 amino of CRH is detected at the level of protein expression using, acids that is an endogenous ligand for the growth hormone 10 secretagogue receptor (GHSR) and is involved in regulating for example, an immunoassay (e.g., ELISA) or an immu growth hormone release. Ghrelin can be acylated, typically nohistochemical assay. Suitable ELISA kits for determining with an n-octanoyl group at serine residue three, to form the presence or level of CRH in a serum, plasma, saliva, or active ghrelin. Alternatively, ghrelin can exist as an unacy urine sample are available from, e.g., Alpco Diagnostics lated form (i.e., desacyl-ghrelin). Ghrelin is primarily 15 (Salem, N.H.) and Cosmo Bio Co., Ltd. (Tokyo, Japan). expressed in specialized enterochromaflin cells located P. Anti-Neutrophil Antibodies mainly in the mucosa of the fundus of the stomach and has The determination of ANCA levels and/or the presence or metabolic effects opposite to those of leptin. Ghrelin stimu absence of pANCA in a sample is also useful in the present lates food intake, enhances the use of carbohydrates and invention. As used herein, the term “anti-neutrophil cyto reduces fat utilization, increases gastric motility and acid plasmic antibody' or “ANCA includes antibodies directed secretion, and reduces locomotor activity. to cytoplasmic and/or nuclear components of neutrophils. In certain instances, the presence or level of ghrelin is ANCA activity can be divided into several broad categories detected at the level of mRNA expression with an assay such based upon the ANCA staining pattern in neutrophils: (1) as, for example, a hybridization assay or an amplification cytoplasmic neutrophil staining without perinuclear high based assay. In certain other instances, the presence or level 25 lighting (cANCA); (2) perinuclear staining around the out of ghrelin is detected at the level of protein expression using, side edge of the nucleus (pANCA); (3) perinuclear staining for example, an immunoassay (e.g., ELISA) or an immu around the inside edge of the nucleus (NSNA); and (4) nohistochemical assay. Suitable ELISA kits for determining diffuse staining with speckling across the entire neutrophil the presence or level of active ghrelin or desacyl-ghrelin in (SAPPA). In certain instances, p ANCA staining is sensitive a serum, plasma, Saliva, or urine sample are available from, 30 to DNase treatment. The term ANCA encompasses all e.g., Alpco Diagnostics (Salem, N.H.), Cayman Chemical varieties of anti-neutrophil reactivity, including, but not Co. (Ann Arbor, Mich.), LINCO Research, Inc. (St. Charles, limited to, cANCA, p-ANCA, NSNA, and SAPPA. Similarly, Mo.), and Diagnostic Systems Laboratories, Inc. (Webster, the term ANCA encompasses all immunoglobulin isotypes Tex.). including, without limitation, immunoglobulin A and G. N. Neurotensin 35 ANCA levels in a sample from an individual can be The determination of the presence or level of neurotensin determined, for example, using an immunoassay Such as an in a sample is also useful in the present invention. As used enzyme-linked immunosorbent assay (ELISA) with alcohol herein, the term “neurotensin' includes a tridecapeptide that fixed neutrophils. The presence or absence of a particular is widely distributed throughout the central nervous system category of ANCA such as paNCA can be determined, for and the gastrointestinal tract. Neurotensin has been identi 40 example, using an immunohistochemical assay Such as an fied as an important mediator in the development and indirect fluorescent antibody (IFA) assay. Preferably, the progression of several gastrointestinal functions and disease presence or absence of p ANCA in a sample is determined conditions, exerting its effects by interacting with specific using an immunofluorescence assay with DNase-treated, receptors that act directly or indirectly on nerves, epithelial fixed neutrophils. In addition to fixed neutrophils, antigens cells, and/or cells of the immune and inflammatory systems 45 specific for ANCA that are suitable for determining ANCA (see, e.g., Zhao et al., Peptides, 27:2434-2444 (2006)). levels include, without limitation, unpurified or partially In certain instances, the presence or level of neurotensin purified neutrophil extracts; purified proteins, protein frag is detected at the level of mRNA expression with an assay ments, or synthetic peptides such as histone H1 or ANCA Such as, for example, a hybridization assay or an amplifi reactive fragments thereof (see, e.g., U.S. Pat. No. 6,074, cation-based assay. In certain other instances, the presence 50 835); histone H1-like antigens, porin antigens, Bacteroides or level of neurotensin is detected at the level of protein antigens, or ANCA-reactive fragments thereof (see, e.g., expression using, for example, an immunoassay (e.g., U.S. Pat. No. 6,033,864); secretory vesicle antigens or ELISA) or an immunohistochemical assay. Suitable ELISA ANCA-reactive fragments thereof (see, e.g., U.S. patent techniques for determining the presence or level of neuro application Ser. No. 08/804,106); and anti-ANCA idiotypic tensin in a sample are described in, e.g., Davis et al., J. 55 antibodies. One skilled in the art will appreciate that the use Neurosci. Methods, 14:15-23 (1985); and Williams et al., J. of additional antigens specific for ANCA is within the scope Histochem. Cytochem., 37:831-841 (1989). of the present invention. O. Corticotropin-Releasing Hormone Q. Anti-Saccharomyces cerevisiae Antibodies The determination of the presence or level of corticotro The determination of ASCA (e.g., ASCA-IgA and/or pin-releasing hormone (CRH; also known as corticotropin 60 ASCA-IgG) levels in a sample is also useful in the present releasing factor or CRF) in a sample is also useful in the invention. As used herein, the term “anti-Saccharomyces present invention. As used herein, the term “corticotropin cerevisiae immunoglobulin A' or “ASCA-IgA' includes releasing hormone, “CRH,” “corticotropin-releasing fac antibodies of the immunoglobulin A isotype that react spe tor,” or “CRF includes a 41-amino acid peptide secreted by cifically with S. cerevisiae. Similarly, the term “anti-Sac the paraventricular nucleus of the hypothalamus that medi 65 charomyces cerevisiae immunoglobulin G’ or "ASCA-IgG” ates the proximal part of the response to stress in mammals includes antibodies of the immunoglobulin G isotype that such as humans. CRH typically binds to one or more react specifically with S. cerevisiae. US 9,482,672 B2 57 58 The determination of whether a sample is positive for PPM as described in, e.g., Faille et al., supra. An exemplary ASCA-IgA or ASCA-IgG is made using an antigen specific neoglycolipid specific for ASCA can be constructed by for ASCA. Such an antigen can be any antigen or mixture of releasing the oligomannoside from its respective PPM and antigens that is bound specifically by ASCA-IgA and/or Subsequently coupling the released oligomannoside to ASCA-IgG. Although ASCA antibodies were initially char 4-hexadecylaniline or the like. acterized by their ability to bind S. cerevisiae, those of skill R. Anti-Microbial Antibodies in the art will understand that an antigen that is bound The determination of anti-Ompo antibody levels in a specifically by ASCA can be obtained from S. cerevisiae or sample is also useful in the present invention. As used from a variety of other sources so long as the antigen is herein, the term “anti-outer membrane protein C antibody” capable of binding specifically to ASCA antibodies. Accord 10 or “anti-Ompo antibody' includes antibodies directed to a ingly, exemplary Sources of an antigen specific for ASCA, bacterial outer membrane porin as described in, e.g., PCT which can be used to determine the levels of ASCA-IgA Patent Publication No. WO 01/893.61. The term “outer and/or ASCA-IgG in a sample, include, without limitation, membrane protein C or "Ompo” refers to a bacterial porin whole killed yeast cells Such as Saccharomyces or Candida that is immunoreactive with an anti-Ompo antibody. cells; yeast cell wall mannan Such as phosphopeptidoman 15 The level of anti-Ompo antibody present in a sample nan (PPM); oligosachharides Such as oligomannosides; neo from an individual can be determined using an OmpC glycolipids; anti-ASCA idiotypic antibodies; and the like. protein or a fragment thereof Such as an immunoreactive Different species and strains of yeast, such as S. cerevisiae fragment thereof. Suitable OmpC antigens useful in deter strain Sul, Su2, CBS 1315, or BM 156, or Candida albicans mining anti-Ompo antibody levels in a sample include, strain VW32, are suitable for use as an antigen specific for without limitation, an OmpC protein, an OmpC polypeptide ASCA-IgA and/or ASCA-IgG. Purified and synthetic anti having Substantially the same amino acid sequence as the gens specific for ASCA are also suitable for use in deter OmpC protein, or a fragment thereof Such as an immuno mining the levels of ASCA-IgA and/or ASCA-IgG in a reactive fragment thereof. As used herein, an OmpC poly sample. Examples of purified antigens include, without peptide generally describes polypeptides having an amino limitation, purified oligosaccharide antigens such as oligo 25 acid sequence with greater than about 50% identity, prefer mannosides. Examples of synthetic antigens include, with ably greater than about 60% identity, more preferably out limitation, synthetic oligomannosides Such as those greater than about 70% identity, still more preferably greater described in U.S. Patent Publication No. 20030105060, e.g., than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% D-Man B(1-2) D-Man B(1-2) D-Man B(1-2) D-Man-OR, amino acid sequence identity with an OmpC protein, with D-Man O.(1-2) D-Man C.(1-2) D-Man C.(1-2) D-Man-OR, 30 the amino acid identity determined using a sequence align and D-Man C.(1-3) D-Man C.(1-2) D-Man C.(1-2) D-Man ment program such as CLUSTALW. Such antigens can be OR, wherein R is a hydrogen atom, a C to Co alkyl, or an prepared, for example, by purification from enteric bacteria optionally labeled connector group. Such as E. coli, by recombinant expression of a nucleic acid Preparations of yeast cell wall mannans, e.g., PPM, can be such as Genbank Accession No. K00541, by synthetic used in determining the levels of ASCA-IgA and/or ASCA 35 means such as solution or Solid phase peptide synthesis, or IgG in a sample. Such water-soluble Surface antigens can be by using phage display. prepared by any appropriate extraction technique known in The determination of anti-I2 antibody levels in a sample the art, including, for example, by autoclaving, or can be is also useful in the present invention. As used herein, the obtained commercially (see, e.g., Lindberg et al., Gut, term “anti-I2 antibody' includes antibodies directed to a 33:909-913 (1992)). The acid-stable fraction of PPM is also 40 microbial antigen sharing homology to bacterial transcrip useful in the statistical algorithms of the present invention tional regulators as described in, e.g., U.S. Pat. No. 6,309, (Sendid et al., Clin. Diag. Lab. Immunol., 3:219-226 643. The term “12 refers to a microbial antigen that is (1996)). An exemplary PPM that is useful in determining immunoreactive with an anti-I2 antibody. The microbial 12 ASCA levels in a sample is derived from S. uvarum strain protein is a polypeptide of 100 amino acids sharing some ATCC H38926. 45 similarity weak homology with the predicted protein 4 from Purified oligosaccharide antigens Such as oligomanno C. pasteurianum, Rv3557c from Mycobacterium tuberculo sides can also be useful in determining the levels of ASCA sis, and a transcriptional regulator from Aquifex aeolicus. IgA and/or ASCA-IgG in a sample. The purified oligoman The nucleic acid and protein sequences for the I2 protein are noside antigens are preferably converted into neoglycolipids described in, e.g., U.S. Pat. No. 6,309,643. as described in, for example, Faille et al., Eur: J. Microbiol. 50 The level of anti-I2 antibody present in a sample from an Infect. Dis., 11:438-446 (1992). One skilled in the art individual can be determined using an I2 protein or a understands that the reactivity of Such an oligomannoside fragment thereof Such as an immunoreactive fragment antigen with ASCA can be optimized by varying the man thereof. Suitable I2 antigens useful in determining anti-I2 nosyl chain length (Frosh et al., Proc Natl. Acad. Sci. USA, antibody levels in a sample include, without limitation, an I2 82:1194-1198 (1985)); the anomeric configuration (Fuka 55 protein, an I2 polypeptide having Substantially the same Zawa et al. In "Immunology of Fungal Disease. E. Kurstak amino acid sequence as the I2 protein, or a fragment thereof (ed.), Marcel Dekker Inc., New York, pp. 37-62 (1989); Such as an immunoreactive fragment thereof. Such I2 poly Nishikawa et al., Microbiol. Immunol., 34:825-840 (1990); peptides exhibit greater sequence similarity to the I2 protein Poulain et al., Eur: J. Clin. Microbiol., 23:46-52 (1993); than to the C. pasteurianum protein 4 and include isotype Shibata et al., Arch. Biochem. Biophys., 243:338-348 60 variants and homologs thereof. As used herein, an I2 poly (1985); Trinel et al., Infect. Immun., 60:3845-3851 (1992)); peptide generally describes polypeptides having an amino or the position of the linkage (Kikuchi et al., Planta, acid sequence with greater than about 50% identity, prefer 190:525-535 (1993)). ably greater than about 60% identity, more preferably Suitable oligomannosides for use in the methods of the greater than about 70% identity, still more preferably greater present invention include, without limitation, an oligoman 65 than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% noside having the mannotetraose Man (1-3) Man(1-2) Man amino acid sequence identity with a naturally-occurring I2 (1-2) Man. Such an oligomannoside can be purified from protein, with the amino acid identity determined using a US 9,482,672 B2 59 60 sequence alignment program such as CLUSTALW. Such I2 Immunoassays such as ELISA are also particularly useful antigens can be prepared, for example, by purification from for determining the presence or level of C-reactive protein microbes, by recombinant expression of a nucleic acid (CRP) in a sample. For example, a sandwich colorimetric encoding an I2 antigen, by synthetic means such as Solution ELISA assay available from Alpco Diagnostics (Salem, or Solid phase peptide synthesis, or by using phage display. N.H.) can be used to determine the level of CRP in a serum, The determination of anti-flagellin antibody levels in a plasma, urine, or stool sample. Similarly, an ELISA kit sample is also useful in the present invention. As used available from Biomeda Corporation (Foster City, Calif.) herein, the term “anti-flagellin antibody' includes antibodies can be used to detect CRP levels in a sample. Other methods directed to a protein component of bacterial flagella as for determining CRP levels in a sample are described in, 10 e.g., U.S. Pat. Nos. 6,838,250 and 6,406,862; and U.S. described in, e.g., PCT Patent Publication No. WO Patent Publication Nos. 2006.0024682 and 200600 19410. 