US007951547B2
(12) United States Patent (10) Patent No.: US 7,951,547 B2 Elsem0re et al. (45) Date of Patent: May 31, 2011
(54) METHODS, DEVICES, KITS AND S. 3. f 39: s et al COMPOSITIONS FOR DETECTING 6,596,502 B2 7/2003 Leereve et al. ROUNDWORM, WHIPWORM, AND 7,303,752 B2 12/2007 Hotez et al. HOOKWORM 7,736,660 B2 * 6/2010 Elsemore et al...... 424,265.1 2002/0132270 A1 9, 2002 Lee (75) Inventors: paid Ally RE, Sh Ital 2003/01296802003/0202980 A1A1* 10/20037/2003 CaplanetO’Connor, al. Jr...... 424,185.1 (US); Jinming Geng, Scarborough, 2004/0014087 A1 1/2004 Hodgson et al. ME (US); Laurie A. Flynn, Raymond, 2004/0214244 A1 10, 2004 Tonelli ME (US); Michael Crawford, St. Louis, 2005/0042232 A1 2/2005 Hotez et al. MO (US) 2006/0198844 A1 9/2006 Langenfeld 2007/0053920 A1 3/2007 Heath et al. (73) Assignees: Idexx Laboratories, Inc., Westbrook, 39. A. 23. SN a al. ME (US); Divergence, Inc., St. Louis, MO (US) FOREIGN PATENT DOCUMENTS WO WO 98.12563 3, 1998 (*) Notice: Subject to any disclaimer, the term of this WO WO O2/O753.13 9, 2002 patent is extended or adjusted under 35 WO WO 2004/097412 11, 2004 U.S.C. 154(b) by 0 days. OTHER PUBLICATIONS (21) Appl. No.: 12/467,826 Bowie et al (Science vol. 257. pp. 1306-1310, 1990).* Uniprotsubmission P07852. Aug. 1988. Retrieved from the Internet (22) Filed: May 18, 2009 Mar. 30, 2010: OTHER PUBLICATIONS GenBank Accession No. CB189034. Feb. 5, 2003. Retrieved from Uniprotsubmission Q0681 1. Nov. 1997. Retrieved from the Internet the Internet Mar. 30, 2010: Uniprot submission Q962V8. Dec. 1, 2001. 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NCBI Blast: SEQ ID No. 4 (Performed Aug. 27, 2009 using http:// Martinez-Maya et al., Taeniosis and detection of antibodies against blast.ncbi.nlm.nih.gov/blast.cgi). Cysticeri among inhabitants of a rural community in Guerro State, Abdel-Rahman et al., Evaluation of a diagnostic monoclonal anti Mexico, Salud Publica de Mexico 45:84-89 (2003). body-based capture enzyme-linked immunosorbent assay for detec Ott et al., Demonstration of both immunologically unique and com tion of a 26- to 28-kd Fasciola hepatica coproantigen in cattle, mon antigenic determinants in Dirofilaria immitis and Toxocara American Journal of Veterinary Research 59:533-537 (1998). canis using monoclonal antibodies, Veterinary Immunology and Bungiro and Cappello, “Detection of Excretory/Secretory Immunopathology 10:147-153 (1985). Roberts, L.S., et al., Foundations of Parasitology, Fifth Edition, 1996, Coproantigens in Hookworm infection.” Am. J. Trop. Med. Hyg. Library of Congress Card Catalog No. 94-72939, ISBN 0-687 73(5):915-920 (2005). 26071-S, pp. 1-4. Bungiro, Jr., et al., “Purification and Molecular Cloning of and Southworth, Exine development in Gerbera jamesonii (Asteraceae: Immunization with Ancylostoma ceylancium Excretory-Secretory Mutisieae), American Journal of Botany, 70:1038-1047 (1983). Protein 2, an Immunocreactive Immunoreactive Protein Produced by Voller, “The Enzyme Linked Immunosorbent Assay”. Diagnostic Adult Hookworms.” Infection and Immunity 72(4):2203-2213 Horizon, vol. 2, No. 1, pp. 1-7, Feb. 1978. (2004). Willard et al., Diagnosis of Aelurostrongylus abstrusus and Carleton et al., Prevalence of Dirofilaria immitis and gastrointestinal Dirofilaria immitis infections in cats from a human shelter, Journal of helminths in cats euthanized at animal control agencies in northwest the American Veterinary Medical Association 192:913-916 (1988). Georgia, Veterinary Parisitology 1 19:319-326 (2004). Yamasaki et al., “Development of Highly Specific Recombinant Coulaud, J.P. et al., Albendazole: a new single dose anthelmintic. Toxocara canis Second-Stage Larva Excretory-Secretory Antigen for Study in 1455 patients, Acta Tropica 41:87-90 (1984). Immunodiagnosis of Human Toxocariasis,” Journal of Clinical De Oliveira et al., IgM-ELISA for diagnosis of schistosomiasis Microbiology 38 (4): 1409-1413 (2000). mansoni in low endemic areas, Cadernos de Saude publica f Zhan et at, “Molecular characterisation of the Ancylostoma-secreted Ministerio da Saude, Fundacao Oswaldo Cruz, Escola Nacional de protein family from the adult stage of Ancylostoma caninum,' Inter Saude Publica 19:255-261 (2003). national Journal for Parisitology 33:897-907 (2003). Deplazes