03/053220 and U.S. Patent Publication No. 2004.0043931. In addition, hemoccult, fecal occult blood, is often indica The term “flagellin' refers to a bacterial flagellum protein tive of gastrointestinal illness and various kits have been that is immunoreactive with an anti-flagellin antibody. developed to monitor gastrointestinal bleeding. For Microbial flagellins are proteins found in bacterial flagellum 15 example, Hemoccult SENSA, a Beckman Coulter product, that arrange themselves in a hollow cylinder to form the is a diagnostic aid for gastrointestinal bleeding, iron defi filament. ciency, peptic ulcers, ulcerative colitis, and, in some The level of anti-flagellin antibody present in a sample instances, in Screening for colorectal cancer. This particular from an individual can be determined using a flagellin assay is based on the oxidation of guaiac by hydrogen protein or a fragment thereof Such as an immunoreactive peroxide to produce a blue color. A similar colorimetric fragment thereof. Suitable flagellin antigens useful in deter assay is commercially available from Helena Laboratories mining anti-flagellin antibody levels in a sample include, (Beaumont, Tex.) for the detection of blood in stool samples. without limitation, a flagellin protein such as Cbir-1 flagel Other methods for detecting occult blood in a stool sample lin, flagellin X, flagellin A, flagellin B, fragments thereof, by determining the presence or level of hemoglobin or heme and combinations thereof, a flagellin polypeptide having 25 activity are described in, e.g., U.S. Pat. Nos. 4.277,250. Substantially the same amino acid sequence as the flagellin 4,920,045, 5,081,040, and 5,310,684. protein, or a fragment thereof Such as an immunoreactive The determination of the presence or level of fibrinogen fragment thereof. As used herein, a flagellin polypeptide or a proteolytic product thereof Such as a fibrinopeptide in a generally describes polypeptides having an amino acid sample is also useful in the present invention. Fibrinogen is sequence with greater than about 50% identity, preferably 30 a plasma glycoprotein synthesized in the liver composed of greater than about 60% identity, more preferably greater 3 structurally different subunits: alpha (FGA); beta (FGB); than about 70% identity, still more preferably greater than and gamma (FGG). Thrombin causes a limited proteolysis of about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino the fibrinogen molecule, during which fibrinopeptides A and acid sequence identity with a naturally-occurring flagellin B are released from the N-terminal regions of the alpha and protein, with the amino acid identity determined using a 35 beta chains, respectively. Fibrinopeptides A and B, which sequence alignment program Such as CLUSTALW. Such have been sequenced in many species, may have a physi flagellin antigens can be prepared, e.g., by purification from ological role as vasoconstrictors and may aid in local bacterium such as Helicobacter Bilis, Helicobacter muste hemostasis during blood clotting. In one embodiment, lae, Helicobacter pylori, Butyrivibriofibrisolvens, and bac human fibrinopeptide A comprises the sequence: Ala-Asp terium found in the cecum, by recombinant expression of a 40 Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val nucleic acid encoding a flagellin antigen, by synthetic means Arg (SEQ ID NO:1). In another embodiment, human Such as solution or Solid phase peptide synthesis, or by using fibrinopeptide B comprises the sequence: Glp-Gly-Val-Asn phage display. Asp-Asn-Glu-Glu-Gly-Phe-Phe-Ser-Ala-Arg (SEQ ID S. Other Diagnostic Markers NO:2). An ELISA kit available from American Diagnostica The determination of the presence or level of lactoferrin 45 Inc. (Stamford, Conn.) can be used to detect the presence or in a sample is also useful in the present invention. In certain level of human fibrinopeptide A in plasma or other biologi instances, the presence or level of lactoferrin is detected at cal fluids. the level of mRNA expression with an assay such as, for In certain embodiments, the determination of the presence example, a hybridization assay or an amplification-based or level of calcitonin gene-related peptide (CGRP) in a assay. In certain other instances, the presence or level of 50 sample is useful in the present invention. Calcitonin is a lactoferrin is detected at the level of protein expression 32-amino acid peptide hormone synthesized by the parafol using, for example, an immunoassay (e.g., ELISA) or an licular cells of the thyroid. It causes reduction in serum immunohistochemical assay. A lactoferrin ELISA kit avail calcium, an effect opposite to that of parathyroid hormone. able from Calbiochem (San Diego, Calif.) can be used to CGRP is derived, with calcitonin, from the CT/CGRP gene detect human lactoferrin in a plasma, urine, bronchoalveolar 55 located on chromosome 11. CGRP is a 37-amino acid lavage, or cerebrospinal fluid sample. Similarly, an ELISA peptide and is a potent endogenous vasodilator. CGRP is kit available from U.S. Biological (Swampscott, Mass.) can primarily produced in nervous tissue; however, its receptors be used to determine the level of lactoferrin in a plasma are expressed throughout the body. An ELISA kit available sample. U.S. Patent Publication No. 2004.0137536 describes from Cayman Chemical Co. (Ann Arbor, Mich.) can be used an ELISA assay for determining the presence of elevated 60 to detect the presence or level of human CGRP in a variety lactoferrin levels in a stool sample. Likewise, U.S. Patent of samples including plasma, serum, nervous tissue, CSF, Publication No. 20040033537 describes an ELISA assay for and culture media. determining the concentration of endogenous lactoferrin in In other embodiments, the determination of the presence a stool, mucus, or bile sample. In some embodiments, then or level of an anti-tissue transglutaminase (tTG) antibody in presence or level of anti-lactoferrin antibodies can be 65 a sample is useful in the present invention. As used herein, detected in a sample using, e.g., lactoferrin protein or a the term “anti-tTG antibody” includes any antibody that fragment thereof. recognizes tissue transglutaminase (tTG) or a fragment US 9,482,672 B2 61 62 thereof. Transglutaminases are a diverse family of Ca"- claudin-8, Zonulin, corticotropin-releasing hormone recep dependent enzymes that are ubiquitous and highly conserved tor-1 (CRHR1), corticotropin-releasing hormone receptor-2 across species. Of all the transglutaminases, tTG is the most (CRHR2), and the like. widely distributed. In certain instances, the anti-tTG anti For instance. Examples 1 and 2 below illustrate that body is an anti-tTG IgA antibody, anti-tTG IgG antibody, or 5 measuring B-tryptase levels is particularly useful for distin mixtures thereof. An ELISA kit available from ScheBo guishing IBS-C patient samples from IBS-A and IBS-D Biotech USA Inc. (Marietta, Ga.) can be used to detect the patient samples. Similarly, Example 1 from US Patent presence or level of human anti-tTG IgA antibodies in a Publication No. 2008/0085524, filed Aug. 14, 2007, which blood sample. is herein incorporated by reference in its entirety for all The determination of the presence of polymorphisms in 10 purposes, illustrates that measuring leptin levels is particu larly useful for distinguishing IBS-C patient samples from the NOD2/CARD15 gene in a sample is also useful in the IBS-A and IBS-D patient samples. In addition, mucosal present invention. For example, polymorphisms in the SERT and tryptophan hydroxylase-1 expression have been NOD2 gene such as a C2 107T nucleotide variant that results shown to be decreased in IBS-C and IBS-D (see, e.g., in a R703W protein variant can be identified in a sample 15 Gershon, J. Clin. Gastroenterol., 39(5 Suppl):S184-193 from an individual (see, e.g., U.S. Patent Publication No. (2005)). Furthermore, IBS-C patients show impaired post 20030190639). In an alternative embodiment, NOD2 prandial 5-HT release, whereas IBS-PI patients have higher mRNA levels can be used as a diagnostic marker of the peak levels of 5-HT (see, e.g., Dunlop, Clin Gastroenterol present invention to aid in classifying IBS. Hepatol., 3:349-357 (2005)). Furthermore, as can be seen in The determination of the presence of polymorphisms in FIG. 11, serum levels of histamine and PGE is also par the serotonin reuptake transporter (SERT) gene in a sample ticularly useful for distinguishing IBS-D patient samples is also useful in the present invention. For example, poly from IBS-A, IBS-C, and healthy control patient samples. morphisms in the promoter region of the SERT gene have effects on transcriptional activity, resulting in altered 5-HT VII. Assays and Kits reuptake efficiency. It has been shown that a strong geno 25 typic association was observed between the SERT-P dele Any of a variety of assays, techniques, and kits known in tion/deletion genotype and the IBS phenotype (see, e.g., Yeo the art can be used to determine the presence or level of one Gut, 53:1396-1399 (2004)). In an alternative embodiment, or more markers in a sample to classify whether the sample SERT mRNA levels can be used as a diagnostic marker of is associated with IBS. the present invention to aid in classifying IBS (see, e.g., 30 The present invention relies, in part, on determining the presence or level of at least one marker in a sample obtained Gershon, J. Clin. Gastroenterol., 39(5 Suppl.):S184-193 from an individual. As used herein, the term "determining (2005)). the presence of at least one marker” includes determining In certain aspects, the level of tryptophan hydroxylase-1 the presence of each marker of interest by using any quan mRNA is a diagnostic marker. For example, tryptophan 35 titative or qualitative assay known to one of skill in the art. hydroxylase-1 mRNA has been shown to be significantly In certain instances, qualitative assays that determine the reduced in IBS (see, e.g., Coats, Gastroenterology, 126: presence or absence of a particular trait, variable, or bio 1897-1899 (2004)). In certain other aspects, a lactulose chemical or serological Substance (e.g., protein or antibody) breath test to measure methane, which is indicative of are Suitable for detecting each marker of interest. In certain bacterial overgrowth, can be used as a diagnostic marker for 40 other instances, quantitative assays that determine the pres IBS. ence or absence of RNA, protein, antibody, or activity are Additional diagnostic markers include, but are not limited Suitable for detecting each marker of interest. As used to, L-selectin/CD62L, anti-U1-70 kDa autoantibodies, Zona herein, the term “determining the level of at least one occludens 1 (ZO-1), vasoactive intestinal peptide (VIP), marker” includes determining the level of each marker of serum amyloid A, gastrin, NB3 gene polymorphisms, NCI1 45 interest by using any direct or indirect quantitative assay gene polymorphisms, fecal leukocytes, C.2A and C2C known to one of skill in the art. In certain instances, adrenoreceptor gene polymorphisms, IL-10 gene polymor quantitative assays that determine, for example, the relative phisms, TNF-C. gene polymorphisms, TGF-B1 gene poly or absolute amount of RNA, protein, antibody, or activity are morphisms, C.-adrenergic receptors, G-proteins, 5-HT suitable for determining the level of each marker of interest. gene polymorphisms, 5-HTT LPR gene polymorphisms, 50 One skilled in the art will appreciate that any assay useful for 5-HT, receptor gene polymorphisms, Zonulin, and the determining the level of a marker is also useful for deter 33-mer peptide (Shan et al., Science, 297:2275-2279 (2002); mining the presence or absence of the marker. PCT Patent Publication No. WO 03/068.170). As used herein, the term “antibody' includes a population of immunoglobulin molecules, which can be polyclonal or VI. Classification Markers 55 monoclonal and of any isotype, or an immunologically active fragment of an immunoglobulin molecule. Such an A variety of classification markers are suitable for use in immunologically active fragment contains the heavy and the methods, systems, and code of the present invention for light chain variable regions, which make up the portion of classifying IBS into a category, form, or clinical Subtype the antibody molecule that specifically binds an antigen. For such as, for example, IBS-constipation (IBS-C), IBS-diar 60 example, an immunologically active fragment of an immu rhea (IBS-D), IBS-mixed (IBS-M), IBS-alternating (IBS noglobulin molecule known in the art as Fab, Fab' or F(ab'), A), or post-infectious IBS (IBS-PI). Examples of classifi is included within the meaning of the term antibody. cation markers include, without limitation, any of the Flow cytometry can be used to determine the presence or diagnostic markers described above (e.g., leptin, serotonin level of one or more markers in a sample. Such flow reuptake transporter (SERT), tryptophan hydroxylase-1,5- 65 cytometric assays, including bead based immunoassays, can hydroxytryptamine (5-HT), tryptase, PGE, histamine, and be used to determine, e.g., antibody marker levels in the the like), as well as antrum mucosal protein 8, keratin-8, same manner as described for detecting serum antibodies to US 9,482,672 B2 63 64 Candida albicans and HIV proteins (see, e.g., Bishop and into the test sample and processed quickly through washes Davis, J. Immunol. Methods, 210:79-87 (1997); McHugh et and detection steps to generate a measurable signal. Such as al., J. Immunol. Methods, 116:213 (1989); Scillian et al., a colored spot. Blood, 73:2041 (1989)). A radioimmunoassay using, for example, an iodine-125 Phage display technology for expressing a recombinant ('I) labeled secondary antibody (Harlow and Lane, supra) antigen specific for a marker can also be used to determine is also suitable for determining the presence or level of one the presence or level of one or more markers in a sample. or more markers in a sample. A secondary antibody labeled Phage particles expressing an antigen specific for, e.g., an with a chemiluminescent marker can also be suitable for use antibody marker can be anchored, if desired, to a multi-well in the present invention. A chemiluminescence assay using plate using an antibody such as an anti-phage monoclonal 10 a chemiluminescent secondary antibody is suitable for sen antibody (Felici et al., “Phage-Displayed Peptides as Tools sitive, non-radioactive detection of marker levels. Such for Characterization of Human Sera’ in Abelson (Ed.). secondary antibodies can be obtained commercially from Methods in Enzymol., 267, San Diego: Academic Press, Inc. various sources, e.g., Amersham LifeSciences, Inc. (Arling (1996)). ton Heights, Ill.). A variety of immunoassay techniques, including competi 15 The immunoassays described above are particularly use tive and non-competitive immunoassays, can be used to ful for determining the presence or level of one or more determine the presence or level of one or more markers in a markers in a sample. As a non-limiting example, an ELISA sample (see, e.g., Self and Cook, Curr: Opin. Biotechnol., using an IL-8-binding molecule Such as an anti-IL-8 anti 7:60-65 (1996)). The term immunoassay encompasses tech body or an extracellular IL-8-binding protein (e.g., IL-8 niques including, without limitation, enzyme immunoassays receptor) is useful for determining whether a sample is (EIA) Such as enzyme multiplied immunoassay technique positive for IL-8 protein or for determining IL-8 protein (EMIT), enzyme-linked immunosorbent assay (ELISA), levels in a sample. A fixed neutrophil ELISA is useful for antigen capture ELISA, sandwich ELISA, IgM antibody determining whether a sample is positive for ANCA or for capture ELISA (MAC ELISA), and microparticle enzyme determining ANCA levels in a sample. Similarly, an ELISA immunoassay (MEIA); capillary electrophoresis immunoas 25 using yeast cell wall phosphopeptidomannan is useful for says (CEIA); radioimmunoassays (RIA); immunoradiomet determining whether a sample is positive for ASCA-IgA ric assays (IRMA); fluorescence polarization immunoassays and/or ASCA-IgG, or for determining ASCA-IgA and/or (FPIA); and chemiluminescence assays (CL). If desired, ASCA-IgG levels in a sample. An ELISA using OmpC Such immunoassays can be automated. Immunoassays can protein or a fragment thereof is useful for determining also be used in conjunction with laser induced fluorescence 30 whether a sample is positive for anti-Ompo antibodies, or (see, e.g., Schmalzing and Nashabeh, Electrophoresis, for determining anti-Ompo antibody levels in a sample. An 18:2184-2193 (1997); Bao, J. Chromatogr: B. Biomed. Sci., ELISA using I2 protein or a fragment thereof is useful for 699:463-480 (1997)). Liposome immunoassays, such as determining whether a sample is positive for anti-I2 anti flow-injection liposome immunoassays and liposome immu bodies, or for determining anti-I2 antibody levels in a nosensors, are also Suitable for use in the present invention 35 sample. An ELISA using flagellin protein (e.g., Cbir-1 (see, e.g., Rongen et al., J. Immunol. Methods, 204:105-133 flagellin) or a fragment thereof is useful for determining (1997)). In addition, nephelometry assays, in which the whether a sample is positive for anti-flagellin antibodies, or formation of protein/antibody complexes results in for determining anti-flagellin antibody levels in a sample. In increased light scatter that is converted to a peak rate signal addition, the immunoassays described above are particularly as a function of the marker concentration, are Suitable for 40 useful for determining the presence or level of other diag use in the present invention. Nephelometry assays are com nostic markers in a sample. mercially available from Beckman Coulter (Brea, Calif.; Kit Specific immunological binding of the antibody to the #449430) and can be performed using a Behring Nephelo marker of interest can be detected directly or indirectly. meter Analyzer (Finket al., J. Clin. Chem. Clin. Biol. Chem., Direct labels include fluorescent or luminescent tags, metals, 27:261-276 (1989)). 45 dyes, radionuclides, and the like, attached to the antibody. Antigen capture ELISA can be useful for determining the An antibody labeled with iodine-125 (I) can be used for presence or level of one or more markers in a sample. For determining the levels of one or more markers in a sample. example, in an antigen capture ELISA, an antibody directed A chemiluminescence assay using a chemiluminescent anti to a marker of interest is bound to a solid phase and sample body specific for the marker is suitable for sensitive, non is added such that the marker is bound by the antibody. After 50 radioactive detection of marker levels. An antibody labeled unbound proteins are removed by washing, the amount of with fluorochrome is also suitable for determining the levels bound marker can be quantitated using, e.g., a radioimmu of one or more markers in a sample. Examples of fluoro noassay (see, e.g., Harlow and Lane, Antibodies: A Labo chromes include, without limitation, DAPI, fluorescein, ratory Manual, Cold Spring Harbor Laboratory, New York, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-phyco 1988)). Sandwich ELISA can also be suitable for use in the 55 erythrin, rhodamine, Texas red, and lissamine. Secondary present invention. For example, in a two-antibody sandwich antibodies linked to fluorochromes can be obtained com assay, a first antibody is bound to a solid Support, and the mercially, e.g., goat F(ab') anti-human IgG-FITC is avail marker of interest is allowed to bind to the first antibody. The able from Tago Immunologicals (Burlingame, Calif.). amount of the marker is quantitated by measuring the Indirect labels include various enzymes well-known in the amount of a second antibody that binds the marker. The 60 art, such as horseradish peroxidase (HRP), alkaline phos antibodies can be immobilized onto a variety of solid phatase (AP), B-galactosidase, urease, and the like. A horse Supports, such as magnetic or chromatographic matrix par radish-peroxidase detection system can be used, for ticles, the Surface of an assay plate (e.g., microtiter wells), example, with the chromogenic Substrate tetramethylbenzi pieces of a Solid Substrate material or membrane (e.g., dine (TMB), which yields a soluble product in the presence plastic, nylon, paper), and the like. An assay strip can be 65 of hydrogen peroxide that is detectable at 450 nm. An prepared by coating the antibody or a plurality of antibodies alkaline phosphatase detection system can be used with the in an array on a solid Support. This strip can then be dipped chromogenic Substrate p-nitrophenyl phosphate, for US 9,482,672 B2 65 66 example, which yields a soluble product readily detectable the purified marker. Purification of the marker can be at 405 nm. Similarly, a B-galactosidase detection system can achieved, for example, by high pressure liquid chromatog be used with the chromogenic substrate o-nitrophenyl-f-D- raphy (HPLC), alone or in combination with mass spec galactopyranoside (ONPG), which yields a soluble product trometry (e.g., MALDI/MS, MALDI-TOF/MS, SELDI detectable at 410 nm. An urease detection system can be TOF/MS, tandem MS, etc.). Qualitative or quantitative used with a Substrate such as urea-bromocresol purple detection of a marker of interest can also be determined by (Sigma Immunochemicals; St. Louis, Mo.). A useful sec well-known methods including, without limitation, Bradford ondary antibody linked to an enzyme can be obtained from assays, Coomassie blue staining, silver staining, assays for a number of commercial sources, e.g., goat F(ab') anti radiolabeled protein, and mass spectrometry. human IgG-alkaline phosphatase can be purchased from 10 The analysis of a plurality of markers may be carried out Jackson ImmunoResearch (West Grove, Pa.). separately or simultaneously with one test sample. For A signal from the direct or indirect label can be analyzed, separate or sequential assay of markers, Suitable apparatuses for example, using a spectrophotometer to detect color from include clinical laboratory analyzers such as the ElecSys a chromogenic Substrate; a radiation counter to detect radia (Roche), the AxSym (Abbott), the Access (Beckman), the tion such as a gamma counter for detection of ''I; or a 15 ADVIAR), the CENTAURR) (Bayer), and the NICHOLS fluorometer to detect fluorescence in the presence of light of ADVANTAGE(R) (Nichols Institute) immunoassay systems. a certain wavelength. For detection of enzyme-linked anti Preferred apparatuses or protein chips perform simultaneous bodies, a quantitative analysis of the amount of marker assays of a plurality of markers on a single Surface. Particu levels can be made using a spectrophotometer Such as an larly useful physical formats comprise Surfaces having a EMAX Microplate Reader (Molecular Devices: Menlo Park, plurality of discrete, addressable locations for the detection Calif.) in accordance with the manufacturers instructions. If of a plurality of different markers. Such formats include desired, the assays of the present invention can be automated protein microarrays, or “protein chips' (see, e.g., Ng et al., or performed robotically, and the signal from multiple J. Cell Mol. Med., 6:329-340 (2002)) and certain capillary samples can be detected simultaneously. devices (see, e.g., U.S. Pat. No. 6,019,944). In these embodi Quantitative western blotting can also be used to detect or 25 ments, each discrete surface location may comprise antibod determine the presence or level of one or more markers in a ies to immobilize one or more markers for detection at each sample. Western blots can be quantitated by well-known location. Surfaces may alternatively comprise one or more methods such as scanning densitometry or phosphorimag discrete particles (e.g., microparticles or nanoparticles) ing. As a non-limiting example, protein samples are elec immobilized at discrete locations of a surface, where the trophoresed on 10% SDS-PAGE Laemmli gels. Primary 30 microparticles comprise antibodies to immobilize one or murine monoclonal antibodies are reacted with the blot, and more markers for detection. antibody binding can be confirmed to be linear using a In addition to the above-described assays for determining preliminary slot blot experiment. Goat anti-mouse horserad the presence or level of various markers of interest, analysis ish peroxidase-coupled antibodies (BioRad) are used as the of marker mRNA levels using routine techniques such as secondary antibody, and signal detection performed using 35 Northern analysis, reverse-transcriptase polymerase chain chemiluminescence, for example, with the Renaissance reaction (RT-PCR), or any other methods based on hybrid chemiluminescence kit (New England Nuclear; Boston, ization to a nucleic acid sequence that is complementary to Mass.) according to the manufacturers instructions. Auto a portion of the marker coding sequence (e.g., slot blot radiographs of the blots are analyzed using a scanning hybridization) are also within the scope of the present densitometer (Molecular Dynamics; Sunnyvale, Calif.) and 40 invention. Applicable PCR amplification techniques are normalized to a positive control. Values are reported, for described in, e.g., Ausubel et al., Current Protocols in example, as a ratio between the actual value to the positive Molecular Biology, John Wiley & Sons, Inc. New York control (densitometric index). Such methods are well known (1999), Chapter 7 and Supplement 47: Theophilus et al., in the art as described, for example, in Parra et al., J. Vasc. “PCR Mutation Detection Protocols. Humana Press, Surg., 28:669-675 (1998). 45 (2002); and Innis et al., PCR Protocols, San Diego, Aca Alternatively, a variety of immunohistochemical assay demic Press, Inc. (1990). General nucleic acid hybridization techniques can be used to determine the presence or level of methods are described in Anderson, "Nucleic Acid Hybrid one or more markers in a sample. The term immunohisto ization.” BIOS Scientific Publishers, 1999. Amplification or chemical assay encompasses techniques that utilize the hybridization of a plurality of transcribed nucleic acid visual detection of fluorescent dyes or enzymes coupled 50 sequences (e.g., mRNA or cDNA) can also be performed (i.e., conjugated) to antibodies that react with the marker of from mRNA or cDNA sequences arranged in a microarray. interest using fluorescent microscopy or light microscopy Microarray methods are generally described in Hardiman, and includes, without limitation, direct fluorescent antibody “Microarrays Methods and Applications: Nuts & Bolts.” assay, indirect fluorescent antibody (IFA) assay, anticomple DNA Press, 2003; and Baldi et al., “DNA Microarrays and ment immunofluorescence, avidin-biotin immunofluores 55 Gene Expression: From Experiments to Data Analysis and cence, and immunoperoxidase assays. An IFA assay, for Modeling.' Cambridge University Press, 2002. example, is useful for determining whether a sample is Analysis of the genotype of a marker Such as a genetic positive for ANCA, the level of ANCA in a sample, whether marker can be performed using techniques known in the art a sample is positive for p ANCA, the level of p ANCA in a including, without limitation, polymerase chain reaction sample, and/or an ANCA staining pattern (e.g., cANCA, 60 (PCR)-based analysis, sequence analysis, and electropho pANCA, NSNA, and/or SAPPA staining pattern). The con retic analysis. A non-limiting example of a PCR-based centration of ANCA in a sample can be quantitated, e.g., analysis includes a Taqman(R) allelic discrimination assay through endpoint titration or through measuring the visual available from Applied Biosystems. Non-limiting examples intensity of fluorescence compared to a known reference of sequence analysis include Maxam-Gilbert sequencing, standard. 