et al., Detection of Taenia hydatigena copro-antigens by ELISA in dogs, Veterinary Parisitology 36:91-103 (1990). * cited by examiner U.S. Patent May 31, 2011 Sheet 1 of 26 US 7,951,547 B2 FIG. 1 Diluent Solution Negative Fecal Pool HOOK POSitive ROUnd POSitive Whip Positive U.S. Patent May 31, 2011 Sheet 2 of 26 US 7,951,547 B2 FIG. 2 Three nematode assays Fecal Samples Cognate rProteins Buffer HW pos Hook OOS Round pos Wii OS A Hookworm Assay B C D Roundworm Assay E F G Whipworm Assay H U.S. Patent May 31, 2011 Sheet 16 of 26 US 7,951,547 B2 188 98 62 49 38 28 17 14 FIG. 16 U.S. Patent May 31, 2011 Sheet 17 of 26 US 7,951,547 B2 188 98 62 49 38 28 17 14 FIG 17 U.S. Patent May 31, 2011 Sheet 18 of 26 US 7,951,547 B2 MGPESCGPNEVWTECTGCELKCGQDENT SCECSPGRGMRRTND QHRAKREEQCKPNEQWSPCRGCEGTCAQ CRPPGCECVAGAGFV EDDCPK (SEQ ID NO: 21) FIG. 18 U.S. Patent May 31, 2011 Sheet 19 of 26 US 7,951,547 B2 :ONCIIÕES) :ONCIIÕES) ·×°**·****ºx: N8ZL9 08Z1,9 U.S. Patent May 31, 2011 Sheet 20 of 26 US 7,951,547 B2 1. marker 2 load(Wash) 3 Void H2 4 Wash#2 5 20 mM H2 6 40mM H2a 7 40mM H2b 8 80 mMH2 9 500 mM H2 FIG. 20 U.S. Patent May 31, 2011 Sheet 21 of 26 US 7,951,547 B2 1 marker load(wash) 3 Voidi2 4 Washii.2 5 20 mM #2 6 40mM #2a 7 40mM #2b 8 80 mMi2 9 500 mM #2a 10 500 mM #2b FIG 21 U.S. Patent May 31, 2011 Sheet 22 of 26 US 7,951,547 B2 Roundworm fecal ELISA capture a DIV6728 conjugate (1 ug/ml) 1200 1.OOO round O.800 round (1:5) + is HOOK + whip + 0.400 - Benegative O.2OO - O.OOO X463 X464 DX467 X468 DM6728 (DIV6728C) (DIV6728C) (DIV6728N) (DIV6728N) Coated rabbit pAB (2 uglml) FIG. 22 U.S. Patent May 31, 2011 Sheet 23 of 26 US 7,951,547 B2 Recombinant Ag ELISA assay aDIW6728 conjugate (1 uglml) DM 872BFL DIW672BN DV62BC ASP5-1 X463 X464 X48 X488 DM6728 (DM6728C) (DM6728C) (DV6728N) (DM6728N) Coated rabbit pAb (2 ugln) FG, 23 U.S. Patent May 31, 2011 Sheet 24 of 26 US 7,951,547 B2 98 62 49 38 28 17 14 FIG. 24 U.S. Patent May 31, 2011 Sheet 25 of 26 US 7,951,547 B2 FIG. 25 U.S. Patent US 7,951,547 B2 FIG. 26 US 7,951,547 B2 1. 2 METHODS, DEVICES, KITS AND has immobilized thereon at least two antibodies selected from COMPOSITIONS FOR DETECTING the group consisting of (a) a first antibody capable of specifi ROUNDWORM, WHIPWORM, AND cally binding a roundworm coproantigen, but not a whip HOOKWORM worm or hookworm coproantigen; (b) a second antibody capable of specifically binding a whipworm coproantigen, CROSS REFERENCE but not a roundworm or hookworm coproantigen; and (c) a third antibody capable of specifically binding a hookworm This application claims priority to U.S. Provisional Patent coproantigen, but not a whipworm or roundworm coproanti Application Ser. Nos. 61/122,260, filed Dec. 12, 2008: gen. The device, may be, but is not limited to being, for 61/128,077, filed May 19, 2008: 61/128,079, filed May 19, 10 example, an ELISA device. Such as a lateral flow immunoas 2008: 61/128,076, filed May 19, 2008: 61/128,099, filed May say device or microtiterplate device. Samples that may be 19, 2008; and 61/122,254, filed Dec. 12, 2008; and U.S. tested for roundworm, whipworm and hookworm by the patent application Ser. Nos. 1 1/763,592, filed Jun. 15, 2007 device include, but are not limited to being, feces, digestive and 1 1/763,583, filed Jun. 15, 2007, which are incorporated tract mucous, urine, whole blood, serum, mammary milk and by reference herein in their entirety. 15 whole tissue. Such as tissue from mammary gland, intestine, liver, heart, lung, esophagus, brain, muscle, and eye, for BACKGROUND OF THE INVENTION example. The device further may include, but need not include, one or more reagents for the detection of one or more 1. Field of the Invention of the group consisting of one or more non-worm parasites, The present invention relates to compositions, devices, kits one or more viruses, one or more fungi, and one or more and methods for the detection of and distinguishing between bacteria. roundworm, whipworm and hookworm in mammals. More In yet another aspect, the invention provides a method of particularly, the present invention relates to antibodies and detecting the presence or absence of one or more helminthic antibody compositions, devices, kits, and methods for detect antigens in a sample, for example coproantigens from a fecal ing the presence or absence of roundworm antigen, whip 25 sample, the method comprising: (a) contacting a sample from worm antigen and hookworm antigen in a sample from a a mammal with at least two antibodies selected from the mammal and for distinguishing between roundworm, whip group consisting of: (i) a first antibody capable of specifically worm and hookworm antigens. binding a roundworm coproantigen, but not a whipworm or 2. Description of the Prior Art hookworm coproantigen; (ii) a second antibody capable of Parasitic worm (helminth) infections are common in ani 30 specifically binding a whipworm coproantigen, but not a mals and, if not diagnosed and treated, can cause serious roundworm or hookworm coproantigen; and (iii) a third anti disease or death. Current methods for diagnosis of parasitic body capable of specifically binding a hookworm coproanti worm infections primarily involve microscopic examination gen, but not a whipworm or roundworm coproantigen (b) of fecal samples, either directly in fecal smears or following forming antibody-coproantigen complexes in the presence of concentration of ova and parasites by flotation in density 35 the coproantigens, if any, in the sample; and (c) detecting the media. Despite this procedure's high adoption, the method presence or absence of the antibody-coproantigen complexes, has significant shortcomings. These microscopic methods are if any. The one or more helminthic coproantigens include time consuming and require specialized equipment. In addi coproantigens of roundworm, such as Toxocara Canis (T. tion, the accuracy of results of these methods is highly depen canis), Toxocara Cati (T. cati), Toxocara vitulorum (T. vitu dent upon the skill and expertise of the operator. For example, 40 lorum), Toxascaris leonina (T. leonina), Baylisascaris pro the presence of whipworms is determined by looking for cyonis (B. procyonis), Ascaridia galli (A. galli), Parascaris eggs, but these are excreted intermittently and in Small num equorum(Pequorum), Ascaris suum (A. suum), or Ascaris bers. Hookworms are difficult for the average practitioner to lumbricoides (A. lumbricoides), Anisakis simplex (A. Sim detect either early in infection or in young animals. The plex), or Pseudoterranova decipiens (P decipiens), whip specificity of roundworm diagnosis using microscopic 45 worm Such as Trichuris vulpis, Trichuris campanula, Trichu examination is approximately 50%. ris Serrata, Trichuris Suis, Trichuris trichiura, Trichuris Stool handling is disagreeable and hazardous. Sanitary and discolor, and hookworm, such as Ancylostoma Caninum, inoffensive procedures for processing stool are awkward and Ancylostoma braziliense, Ancylostoma duodenal, Ancylos often complex. Such procedures may include weighing, cen toma ceylanicum, Ancylostoma tubaeforme and Ancylostoma trifuging and storing, and are difficult except in a clinical 50 pluridentatum, Necator americanus, and Uncinaria Steno laboratory equipped with a suitable apparatus, protective cephala, for example, in a sample obtained from a mammal, equipment, and a skilled technician. Therefore, any reduction Such as a canine, feline, porcine, bovine, or human and dis in the number of steps required to perform a fecal test and any tinguishing between roundworm, whipworm and hookworm. reduction in contact between test operator and the test mate In one aspect, the method is carried out to test a fecal mam rial is desirable. Clinical laboratories have been using the 55 malian sample for roundworm coproantigen, whipworm immunoassay methods for the detection of various viruses, coproantigen, and hookworm coproantigen. The method, bacteria and non-helminth parasites and organisms in feces. however, is not limited to being carried out to test a fecal However, there remains a need for a simple immunoassay sample. In addition to feces, the sample therefore may be, but method for the detection of a parasitic worm infection in is not limited to being whole blood, serum, mammary milk feces, whole blood or in serum. 