65 Sanger sequencing, capillary array DNA sequencing, ther Alternatively, the presence or level of a marker of interest mal cycle sequencing (Sears et al., Biotechniques, 13:626 can be determined by detecting or quantifying the amount of 633 (1992)), Solid-phase sequencing (Zimmerman et al., US 9,482,672 B2 67 68 Methods Mol. Cell. Biol., 3:39-42 (1992)), sequencing with preferred embodiment, the methods, assays, systems, and mass spectrometry Such as matrix-assisted laser desorption/ code provided herein use a combination of at least two ionization time-of-flight mass spectrometry (MALDI-TOF/ statistical algorithms. MS: Fu et al., Nature Biotech., 16:381-384 (1998)), and In some embodiments, the first statistical algorithm is a sequencing by hybridization (Chee et al., Science, 274:610 learning statistical classifier system selected from the group 614 (1996); Drmanac et al., Science, 260:1649-1652 (1993); consisting of a random forest (RF), classification and regres Drmanac et al., Nature Biotech., 16:54-58 (1998)). Non sion tree (C&RT), boosted tree, neural network (NN), Sup limiting examples of electrophoretic analysis include slab port vector machine (SVM), general chi-squared automatic gel electrophoresis Such as agarose or polyacrylamide gel interaction detector model, interactive tree, multiadaptive electrophoresis, capillary electrophoresis, and denaturing 10 regression spline, machine learning classifier, and combina gradient gel electrophoresis. Other methods for genotyping tions thereof. In certain instances, the first statistical algo an individual at a polymorphic site in a marker include, e.g., rithm is a single learning statistical classifier system. Pref the INVADER(R) assay from Third Wave Technologies, Inc., erably, the single learning statistical classifier system restriction fragment length polymorphism (RFLP) analysis, comprises a tree-based statistical algorithm such as a RF or allele-specific oligonucleotide hybridization, a heteroduplex 15 C&RT. In certain other instances, the first statistical algo mobility assay, and single strand conformational polymor rithm is a combination of at least two learning statistical phism (SSCP) analysis. classifier systems, e.g., used in tandem or parallel. As a Several markers of interest may be combined into one test non-limiting example, a RF can first be used to generate a for efficient processing of a multiple of samples. In addition, prediction or probability value based upon the diagnostic one skilled in the art would recognize the value of testing marker profile, alone or in combination with a symptom multiple samples (e.g., at Successive time points, etc.) from profile, and a NN (e.g., artificial NN) can then be used to the same Subject. Such testing of serial samples can allow classify the sample as a non-IBD sample or IBD sample the identification of changes in marker levels over time. based upon the prediction or probability value and the same Increases or decreases in marker levels, as well as the or different diagnostic marker profile or combination of absence of change in marker levels, can also provide useful 25 profiles. The hybrid RF/NN learning statistical classifier information to classify IBS or to rule out diseases and system of the present invention typically classifies the disorders associated with IBS-like symptoms. sample as a non-IBD sample with a sensitivity, specificity, A panel for measuring one or more of the markers positive predictive value, negative predictive value, and/or described above may be constructed to provide relevant overall accuracy of at least about 75%, 76%, 77%, 78%, information related to the approach of the present invention 30 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, for classifying a sample as being associated with IBS. Such 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or a panel may be constructed to determine the presence or 99%. level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, In certain embodiments, the second statistical algorithm 18, 19, 20, 25, 30, 35, 40, or more individual markers. The comprises any of the learning statistical classifier systems analysis of a single marker or Subsets of markers can also be 35 described above. In certain instances, the second statistical carried out by one skilled in the art in various clinical algorithm is a single learning statistical classifier system settings. These include, but are not limited to, ambulatory, Such as, for example, a tree-based statistical algorithm (e.g., urgent care, critical care, intensive care, monitoring unit, RF or C&RT). In certain other instances, the second statis inpatient, outpatient, physician office, medical clinic, and tical algorithm is a combination of at least two learning health screening settings. 40 statistical classifier systems, e.g., used in tandem or parallel. The analysis of markers could be carried out in a variety As a non-limiting example, a RF can first be used to generate of physical formats as well. For example, the use of micro a prediction or probability value based upon the diagnostic titer plates or automation could be used to facilitate the marker profile, alone or in combination with a symptom processing of large numbers of test samples. Alternatively, profile, and a NN (e.g., artificial NN) or SVM can then be single sample formats could be developed to facilitate 45 used to classify the non-IBD sample as a non-IBS sample or treatment and diagnosis in a timely fashion. IBS sample based upon the prediction or probability value and the same or different diagnostic marker profile or VIII. Statistical Algorithms combination of profiles. The hybrid RF/NN or RF/SVM learning statistical classifier system described herein typi In some aspects, the present invention provides methods, 50 cally classifies the sample as an IBS sample with a sensi assays, systems, and code for classifying whether a sample tivity, specificity, positive predictive value, negative predic is associated with IBS using a statistical algorithm or tive value, and/or overall accuracy of at least about 75%, process to classify the sample as an IBS sample or non-IBS 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, sample. In other aspects, the present invention provides 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, methods, systems, and code for classifying whether a sample 55 96%, 97%, 98%, or 99%. is associated with IBS using a first statistical algorithm or In some instances, the data obtained from using the process to classify the sample as a non-IBD sample or IBD learning statistical classifier system or systems can be pro sample (i.e., IBD rule-out step), followed by a second cessed using a processing algorithm. Such a processing statistical algorithm or process to classify the non-IBD algorithm can be selected, for example, from the group sample as an IBS sample or non-IBS sample (i.e., IBS 60 consisting of a multilayer perceptron, backpropagation net rule-in step). Preferably, the statistical algorithms or pro work, and Levenberg-Marquardt algorithm. In other cesses independently comprise one or more learning statis instances, a combination of Such processing algorithms can tical classifier systems. As described herein, a combination be used, such as in a parallel or serial fashion. of learning statistical classifier systems advantageously pro The term “statistical algorithm' or “statistical process” vides improved sensitivity, specificity, negative predictive 65 includes any of a variety of statistical analyses used to value, positive predictive value, and/or overall accuracy for determine relationships between variables. In the present classifying whether a sample is associated with IBS. In a invention, the variables are the presence or level of at least US 9,482,672 B2 69 70 one marker of interest and/or the presence or severity of at Calif.). See, e.g., Breiman, Machine Learning, 45:5-32 least one IBS-related symptom. Any number of markers (2001); and http://stat-www.berkeley.edu/users/breiman/ and/or symptoms can be analyzed using a statistical algo RandomForests/cc home.htm, for a description of random rithm described herein. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9. forests. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, Classification and regression trees represent a computer 50, 55, 60, or more biomarkers and/or symptoms can be intensive alternative to fitting classical regression models included in a statistical algorithm. In one embodiment, and are typically used to determine the best possible model logistic regression is used. In another embodiment, linear for a categorical or continuous response of interest based regression is used. In certain instances, the statistical algo upon one or more predictors. Classification and regression rithms of the present invention can use a quantile measure 10 tree analysis can be performed, e.g., using the CART soft ment of a particular marker within a given population as a ware available from Salford Systems or the Statistica data variable. Quantiles are a set of “cut points' that divide a analysis software available from StatSoft, Inc. (Tulsa, sample of data into groups containing (as far as possible) Okla.). A description of classification and regression trees is equal numbers of observations. For example, quartiles are found, e.g., in Breiman et al. “Classification and Regression values that divide a sample of data into four groups con 15 Trees.” Chapman and Hall, New York (1984); and Steinberg taining (as far as possible) equal numbers of observations. et al., “CART: Tree-Structured Non-Parametric Data Analy The lower quartile is the data value a quarter way up through sis.” Salford Systems, San Diego, (1995). the ordered data set; the upper quartile is the data value a Neural networks are interconnected groups of artificial quarter way down through the ordered data set. Quintiles are neurons that use a mathematical or computational model for values that divide a sample of data into five groups contain information processing based on a connectionist approach to ing (as far as possible) equal numbers of observations. The computation. Typically, neural networks are adaptive sys present invention can also include the use of percentile tems that change their structure based on external or internal ranges of marker levels (e.g., tertiles, quartile, quintiles, information that flows through the network. Specific etc.), or their cumulative indices (e.g., quartile sums of examples of neural networks include feed-forward neural marker levels, etc.) as variables in the algorithms (just as 25 networks such as perceptrons, single-layer perceptrons, with continuous variables). multi-layer perceptrons, backpropagation networks, ADA Preferably, the statistical algorithms of the present inven LINE networks, MADALINE networks, Learnmatrix net tion comprise one or more learning statistical classifier works, radial basis function (RBF) networks, and self systems. As used herein, the term “learning statistical clas organizing maps or Kohonen self-organizing networks: sifier system’ includes a machine learning algorithmic tech 30 recurrent neural networks Such as simple recurrent networks nique capable of adapting to complex data sets (e.g., panel and Hopfield networks; stochastic neural networks Such as of markers of interest and/or list of IBS-related symptoms) Boltzmann machines; modular neural networks such as and making decisions based upon Such data sets. In some committee of machines and associative neural networks; and embodiments, a single learning statistical classifier system other types of networks such as instantaneously trained Such as a classification tree (e.g., random forest) is used. In 35 neural networks, spiking neural networks, dynamic neural other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, networks, and cascading neural networks. Neural network or more learning statistical classifier systems are used, analysis can be performed, e.g., using the Statistica data preferably in tandem. Examples of learning statistical clas analysis software available from StatSoft, Inc. See, e.g., sifier systems include, but are not limited to, those using Freeman et al. In “Neural Networks: Algorithms, Applica inductive learning (e.g., decision/classification trees Such as 40 tions and Programming Techniques.” Addison-Wesley Pub random forests, classification and regression trees (C&RT), lishing Company (1991); Zadeh, Information and Control, boosted trees, etc.), Probably Approximately Correct (PAC) 8:338-353 (1965); Zadeh, “IEEE Trans. on Systems, Man learning, connectionist learning (e.g., neural networks (NN), and Cybernetics.” 3:28-44 (1973); Gersho et al., In “Vector artificial neural networks (ANN), neuro fuzzy networks Quantization and Signal Compression.” Kluywer Academic (NFN), network structures, perceptrons such as multi-layer 45 Publishers, Boston, Dordrecht, London (1992); and Has perceptrons, multi-layer feed-forward networks, applica soun, “Fundamentals of Artificial Neural Networks, MIT tions of neural networks, Bayesian learning in belief net Press, Cambridge, Mass., London (1995), for a description works, etc.), reinforcement learning (e.g., passive learning in of neural networks. a known environment such as naive learning, adaptive Support vector machines are a set of related Supervised dynamic learning, and temporal difference learning, passive 50 learning techniques used for classification and regression learning in an unknown environment, active learning in an and are described, e.g., in Cristianini et al., “An Introduction unknown environment, learning action-value functions, to Support Vector Machines and Other Kernel-Based Learn applications of reinforcement learning, etc.), and genetic ing Methods. Cambridge University Press (2000). Support algorithms and evolutionary programming. Other learning vector machine analysis can be performed, e.g., using the statistical classifier systems include Support vector machines 55 SVM'8" software developed by Thorsten Joachims (Cornell (e.g., Kernel methods), multivariate adaptive regression University) or using the LIBSVM software developed by splines (MARS), Levenberg-Marquardt algorithms, Gauss Chih-Chung Chang and Chih-Jen Lin (National Taiwan Newton algorithms, mixtures of Gaussians, gradient descent University). algorithms, and learning vector quantization (LVO). The learning statistical classifier systems described herein Random forests are learning statistical classifier systems 60 can be trained and tested using a cohort of samples (e.g., that are constructed using an algorithm developed by Leo serological samples) from healthy individuals, IBS patients, Breiman and Adele Cutler. Random forests use a large IBD patients, and/or Celiac disease patients. For example, number of individual decision trees and decide the class by samples from patients diagnosed by a physician, and pref choosing the mode (i.e., most frequently occurring) of the erably by a gastroenterologist as having IBD using a biopsy, classes as determined by the individual trees. Random forest 65 colonoscopy, or an immunoassay as described in, e.g., U.S. analysis can be performed, e.g., using the RandomForests Pat. No. 6,218,129, are suitable for use in training and software