60 and whole tissue. Such as tissue from mammary gland, intes tine, liver, heart, lung, esophagus, brain, muscle, and eye, for SUMMARY OF THE INVENTION example. In yet another aspect, the invention provides a method of In one aspect, the invention provides a device for specifi diagnosing whether a mammal is infected with one or more cally binding and isolating helminthic antigens from a 65 parasitic worms, the method comprising the steps of: (a) sample, for example coproantigens from a fecal sample, the contacting a sample from a mammal with at least two anti device comprising a solid Support, wherein the Solid Support bodies selected from the group consisting of: (i) a first anti US 7,951,547 B2 3 4 body capable of specifically binding a roundworm coproan FIG.3 shows the nucleotide sequence of a 1210-nucleotide tigen, but not a whipworm or hookworm coproantigen; (ii) a cDNA sequence from whole adult Trichuris vulpis (SEQ ID second antibody capable of specifically binding a whipworm NO: 1). coproantigen, but not a roundworm or hookworm coproanti FIG. 4 shows the nucleotide sequence of a 1059-nucleotide gen; and (iii) a third antibody capable of specifically binding cDNA sequence from whole adult Trichuris vulpis. (SEQID a hookworm coproantigen, but not a whipworm or round NO:2). worm coproantigen (b) forming antibody-coproantigen com FIG.5 shows a comparison alignment of SEQID NO:3 and plexes in the presence of the coproantigens, if any, in the SEQID NO:4. The consensus sequence of SEQID NO:3 and sample; (c) detecting the presence or absence of the antibody SEQID NO:4 is shown as SEQID NO:9. coproantigen complexes, if any; and (d) diagnosing the mam 10 FIG. 6 shows the nucleotide sequence of an 865-nucleotide mal as having: (i) a roundworm infection if a roundworm cDNA sequence from whole adult Toxocara canis. (SEQ ID antibody-coproantigen complex is present; (ii) a whipworm NO: 10). infection if a whipworm antibody-coproantigen complex is FIG. 7 shows the nucleotide sequence of a 632-nucleotide present; and (iii) a hookworm infection if a hookworm anti 15 cDNA sequence from whole adult Toxocara cati. (SEQ ID body-coproantigen complex is present. The method may also NO:11). be used to test for and distinguish between environmental FIG. 8 shows a comparison alignment of SEQID NO: 13 contamination with roundworm, whipworm and/or hook and SEQID NO: 14. The consensus sequence of SEQID NO: worm. Environmental samples that may be tested for round 13 and SEQID NO: 14 is shown as SEQID NO: 16. worm, whipworm and/or hookworm by the device include, FIG. 9 shows the nucleotide sequence of a 535-nucleotide but are not limited to Soil, decomposing material, or fecal cDNA sequence from whole adult Toxocara canis. (SEQ ID matter from residential settings including yards, gardens, NO:17). sandboxes, playgrounds. Testing locations may also include FIG.10 shows the nucleotide sequence of a 536-nucleotide parks, beaches, forests, farms, or other locations exposed to cDNA sequence from whole adult Toxocara cati. (SEQ ID fecal material from dogs, cats, or other mammalian hosts of 25 NO:18). roundworms. Feces from indoor and outdoor litterboxes may FIG. 11 shows a comparison alignment of SEQID NO:20 also be tested. and SEQ ID NO:21. The consensus sequence of SEQ ID In yet another aspect, the present invention includes a kit NO:20 and SEQID NO:21 is shown as SEQID NO:23. for carrying out one or more steps of the method of the FIG. 12 shows the nucleotide sequence of a 469-nucleotide invention. The kit may optionally include, for example, the 30 cDNA sequence from whole adult Toxocara canis. (SEQ ID device and one or more of the compositions of the present NO:24). invention and instructions for carrying out the method of the FIG.13 shows the nucleotide sequence of a 548-nucleotide present invention. The kit may further optionally include, for cDNA sequence from whole adult Toxocara cati. (SEQ ID example, one or more indicator reagents, one or more anti NO:25). body labeling compounds, one or more antibodies, one or 35 FIG. 14 shows a comparison alignment of SEQID NO:27 more antigen capture reagents, one or more inhibitors, and and SEQ ID NO:28. The consensus sequence of SEQ ID one or more wash reagents to be used as part of the device NO:27 and SEQID NO:28 is shown as SEQID NO:30. and/or to be used in carrying out the method. FIG. 15 shows an ELISA with elution fractions from SP In yet another aspect, the present invention includes a columns