available from Salford Systems (San Diego, testing the learning statistical classifier systems of the pres US 9,482,672 B2 71 72 ent invention. Samples from patients diagnosed with IBD the negative predictive value in a population having a can also be stratified into Crohn's disease or ulcerative disease prevalence is in the range of about 70% to about colitis using an immunoassay as described in, e.g., U.S. Pat. 99% and can be, for example, at least about 70%, 75%, 76%, Nos. 5,750,355 and 5,830,675. Samples from patients diag 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, nosed with IBS using a published criteria such as the 5 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, Manning, Rome I, Rome II, or Rome III diagnostic criteria 97%, 98%, or 99%. In preferred embodiments, the negative are suitable for use in training and testing the learning predictive value of classifying IBS is at least about 87% statistical classifier systems of the present invention. when a combination of learning statistical classifier systems Samples from healthy individuals can include those that is used (see, Example 10 from US Patent Publication No. were not identified as IBD and/or IBS samples. One skilled 10 2008/0085524, which is incorporated herein by reference in in the art will know of additional techniques and diagnostic its entirety for all purposes). criteria for obtaining a cohort of patient samples that can be The term “positive predictive value” or “PPV refers to used in training and testing the learning statistical classifier the probability that an individual identified as having IBS systems of the present invention. actually has the disease. Positive predictive value can be As used herein, the term “sensitivity” refers to the prob- 15 calculated as the number of true positives divided by the sum ability that a diagnostic method, system, or code of the of the true positives and false positives. Positive predictive present invention gives a positive result when the sample is value is determined by the characteristics of the diagnostic positive, e.g., having IBS. Sensitivity is calculated as the method, system, or code as well as the prevalence of the number of true positive results divided by the sum of the true disease in the population analyzed. The statistical algorithms positives and false negatives. Sensitivity essentially is a can be selected such that the positive predictive value in a measure of how well a method, system, or code of the population having a disease prevalence is in the range of present invention correctly identifies those with IBS from about 80% to about 99% and can be, for example, at least those without the disease. The statistical algorithms can be about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, selected such that the sensitivity of classifying IBS is at least 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In preferred about 60%, and can be, for example, at least about 65%, 25 embodiments, the positive predictive value of classifying 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, IBS is at least about 90% when a combination of learning 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, statistical classifier systems is used (see, Example 10 from 94%. 95%, 96%, 97%, 98%, or 99%. In preferred embodi US Patent Publication No. 2008/0085524, which is incor ments, the sensitivity of classifying IBS is at least about 90% porated herein by reference in its entirety for all purposes). when a combination of learning statistical classifier systems 30 Predictive values, including negative and positive predic is used (see, Example 10 from US Patent Publication No. tive values, are influenced by the prevalence of the disease 2008/0085524, which is incorporated herein by reference in in the population analyzed. In the methods, systems, and its entirety for all purposes) or at least about 85% when a code of the present invention, the statistical algorithms can single learning statistical classifier system is used (see, be selected to produce a desired clinical parameter for a Example 11 from US Patent Publication No. 2008/0085524, 35 clinical population with a particular IBS prevalence. For which is incorporated herein by reference in its entirety for example, learning statistical classifier systems can be all purposes). selected for an IBS prevalence of up to about 1%, 2%. 3%, The term “specificity' refers to the probability that a 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, diagnostic method, system, or code of the present invention 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, which can gives a negative result when the sample is not positive, e.g., 40 be seen, e.g., in a clinicians office Such as a gastroenter not having IBS. Specificity is calculated as the number of ologists office or a general practitioners office. true negative results divided by the sum of the true negatives As used herein, the term “overall agreement” or "overall and false positives. Specificity essentially is a measure of accuracy” refers to the accuracy with which a method, how well a method, system, or code of the present invention system, or code of the present invention classifies a disease excludes those who do not have IBS from those who have 45 state. Overall accuracy is calculated as the sum of the true the disease. The statistical algorithms can be selected Such positives and true negatives divided by the total number of that the specificity of classifying IBS is at least about 70%, sample results and is affected by the prevalence of the for example, at least about 75%, 80%, 85%, 86%, 87%, disease in the population analyzed. For example, the statis 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, tical algorithms can be selected Such that the overall accu 98%, or 99%. In preferred embodiments, the specificity of 50 racy in a patient population having a disease prevalence is at classifying IBS is at least about 86% when a combination of least about 60%, and can be, for example, at least about learning statistical classifier systems is used (see, Example 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 10 from US Patent Publication No. 2008/0085524, which is 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, incorporated herein by reference in its entirety for all pur 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In preferred poses) or at least about 84% when a single learning statis- 55 embodiments, the overall accuracy of classifying IBS is at tical classifier system is used (see, Example 11 from US least about 80% when a combination of learning statistical Patent Publication No. 2008/0085524, which is incorporated classifier systems is used (see, Example 10 from US Patent herein by reference in its entirety for all purposes). Publication No. 2008/0085524, which is incorporated herein As used herein, the term “negative predictive value” or by reference in its entirety for all purposes). “NPV refers to the probability that an individual identified 60 as not having IBS actually does not have the disease. IX. Disease Classification System Negative predictive value can be calculated as the number of true negatives divided by the Sum of the true negatives and FIG. 13 illustrates a disease classification system (DCS) false negatives. Negative predictive value is determined by (200) according to one embodiment of the present invention. the characteristics of the diagnostic method, system, or code 65 As shown therein, a DCS includes a DCS intelligence as well as the prevalence of the disease in the population module (205), such as a computer, having a processor (215) analyzed. The statistical algorithms can be selected Such that and memory module (210). The intelligence module also US 9,482,672 B2 73 74 includes communication modules (not shown) for transmit According to one embodiment, each client system and all ting and receiving information over one or more direct of its components are operator configurable using applica connections (e.g., USB, Firewire, or other interface) and one tions, such as a browser, including computer code run using or more network connections (e.g., including a modem or a central processing unit Such as an Intel(R) Pentium R other network interface device). The memory module may 5 processor or the like. Similarly, the intelligence module and include internal memory devices and one or more external all of its components might be operator configurable using memory devices. The intelligence module also includes a application(s) including computer code run using a central processing unit (215) such as an Intel Pentium processor or display module (225). Such as a monitor or printer. In one the like, or multiple processor units. Computer code for aspect, the intelligence module receives data Such as patient operating and configuring the intelligence module to process test results from a data acquisition module such as a test 10 data and test results as described herein is preferably down system (250), either through a direct connection or over a loaded and stored on a hard disk, but the entire program network (240). For example, the test system may be con code, or portions thereof, may also be stored in any other figured to run multianalyte tests on one or more patient volatile or non-volatile memory medium or device as is well samples (255) and automatically provide the test results to known, such as a ROM or RAM, or provided on any other the intelligence module. The data may also be provided to 15 computer readable medium (260) capable of storing pro the intelligence module via direct input by a user or it may gram code, such as a compact disk (CD) medium, digital be downloaded from a portable medium such as a compact versatile disk (DVD) medium, a floppy disk, ROM, RAM, disk (CD) or a digital versatile disk (DVD). The test system and the like. may be integrated with the intelligence module, directly The computer code for implementing various aspects and coupled to the intelligence module, or it may be remotely embodiments of the present invention can be implemented coupled with the intelligence module over the network. The in any programming language that can be executed on a intelligence module may also communicate data to and from computer system such as, for example, in C, C++, C#, one or more client systems (230) over the network as is well HTML, Java, JavaScript, or any other scripting language, known. For example, a requesting physician or healthcare such as VBScript. Additionally, the entire program code, or provider may obtain and view a report from the intelligence 25 portions thereof, may be embodied as a carrier signal, which module, which may be resident in a laboratory or hospital, may be transmitted and downloaded from a software source using a client system (230). (e.g., server) over the Internet, or over any other conven tional network connection as is well known (e.g., extranet, The network can be a LAN (local area network), WAN VPN, LAN, etc.) using any communication medium and (wide area network), wireless network, point-to-point net protocols (e.g., TCP/IP, HTTP, HTTPS, Ethernet, etc.) as work, star network, token ring network, hub network, or 30 are well known. other configuration. As the most common type of network in According to one embodiment, the intelligence module current use is a TCP/IP (Transfer Control Protocol and implements a disease classification process for analyzing Internet Protocol) network such as the global internetwork patient test results and/or questionnaire responses to deter of networks often referred to as the “Internet” with a capital mine whether a patient sample is associated with IBS. The “I,” that will be used in many of the examples herein, but it 35 data may be stored in one or more data tables or other logical should be understood that the networks that the present data structures in memory (210) or in a separate storage or invention might use are not so limited, although TCP/IP is database system coupled with the intelligence module. One the currently preferred protocol. or more statistical processes are typically applied to a data Several elements in the system shown in FIG. 2 from US set including test data for a particular patient. For example, Patent Publication No. 2008/0085524 may include conven 40 the test data might include a diagnostic marker profile, tional, well-known elements that need not be explained in which comprises data indicating the presence or level of at detail here. For example, the intelligence module could be least one marker in a sample from the patient. The test data implemented as a desktop personal computer, workstation, might also include a symptom profile, which comprises data mainframe, laptop, etc. Each client system could include a indicating the presence or severity of at least one symptom desktop personal computer, workstation, laptop, PDA, cell 45 associated with IBS that the patient is experiencing or has recently experienced. In one aspect, a statistical process phone, or any WAP-enabled device or any other computing produces a statistically derived decision classifying the device capable of interfacing directly or indirectly to the patient sample as an IBS sample or non-IBS sample based Internet or other network connection. A client system typi upon the diagnostic marker profile and/or symptom profile. cally runs an HTTP client, e.g., a browsing program, Such as In another aspect, a first statistical process produces a first Microsoft’s Internet ExplorerTM browser, Netscape's Navi 50 statistically derived decision classifying the patient sample gatorTM browser, Opera's browser, or a WAP-enabled as an IBD sample or non-IBD sample based upon the browser in the case of a cell phone, PDA or other wireless diagnostic marker profile and/or symptom profile. If the device, or the like, allowing a user of the client system to patient sample is classified as a non-IBD sample, a second access, process, and view information and pages available to statistical process is applied to the same or a different data it from the intelligence module over the network. Each client 55 set to produce a second statistically derived decision clas system also typically includes one or more user interface sifying the non-IBD sample as an IBS sample or non-IBS devices, such as a keyboard, a mouse, touch screen, pen or sample. The first and/or the second statistically derived the like, for interacting with a graphical user interface (GUI) decision may be displayed on a display device associated provided by the browser on a display (e.g., monitor Screen, with or coupled to the intelligence module, or the decision(s) LCD display, etc.) (235) in conjunction with pages, forms, 60 may be provided to and displayed at a separate system, e.g., and other information provided by the intelligence module. a client system (230). The displayed results allow a physi As discussed above, the present invention is Suitable for use cian to make a reasoned diagnosis or prognosis. with the Internet, which refers to a specific global internet work of networks. However, it should be understood that X. Therapy and Therapeutic Monitoring other networks can be used instead of the Internet, such as 65 an intranet, an extranet, a virtual private network (VPN), a Once a sample from an individual has been classified as non-TCP/IP based network, any LAN or WAN, or the like. an IBS sample, the methods, systems, and code of the US 9,482,672 B2 75 76 present invention can further comprise administering to the organic acids and bases; Sweetening agents; and flavoring individual a therapeutically effective amount of a drug agents. The dosage forms may also comprise biodegradable useful for treating one or more symptoms associated with polymer beads, dextran, and cyclodextrin inclusion com IBS (i.e., an IBS drug). For therapeutic applications, the IBS plexes. drug can be administered alone or co-administered in com For oral administration, the therapeutically effective dose bination with one or more additional IBS drugs and/or one can be in the form of tablets, capsules, emulsions, Suspen or more drugs that reduce the side-effects associated with the sions, Solutions, syrups, sprays, lozenges, powders, and IBS drug. sustained-release formulations. Suitable excipients for oral IBS drugs can be administered with a suitable pharma administration include pharmaceutical grades of mannitol, ceutical excipient as necessary and can be carried out via any 10 lactose, starch, magnesium Stearate, sodium saccharine, tal of the accepted modes of administration. Thus, administra cum, cellulose, glucose, gelatin, Sucrose, magnesium car tion can be, for example, intravenous, topical, Subcutaneous, bonate, and the like. transcutaneous, transdermal, intramuscular, oral, buccal, In some embodiments, the therapeutically effective dose Sublingual, gingival, palatal, intra-joint, parenteral, intra takes the form of a pill, tablet, or capsule, and thus, the arteriole, intradermal, intraventricular, intracranial, intrap 15 dosage form can contain, along with an IBS drug, any of the eritoneal, intralesional, intranasal, rectal, vaginal, or by following: a diluent such as lactose. Sucrose, dicalcium inhalation. By “co-administer it is meant that an IBS drug phosphate, and the like; a disintegrant such as starch or is administered at the same time, just prior to, or just after derivatives thereof: a lubricant Such as magnesium Stearate the administration of a second drug (e.g., another IBS drug, and the like; and a binder Such a starch, gum acacia, a drug useful for reducing the side-effects of the IBS drug, polyvinylpyrrolidone, gelatin, cellulose and derivatives etc.). thereof. An IBS drug can also be formulated into a supposi Atherapeutically effective amount of an IBS drug may be tory disposed, for example, in a polyethylene glycol (PEG) administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or carrier. more times, or the dose may be administered by continuous Liquid dosage forms can be prepared by dissolving or infusion. The dose may take the form of solid, semi-solid, 25 dispersing an IBS drug and optionally one or more pharma lyophilized powder, or liquid dosage forms, such as, for ceutically acceptable adjuvants in a carrier Such as, for example, tablets, pills, pellets, capsules, powders, Solutions, example, aqueous saline (e.g., 0.9% w/v Sodium chloride), Suspensions, emulsions, Suppositories, retention enemas, aqueous dextrose, glycerol, ethanol, and the like, to form a creams, ointments, lotions, gels, aerosols, foams, or the like, Solution or Suspension, e.g., for oral, topical, or intravenous preferably in unit dosage forms suitable for simple admin 30 administration. An IBS drug can also be formulated into a istration of precise dosages. retention enema. As used herein, the term “unit dosage form” refers to For topical administration, the therapeutically effective physically discrete units Suitable as unitary dosages for dose can be in the form of emulsions, lotions, gels, foams, human Subjects and other mammals, each unit containing a creams, jellies, solutions, Suspensions, ointments, and trans predetermined quantity of an IBS drug calculated to produce 35 dermal patches. For administration by inhalation, an IBS the desired onset, tolerability, and/or therapeutic effects, in drug can be delivered as a dry powder or in liquid form via association with a suitable pharmaceutical excipient (e.g., an a nebulizer. For parenteral administration, the therapeuti ampoule). In addition, more concentrated dosage forms may cally effective dose can be in the form of sterile injectable be prepared, from which the more dilute unit dosage forms Solutions and sterile packaged powders. Preferably, inject may then be produced. The more concentrated dosage forms 40 able solutions are formulated at a pH of from about 4.5 to thus will contain Substantially more than, e.g., at least 1, 2, about 7.5. 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the IBS The therapeutically effective dose can also be provided in drug. a lyophilized form. Such dosage forms may include a buffer, Methods for preparing Such dosage forms are known to e.g., bicarbonate, for reconstitution prior to administration, those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTI 45 or the buffer may be included in the lyophilized dosage form CAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, Pa. for reconstitution with, e.g., water. The lyophilized dosage (1990)). The dosage forms typically include a conventional form may further comprise a suitable vasoconstrictor, e.g., pharmaceutical carrier or excipient and may additionally epinephrine. The lyophilized dosage form can be provided in include other medicinal agents, carriers, adjuvants, diluents, a Syringe, optionally packaged in combination with the tissue permeation enhancers, Solubilizers, and the like. 50 buffer for reconstitution, such that the reconstituted dosage Appropriate excipients can be tailored to the particular form can be immediately administered to an individual. dosage form and route of administration by methods well In therapeutic use for the treatment of IBS, an IBS drug known in the art (see, e.g., REMINGTON'S PHARMACEUTICAL can be administered at the initial dosage of from about 0.001 SCIENCES, Supra). mg/kg to about 1000 mg/kg daily. A daily dose range of from Examples of suitable excipients include, but are not 55 about 0.01 mg/kg to about 500 mg/kg, from about 0.1 mg/kg limited to, lactose, dextrose, Sucrose, Sorbitol, mannitol, to about 200 mg/kg, from about 1 mg/kg to about 100 Starches, gum acacia, calcium phosphate, alginates, traga mg/kg, or from about 10 mg/kg to about 50 mg/kg, can be canth, gelatin, calcium silicate, microcrystalline cellulose, used. The dosages, however, may be varied depending upon polyvinylpyrrolidone, cellulose, water, saline, syrup, meth the requirements of the individual, the severity of IBS ylcellulose, ethylcellulose, hydroxypropylmethylcellulose, 60 symptoms, and the IBS drug being employed. For example, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, dosages can be empirically determined considering the Carbopol 980, Carbopol 981, etc. The dosage forms can severity of IBS symptoms in an individual classified as additionally include lubricating agents such as talc, magne having IBS according to the methods described herein. The sium Stearate, and mineral oil; wetting agents; emulsifying dose administered to an individual, in the context of the agents: Suspending agents; preserving agents such as 65 present invention, should be sufficient to affect a beneficial methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the therapeutic response in the individual over time. The size of parabens); pH adjusting agents such as inorganic and the dose can also be determined by the existence, nature, and US 9,482,672 B2 77 78 extent of any adverse side-effects that accompany the tricyclic antidepressants include, but are not limited to, administration of a particular IBS drug in an individual. desipramine, nortriptyline, protriptyline, amitriptyline, clo Determination of the proper dosage for a particular situation mipramine, doxepin, imipramine, trimipramine, maproti is within the skill of the practitioner. Generally, treatment is line, amoxapine, clomipramine, free bases thereof, pharma initiated with smaller dosages which are less than the ceutically acceptable salts thereof, derivatives thereof, optimum dose of the IBS drug. Thereafter, the dosage is analogs thereof, and combinations thereof. increased by small increments until the optimum effect Chloride channel activators are useful for the treatment of under circumstances is reached. For convenience, the total IBS symptoms such as constipation. A non-limiting example daily dosage may be divided and administered in portions of a chloride channel activator is lubiprostone (AmitizatM), during the day, if desired. 10 a free base thereof, a pharmaceutically acceptable salt As used herein, the term "IBS drug includes all phar maceutically acceptable forms of a drug that is useful for thereof, a derivative thereof, or an analog thereof. In addi treating one or more symptoms associated with IBS. For tion, chloride channel blockers such as crofelemer are useful example, the IBS drug can be in a racemic or isomeric for the treatment of IBS symptoms such as diarrhea. Gua mixture, a solid complex bound to an ion exchange resin, or 15 nylate cyclase agonists such as MD-1100 are useful for the the like. In addition, the IBS drug can be in a solvated form. treatment of constipation associated with IBS (see, e.g., The term “IBS drug is also intended to include all phar Bryant et al., Gastroenterol., 128:A-257 (2005)). Antibiotics maceutically acceptable salts, derivatives, and analogs of the Such as neomycin can also be suitable for use in treating IBS drug being described, as well as combinations thereof. constipation associated with IBS (see, e.g., Park et al., For example, the pharmaceutically acceptable salts of an Gastroenterol., 128:A-258 (2005)). Non-absorbable antibi IBS drug include, without limitation, the tartrate, succinate, otics like rifaximin (XifaxanTM) are suitable to treat small tartarate, bitartarate, dihydrochloride, Salicylate, hemisucci bowel bacterial overgrowth and/or constipation associated nate, citrate, maleate, hydrochloride, carbamate, Sulfate, with IBS (see, e.g., Sharara et al., Am. J. Gastroenterol., nitrate, and benzoate Salt forms thereof, as well as combi 101:326-333 (2006)). nations thereof and the like. Any form of an IBS drug is 25 Opioids such as kappa opiods (e.g., asimadoline) may be Suitable for use in the methods of the present invention, e.g., useful for treating pain and/or constipation associated with a pharmaceutically acceptable salt of an IBS drug, a free IBS. Neurokinin (NK) antagonists such as talnetant, sare base of an IBS drug, or a mixture thereof. dutant, and other NK2 and/or NK3 antagonists may be Suitable drugs that are useful for treating one or more useful for treating IBS symptoms such as oversensitivity of symptoms associated with IBS include, but are not limited 30 the muscles in the colon, constipation, and/or diarrhea. to, serotonergic agents, antidepressants, chloride channel Antispasmodic or anticholinergic agents such as dicyclo activators, chloride channel blockers, guanylate cyclase ago nists, antibiotics, opioids, neurokinin antagonists, antispas mine may be useful for treating IBS symptoms such as modic or anticholinergic agents, belladonna alkaloids, bar spasms in the muscles of the gut and bladder. Other anti biturates, glucagon-like peptide-1 (GLP-1) analogs, 35 spasmodic or anticholinergic agents such as belladonna corticotropin releasing factor (CRF) antagonists, probiotics, alkaloids (e.g., atropine, Scopolamine, hyoscyamine, etc.) free bases thereof, pharmaceutically acceptable salts thereof, can be used in combination with barbiturates Such as phe derivatives thereof, analogs thereof, and combinations nobarbital to reduce bowel spasms associated with IBS. thereof. Other IBS drugs include bulking agents, dopamine GLP-1 analogs such as GTP-010 may be useful for treating antagonists, carminatives, tranquilizers, dextofisopam, phe 40 IBS symptoms Such as constipation. CRF antagonists Such nyloin, timolol, and diltiazem. as astressin and probiotics such as VSLif3(R) may be useful Serotonergic agents are useful for the treatment of IBS for treating one or more IBS symptoms. One skilled in the symptoms Such as constipation, diarrhea, and/or alternating art will know of additional IBS drugs currently in use or in constipation and diarrhea. Non-limiting examples of sero development that are Suitable for treating one or more tonergic agents are described in Cash et al., Aliment. Phar 45 symptoms associated with IBS. macol. Ther, 22:1047-1060 (2005), and include 5-HT An individual can also be monitored at periodic time receptor agonists (e.g., MKC-733, etc.), 5-HT, receptor intervals to assess the efficacy of a certain therapeutic agonists (e.g., tegaserod (ZelnormTM), prucalopride, AG1 regimen once a sample from the individual has been clas 001, etc.). 5-HT receptor antagonists (e.g., alosetron (Lo sified as an IBS sample. For example, the levels of certain troneXR), cilansetron, ondansetron, granisetron, dolasetron, 50 markers change based on the therapeutic effect of a treat ramosetron, palonosetron, E-3620, DDP-225, DDP-733, ment Such as a drug. The patient is monitored to assess etc.), mixed 5-HT receptor antagonists/5-HT, receptor ago response and understand the effects of certain drugs or nists (e.g., cisapride, mosapride, renzapride, etc.), free bases treatments in an individualized approach. Additionally, thereof, pharmaceutically acceptable salts thereof, deriva patients may not respond to a drug, but the markers may tives thereof, analogs thereof, and combinations thereof. 55 Additionally, amino acids like glutamine and glutamic acid change, Suggesting that these patients belong to a special which regulate intestinal permeability by affecting neuronal population (not responsive) that can be identified by their or glial cell signaling can be administered to treat patients marker levels. These patients can be discontinued on their with IBS. current therapy and alternative treatments prescribed. Antidepressants such as selective serotonin reuptake 60 inhibitor (SSRI) or tricyclic antidepressants are particularly XI. Examples useful for the treatment of IBS symptoms such as abdominal pain, constipation, and/or diarrhea. Non-limiting examples The following examples are offered to illustrate, but not of SSRI antidepressants include citalopram, fluvoxamine, to limit, the claimed invention. paroxetine, fluoxetine, Sertraline, free bases thereof, phar 65 The Examples from US Patent Publication No. 2008/ maceutically acceptable salts thereof, derivatives thereof, 0085524, filed Aug. 14, 2007, are herein incorporated by analogs thereof, and combinations thereof. Examples of reference in their entirety for all purposes. US 9,482,672 B2 79 80 A. Example 1 B. Example 2 Evaluation of the Diagnostic Utility of Serum A Tryptase ELISA for Predicting IBS Tryptase Levels in IBS Background: This example describes a sensitive ELISA for detecting Mast cells play an important role in the pathogenesis of the presence or level of mast cell B-tryptase. See also, FIGS. irritable bowel syndrome (IBS). Increased mast cell infil 1-7. tration and activation in distal gut segments are associated Background: Mast cells play an important role in the with symptom onset and severity of IBS. Mast cells have pathogenesis of irritable bowel syndrome (IBS). Increased 10 been implicated in the elevated response of visceral afferent mast cell infiltration and activation in distal gut segments are nerves to mucosal stimulus in IBS patients. Measurement of associated with symptom onset and severity of IBS. Mast mast cell markers can have important implications in clinical diagnosis of IBS. However, this effort was hindered due to cells have been implicated in the elevated response of lack of sensitive assays. visceral afferent nerves to mucosal stimulus in IBS patients. 15 Methods: Here we report the development and validation Measurement of mast cell markers can have important of a highly sensitive two-site ELISA assay to measure implication in clinical diagnosis of IBS. However this effort tryptase level in human serum samples (detection limit was hindered due to lack of sensitive assays. 0.019 ng/ml). The assay is precise, robust, and reproducible. Methods: Here we report the development and validation Serum tryptase concentrations in healthy controls and IBS patients was measured using this assay. of a highly sensitive two-side ELISA assay to measure Results: The average serum tryptase level in healthy tryptase level in human serum samples (detection limit controls was 7.1+2.5 ng/ml. IBS-D and IBS-A patients 0.019 ng/ml). The assay is precise, robust, and reproducible. showed higher serum tryptase concentrations (10.3+8.7 Serum tryptase concentration in healthy controls and IBS ng/ml for IBS-D and 12.1+10.7 ng/ml for IBS-A) than the patients was measured using this assay. 25 healthy control subjects, while the average tryptase level Results: The average serum tryptase level in healthy was 9.6+9.4 ng/ml for IBS-C. There was a statistical differ controls was 9.32+2.1 ng/ml (n=156). IBS-D and IBS-A ence of serum tryptase levels between the IBS-C and IBS-D patients showed significant higher serum tryptase concen groups (p<0.01). Conclusion: Serum tryptase concentration is the first tration (12.71 ng/ml (n=209) for IBS-D and 11.94 ng/ml biomarker developed so far that differentiates IBS-D from (n=57) for IBS-A, p<0.01); while the average tryptase level 30 IBS-C patients. Serum tryptase levels can be combined with is 9.34 ng/ml (n=118) for IBS-C, which has no significant other IBS biomarkers to improve the accuracy of diagnosing difference from healthy controls for IBS-C. IBS. Conclusion: This is the first biomarker developed so far FIG. 8 shows the dose-dependent responses of human that differentiates IBS-D and IBS-A patients from IBS-C tryptase in the tryptase ELISA described herein. Protocol: A 35 96-well microtiter plate was coated with 100 ml of 2 mg/ml patients and healthy controls. Combining serum tryptase of anti-human tryptase antibody in Sodium carbonate (pH level with other Prometheus IBS biomarkers, we were able 9.6) at 4°C. overnight. After washing with PBST, the plate to improve the accuracy of diagnosing IBS-D and IBS-A was incubated with 300 ml/well of blocking/assay buffer patients. (5% BSA in PBS) at room temperature (RT) for 30 minutes An ELISA assay was developed for the determination of 40 with gentle agitation. After washing, 100 ml/well of serially diluted (1:8 in assay buffer) human tryptase were added to serum mast cell B-tryptase level using rabbit anti-tryptase as the plate. After incubating at RT for another 2 hours, the capture antibody and alkaline phosphatase conjugated G3 as plate was washed, and then incubated with 100 ml of detecting antibody. Luminescent substrate CPSD (disodium AP-conjugated anti-human tryptase at an optimized dilution 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricyclo 45 in assay buffer. The plate was incubated for 2 hours with 3.3.1.13.7 decan-4-yl)phenyl phosphate), was used to gentle agitation and then washed. 100 ml of Tropix. CSPD substrate was added to each well and incubated in the dark enhance the assay sensitivity. Linear dose-response curve for 30 minutes before reading the luminescence with a was observed over the standard range of 1-1000 ng/ml for luminescence plate reader. The Relative Luminescent Unit B-tryptase in buffer with 5% BSA and 10% normal human (RLU) and the tryptase concentration was plotted with the serum. The low limit of the assay was 0.019 ng/ml, the 50 Prism Graphpad Program. Tryptase detection range=0.019 intra-assay and inter-assay coefficients of variation were 5000 ng/ml. EC50=65 ng/ml. Recovery was 81.5% with 20 ng spiked in normal pooled serum. below 15% at 1 ng/ml and 1000 ng/ml tryptase concentra FIG. 9 shows the optimization of the tryptase ELISA tions. The recovery of known amounts of purified tryptase described herein. FIG. 10 shows tryptase levels in serum added to serum was 88%. The immunoassay was utilized to 55 from healthy controls (n=139) and from subjects with IBS examine serum levels of tryptase from healthy controls, (n=378). Tryptase levels were measured by ELISA in serum IBS-C and IBS-D patients. Average serum tryptase level in samples diluted 10 times with assay buffer. IBS patients are healthy control was 7.0+2.1 ng/ml (n=113). The average also shown according to its subtypes: IBS-D (n=206): diar tryptase level in IBS-C and IBS-D were 9.6 ng/ml (n=116) rhea predominant; IBS-C (n=116): constipation predomi 60 nant; IBS-A (n=56): alternating symptoms. Each value is the and 12.7 (n=209) respectively. Using the cutoff value of 11.4 average of duplicate determinations. Solid lines are the ng/ml (average +2SD), the assay specificity for GI healthy median value from each group. The cutoff value of 12 ng/ml control was 82% (n=156), the assay sensitivity for IBS-C was calculated from healthy subjects with median plus 2SD and IBS-D were 21.5% and 24.9% respectively. Combine (dotted line). *p-0.0001 vs healthy subjects; **p<0.0001 vs with symptoms of the patient and other markers for IBS, 65 IBS-D; Mann Whitney U test. Table 2 provides a summary serum tryptase levels may help differentiate IBS-D from of tryptase levels in IBS Subtypes and healthy subjects other types of irritable bowel syndrome. (cutoff=12.0 ng/ml). US 9,482,672 B2 81 82 TABLE 2 all or a subset of the 93 questions set forth on pages 920-936 of the Rome III Diagnostic Questionnaire for the Adult Tryptase levels (ng/ml) in IBS Subtypes and Functional GI Disorders (Appendix C), available at http:// healthy Subjects (cutoff = 12.0 ng/ml). www.romecriteria.org/pdfs/AdultFunctGIQ.pdf. Preferably, Healthy the first section of the questionnaire contains 16 of the 93 Control IBS IBS-D IBS-C IBS-A questions set forth in the Rome III Diagnostic Questionnaire Subjects (n) 139 381 209 116 56 (see, Table 3). Alternatively, the first section of the ques Mean (ng/ml) 7.1 10.3 10.3 9.6 12.1 tionnaire can contain a Subset (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9. STDEV 2.4 9.2 8.7 9.4 10.7 10, 11, 12, 13, 14, or 15) of the 16 questions shown in Table Positive 9 82 44 22 16 10 3. As a non-limiting example, the following 10 questions set forth in Table 3 can be included in the questionnaire: FIG. 11 shows the increased serum level of histamine and Question Nos. 2, 3, 5, 6, 9, 10, 11, 13, 15, and 16. One PGE in IBS patients vs healthy controls. PGE was mea skilled in the art will appreciate that the first section of the sured in 50 ul of serum by ELISA using a kit from Cayman. 15 questionnaire can comprise questions similar to the ques Histamine was measured in 10 ul of serum using an Immu tions shown in Table 3 regarding pain, discomfort, and/or notech EIA kit. The samples were tested in duplicate. The changes in stool consistency. Solid line is the median of each group. As such, in certain aspects, IBS-D may be diagnosed or distinguished from TABLE 3 other clinical subtypes of IBS by detecting a higher level of PGE relative to healthy control, IBS-A, and/or IBS-C Exemplary first section of a questionnaire for identifying samples or standards. In certain other aspects, IBS-D or the presence or severity of IBS-related symptoms. IBS-A may be diagnosed or distinguished from IBS-C by 1. In the last 3 months, Newer how often did you have Less unan one day a monun detecting a higher level of histamine relative to healthy pain or discomfort in the One day a month control and/or IBS-C samples or standards. 25 middle of your chest Two to three days a month Conclusions: (1) A sandwich ELISA method was devel (not related to heart One day a week oped that can measure serum tryptase level with high problems)? More than one day a week sensitivity, accuracy, and precision. The assay has a high Every day 2. In the last 3 months, Newer degree of reproducibility and is suitable for routine testing of how often did you have Less unan one day a monun a large number of human sera. (2) Using this ELISA, we 30 heartburn (a burning ne day a montin found significant differences in serum tryptase levels discomfort or burning Two to three days a month between healthy controls and IBS patients. Among the IBS pain in your chest)? One day a week patients, IBS-D and IBS-A patients had statistically higher More than one day a week tryptase levels compared to IBS-C patients. (3) Additional Every day 35 3. In the last 3 months, Never --> mast cell markers, histamine and PGE, were also found to how often did you feel Less unan one day a monun be aberrant in IBS patient serum samples. Combining these uncomfortably full after ne day a montin markers with tryptase resulted in improved diagnostic accu a regular-sized meal? Two to three days a month racy of IBS. One day a week More than one day a week C. Example 3 40 Every day 4. In the last 3 months, Never --> how often were you Less unan one day a monun Questionnaire for Identifying the Presence or unable to finish a ne day a montin Severity of Symptoms Associated with IBS regular size meal? Two to three days a month One day a week This example illustrates a questionnaire that is useful for 45 More than one day a week Every day identifying the presence or severity of one or more IBS 5. In the last 3 months, Never --> related symptoms in an individual. The questionnaire can be how often did you have Less unan one day a monun completed by the individual at the clinic or physicians pain or burning in the ne day a montin office, or can be brought home and submitted when the middle of your Two to three days a month individual returns to the clinic or physicians office, e.g., to 50 abdomen, above your One day a week have his or her blood drawn. belly button but not in More than one day a week your chest? Every day In some embodiments, the questionnaire comprises a first 6. In the last 3 months, Never --> section containing a set of questions asking the individual to how often did you have Less unan one day a monun provide answers regarding the presence or severity of one or discomfort or pain ne day a montin more symptoms associated with IBS. The questionnaire 55 anywhere in your Two to three days a month generally includes questions directed to identifying the abdomen? One day a week More than one day a week presence, severity, frequency, and/or duration of IBS-related Every day symptoms such as chest pain, chest discomfort, heartburn, 7. In the last 3 months, O) Never or rarely uncomfortable fullness after having a regular-sized meal, how often did you have Sometimes inability to finish a regular-sized meal, abdominal pain, 60 fewer than three bowel (2) Often abdominal discomfort, constipation, diarrhea, bloating, and/ movements (0-2) a (3) Most of the time or abdominal distension. week? (4) Always 8. In the last 3 months, O) Never or rarely In certain instances, the first section of the questionnaire how often did you have O Sometimes (25% of the time) includes all or a Subset of the questions from a questionnaire hard or lumpy stools? (2) Often (50% of the time) developed by the Rome Foundation Board based on the 65 (3) Most of the time (75% of the time) Rome III criteria, available at http://www.romecriteria.org/ (4) Always questionnaires/. For example, the questionnaire can include US 9,482,672 B2 83 84 TABLE 3-continued thoughts or feelings associated with having IBS-related pain or discomfort. As a non-limiting example, an individual can Exemplary first section of a questionnaire for identifying be asked to rate the degree to which he or she has one or the presence or severity of IBS-related symptoms. more of the following thoughts and feelings when experi 9. In the last 3 months, O) Never or rarely encing pain: “I worry all the time about whether the pain will how often did you strain (1) Sometimes during bowel (2) Often end’”: “I feel I can’t stand it anymore”: “I become afraid that movements? Most of the time the pain will get worse”: “I anxiously want the pain to go Always away'; and “I keep thinking about how much it hurts.” One 10. In the last 3 months, Never or rarely skilled in the art will understand that the questionnaire can how often did you have Sometimes 10 comprise similar questions regarding negative thoughts or a feeling of incomplete Often emptying after bowel Most of the time feelings associated with having IBS-related pain or discom movements? Always fort. 11. In the last 3 months, Never or rarely In some embodiments, the questionnaire includes only how often did you have Sometimes questions from the first section of the questionnaire or a a sensation that the stool Often 15 could not be passed, Most of the time subset thereof (see, e.g., Table 3). In other embodiments, the (i.e., blocked), when Always questionnaire includes only questions from the second sec having a bowel tion of the questionnaire or a Subset thereof. movement? Upon completion of the questionnaire by the individual, 12. In the last 3 months, O) Never or rarely how often did you press C1) Sometimes the numbers corresponding to the answers to each question on or around your (2) Often can be Summed and the resulting value can be combined bottom or remove stool (3) Most of the time with the analysis of one or more diagnostic markers in a in order to complete a (4) Always sample from the individual and processed using the statis bowel movement? 13. Did any of the O) No tical algorithms described herein to increase the accuracy of symptoms of (1) Yes predicting IBS. constipation listed in 25 Alternatively, a “Yes” or “No” answer from the individual questions 27-32 above to the following question: “Are you currently experiencing begin more than 6 months ago? any symptoms?’ can be combined with the analysis of one 14. In the last 3 months, Never or rarely --> or more of the biomarkers described herein and processed how often did you have O Sometimes (25% of the time) using a single statistical algorithm or a combination of oose, mushy or watery (2) Often (50% of the time) statistical algorithms to increase the accuracy of predicting stools? (3) Most of the time (75% of the time) (4) Always IBS. 15. In the last 3 months, O) Never -> how often did you have O Less than one day a month D. Example 4 bloating or distension? (2) One day a month G.) Two to three days a month (4) One day a week 35 Blood Based Diagnostic Assay for the Diagnosis of G) More than one day a week Irritable Bowel Syndrome (IBS) (6) Every day 16. Did your symptoms of (0) No The present example describes the first blood-based bio bloating or distention begin more than 6 marker test for Irritable Bowel Syndrom (IBS). This test can months ago? 40 aid clinicians in the diagnosis of IBS. The IBS Diagnostic described below was validated using well-characterized IBS samples collected from recognized IBS experts and GI In other embodiments, the questionnaire comprises a clinics. The samples were either Rome II or Rome III second section containing a set of questions asking the positive and the patients had a diagnosis of IBS for greater individual to provide answers regarding the presence or 45 than one year. The test has a sensitivity of 50%, specificity severity of negative thoughts or feelings associated with of 88% with an overall accuracy of 70%. having IBS-related pain or discomfort. For example, the The total cohort used to develop the present assay con questionnaire can include questions directed to identifying sisted of 1,721 serum samples. The validation cohort used the presence, severity, frequency, and/or duration of anxiety, consisted of 516 serum samples, of which 50% were diag fear, nervousness, concern, apprehension, worry, stress, 50 nosed with IBS according to Rome II or Rome III criteria: depression, hopelessness, despair, pessimism, doubt, and/or 36% were Non-IBS disease controls; and 14% were normal negativity when the individual is experiencing pain or healthy controls. The sensitivity of the assay is 50% and the discomfort associated with one or more symptoms of IBS. specificity is 88%. When ruling in IBS, wherein the physi In certain instances, the second section of the question cian has determined that there is about a 75% probability of naire includes all or a Subset of the questions from a 55 disease in the patient, 94% of the positive test results are true questionnaire described in Sullivan et al., The Pain Cata positives, while 38% of the negative test results are true strophizing Scale: Development and Validation, Psychol. negatives. When ruling out IBS, wherein the physician has Assess., 7:524–532 (1995). For example, the questionnaire determined that there is about a 25% probability of disease can include a set of questions to be answered by an indi in the patient, 86% of the negative test results are true vidual according to a Pain Catastrophizing Scale (PCS), 60 negatives, while 61% of the positive test results are true which indicates the degree to which the individual has positives. certain negative thoughts and feelings when experiencing The assay involves the quantitative analysis of bio-mark pain: 0 not at all; 1=to a slight degree: 2-to a moderate ers combined with a two learning statistical classifier system degree: 3-to a great degree: 4-all the time. The second consisting of a random forest classifier and a neural network section of the questionnaire can contain 1, 2, 3, 4, 5, 6, 7, 8, 65 classifier. 9, 10, 11, 12, 13, 14, 15, or more questions or statements The specimen requirements for the assay consist of the related to identifying the presence or severity of negative physician obtaining 2.0 ml of separated serum in, for US 9,482,672 B2 85 86 example, an SST tube. For best results, the sample should be the diagnosis of IBS. An example of a checklist that may be centrifuges and refrigerates within 2 hours of collection. used in this fashion is provided in Table 5. When samples need to be shipped prior to assay detection, the samples should be refrigerated or frozen. For best TABLE 5 results, the samples should be stored, prior to use, for no more than 7 days at 4°C. or 30 days if frozen. Example patient checklist of symptoms commonly associated with IBS. Briefly, enzyme-linked immunosorbent assays (ELISAs) IBS Symptom Checklist are performed with antibodies specific for the IBS biomark Fill out and bring to your next appointment. Your Name ers ASCA-IgA, CBirl, ANCA, and tTG. Additionally, Appointment chemiluminescent assays are performed for the IBS bio 10 Date markers BDNF, NGAL, TWEAK, GRO-O., IL-1 B, and Place a check mark next to any symptoms you are currently having or TIMP-1. Reference values for the normal range of these have had in the past. Note how often these symptoms are present. Recurrent abdominal pain or discomfort markers in healthy subjects are provided in Table 4. Abnormal stool frequency (greater than 3 bowel movements/day or less than 3 bowel movements/week) TABLE 4 15 Abnormal stool form (lumpy/hard or loosef watery stool) Abnormal stool passage (straining, urgency, or feeling of Reference values for the markers detected in the IBS assay. incomplete bowel movement) Passage of mucus Marker Reference Level Bloating or feeling of abdominal distension Gassiness BDNF (Brain-Derived Neurotrophic Factor) 7536.5-31324.4 pg/ml Feelings of urgency (the need to find a restroom fast) NGAL (Neutrophil Gelatinase-Associated 28.3-272.5 ng/ml Lipocalin) TWEAK (TNF-related Weak Inducer of 351.6-1751.7 pg/ml Apoptosis E. References GRO-C. (Growth-Regulated Oncogene Alpha) 26.4-499.3 pg/ml IL-1B (Interleukin-1 Beta) 279.5-1358.6 fg/ml TIMP-1 (Tissue Inhibitor of Metallo- 156.1-410.6 ng/ml 25 1. Barbara G and Cremon C. Serine proteases: new players proteinase-1) in diarrhoea-predominant irritable bowel syndrome. Gut. ASCA-IgA (Anti-Saccharomyces <20.0 EUml cerevisiae Anitbody) 2008 May: 57(5):591-9. CBirl (Anti-CBirl Antibody) <21.0 EUml 2. Barbara G. Wang B, Stanghellini V. et al. Mast Cell ANCA (Anti-Human Neutrophil Cytoplasmic <12.1 EUml Dependent Excitation of Visceral-Nociceptive Sensory Antibody) 30 Neurons in Irritable Bowel Syndrome. Gastroenterology tTG (Anti-Human Tissue Transglutaminase <4.0 Uml 2007; 132:26-37. IgA) Although the foregoing invention has been described in Some detail by way of illustration and example for purposes In certain instances, information from the detection of the of clarity of understanding, one of skill in the art will IBS biomarkers described above can be combined with 35 appreciate that certain changes and modifications may be symptom information provided by the patient. For example, practiced within the scope of the appended claims. In the patient may fill out a checklist of their symptoms, the addition, each reference provided herein is incorporated by information from which can be combined with the informa reference in its entirety to the same extent as if each tion from the IBS biomarkers to further aid the clinician in reference was individually incorporated by reference.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 3

<21 Oc SEO ID NO 1 <211 LENGTH: 16 <212> TYPE PRT <213> ORGANISM: Homo sapiens <22 Os FEATURE; <223> OTHER INFORMATION: human fibrinopeptide A

<4 OOs SEQUENCE: 1 Ala Asp Ser Gly Glu Gly Asp Phe Lieu Ala Glu Gly Gly Gly Val Arg 1. 5 1O 15

SEO ID NO 2 LENGTH: 14 TYPE PRT ORGANISM: Homo sapiens FEATURE; OTHER INFORMATION: human fibrinopeptide B

<4 OOs SEQUENCE: 2 Glu Gly Val Asn Asp Asn. Glu Glu Gly Phe Phe Ser Ala Arg 1. 5 1O US 9,482,672 B2 87 88 - Continued

SEQ ID NO 3 LENGTH: 5 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: synthetic amidated neuropeptide tachykinin carboxy-terminal sequence FEATURE: NAME/KEY: VARIANT LOCATION: (2) ... (2) OTHER INFORMATION: Xaa = any amino acid FEATURE: NAME/KEY: AMIDATION LOCATION: (5) . . . (5) OTHER INFORMATION: methioninamide

<4 OOs, SEQUENCE: 3 Phe Xaa Gly Lieu Met 1. 5

What is claimed is: 9. The method according to claim 1, wherein the subject 1. A method for aiding in the diagnosis of irritable bowel is suspected of having IBS. syndrome (IBS) in a subject, said method comprising: 10. The method according to claim 1, wherein the method (a) contacting a blood or serum sample from the Subject further comprises determining the level of at least one with a prostaglandin E2 (PGE) antibody under condi 25 tions suitable to transform PGE present in the sample additional biomarker in a biological sample from the Sub into a complex comprising PGE and the PGE anti ject, the biomarker selected from the group consisting of body; Brain-Derived Neurotropic Factor (BDNF), Neutrophil (b) detecting the level of said complex using a sandwich Gelatinase-Associated Lipocalin (NGAL). TNF-related enzyme-linked immunosorbent assay (ELISA) com 30 Weak Inducer of Apoptosis (TWEAK), Growth-Related prising an alkaline phosphatase conjugated anti-PGE Oncogene Alpha (GRO-O.), Interleukin-1 Beta (IL-13), Tis antibody as the detecting antibody and a disodium Sue Inhibitor of Metalloproteinase-1 (TIMP-1), Anti-Sac 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro)tricy charomyces cerevisiae Antibody (ASCA-IgA), Anti-CBir-1 clo3.3.1.13.7 decan-4-yl)phenyl phosphate (CPSD) Antibody (CBirl), Anti-Human Neutrophil Cytoplasmic containing luminescent Substrate to enhance assay sen 35 Antibody (ANCA), Anti-Human Tissue Transglutaminase sitivity, thereby determining the level of PGE present IgA (tTG), and a combination thereof. in the sample; and 11. The method of claim 10, wherein said biological (c) comparing the level of PGE present in the sample to sample is selected from the group consisting of serum, a control level, wherein a difference in the level of plasma, whole blood, and stool. PGE present in the sample relative to the control level 40 12. The method according to claim 1, wherein the method is indicative of an increased likelihood of said subject further comprises: having IBS. (d) determining a symptom profile for the Subject, 2. The method of claim 1, wherein the control level is the wherein said symptom profile is determined by identi level of PGE present in a blood or serum sample from a fying the presence or severity of at least one symptom healthy subject. 45 3. The method of claim 2, wherein an increased level of in said Subject; and PGE present in the sample relative to the control level is (e) diagnosing the Subject as having IBS or not having indicative of an increased likelihood of said subject having IBS using an algorithm based upon the level of PGE IBS. present in the sample and the system profile. 4. The method of claim 2, wherein the same or a reduced 50 13. The method of claim 12, wherein said at least one level of PGE present in the sample relative to the control symptom is selected from the group consisting of chest pain, level is indicative of an increased likelihood of said subject chest discomfort, heartburn, uncomfortable fullness after not having IBS. having a regular-sized meal, inability to finish a regular 5. The method of claim 1, wherein the control level is the sized meal, abdominal pain, abdominal discomfort, consti level of PGE present in a blood or serum sample from a 55 pation, diarrhea, bloating, abdominal distension, negative subject with IBS. thoughts or feelings associated with having pain or discom 6. The method of claim 5, wherein the same or an fort, and a combination thereof. increased level of PGE present in the sample relative to the 14. The method of claim 13, wherein the presence or control level is indicative of an increased likelihood of said severity of said at least one symptom is identified using a subject having IBS. 60 questionnaire. 7. The method of claim 5, wherein a reduced level of 15. The method according to claim 12, wherein said PGE present in the sample relative to the control level is algorithm comprises a statistical algorithm. indicative of an increased likelihood of said subject not 16. The method of claim 15, wherein said statistical having IBS. algorithm comprises a learning statistical classifier system. 8. The method according to claim 1, wherein the method 65 17. The method of claim 16, wherein said statistical further comprises determining the level of B-tryptase and/or algorithm comprises a combination of at least two learning histamine present in the sample. statistical classifier systems. US 9,482,672 B2 89 90 18. The method of claim 17, wherein the combination of at least two learning statistical classifier systems comprises a random forest classifier and a neural network classifier. 19. The method according to claim 12, wherein the method comprises providing a probability that the Subject has IBS. 20. The method according to claim 12, wherein the method further comprises classifying a diagnosis of IBS as IBS-constipation (IBS-C), IBS-diarrhea (IBS-D), IBS mixed (IBS-M), IBS-alternating (IBS-A), or post-infectious 10 IBS (IBS-PI). 21. The method according to claim 12, wherein said method further comprises diagnosing the Subject not having IBS as having IBD, as not having IBD, or as being a healthy Subject. 15