as samples and that CoproG728 can be partially device for specifically binding helminthic antigens from a 40 purified and enriched by eluting the SP column by following sample, for example coproantigens from a fecal sample, the the method of the present invention in the third Example. device comprising a solid Support, wherein the Solid Support FIG. 16 shows that the molecular weight of Copro6728 has immobilized thereon at least two antibodies selected from was about 7 KD using a western Blot probed with rabbit anti the group consisting of: (a) a first antibody capable of spe full-length DIV6728 IgG-HRP following the method of the cifically binding a roundworm coproantigen, but not a whip 45 present invention in the third Example. worm or hookworm coproantigen; (b) a second antibody FIG. 17 shows that the molecular weight of Copro6728 capable of specifically binding a whipworm coproantigen, was about 7 KD using an SDS-PAGE gel stained with Impe but not a roundworm or hookworm coproantigen; and (c) a rial Protein Staining following the method of the present third antibody capable of specifically binding a hookworm invention in the third Example. coproantigen, but not a whipworm or roundworm coproanti 50 FIG. 18. shows the amino acid sequence of the full length gen; and (d) one or more types of roundworm antigen, whip DIV6728 (SEQ ID NO: 21) with the two peptides (SEQ ID worm antigen, and/or hookworm antigen, wherein the one or NO:35 and SEQIDNO:36) identified by Mass Spectrometry more types of roundworm antigen, whipworm antigen, and analysis identified by highlighting them in the shaded boxes hookworm antigen are specifically bound to the antibodies. following the method of the present invention in the third 55 Example. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 19 shows an alignment of the 6728N (SEQ ID NO: 37) and 6728C (SEQ ID NO: 38) amino acid sequences FIG. 1 shows the results of an ELISA assay, which was encoded by the constructs following the method of the present carried out by using a lateral flow device and which tested invention in the fourth Example. fecal samples from canines infected with roundworm, whip 60 FIG. 20 shows a SDS-PAGE gel loaded with different worm and/or hookworm by following the method of the samples to check the purification of the recombinant 6728N present invention in a first Example. following the method of the present invention in the fourth FIG. 2 shows the results of an ELISA assay, which was Example. carried out by using a microtiter plate and which tested fecal FIG. 21 shows a SDS-PAGE gel loaded with different samples from canines infected with either roundworm, hook 65 samples to check the purification of the recombinant 6728C worm, whipworm or heartworm by following the method of following the method of the present invention in the fourth the present invention in a second Example Example. US 7,951,547 B2 5 6 FIG.22 shows the ELISA data obtained with different fecal worms, and hookworms all are related nematodes, and an samples to test the different polyclonal antibodies against antibody raised against a protein isolated from any one of different recombinant 6728 proteins following the method of these worms would be expected to crossreact with one or the present invention in the fourth Example. more of the other worms, host antigens, or other host compo FIG.23 shows the ELISA data obtained with recombinant 5 nentS. proteins to test the different polyclonal antibodies against It was further determined that this antibody can be used to different recombinant 6728 proteins following the method of capture and detect roundworm, whipworm and hookworm the present invention in the fourth Example. antigens in a mammal as early as 9 days after the mammal is FIG. 24 shows Western blotting with different fecal first infected. This ability to detect roundworm, whipworm or samples probed with rabbit anti-full-length DIV6728 IgG 10 hookworm so soon after infection, and before the appearance HRP following the method of the present invention in the of any ova in the feces of the infected mammal, is Surprising fourth Example. because ova generally do not appear in the feces of an infec FIG. 25 shows Western blotting with different fecal tive host until about five-to-eight weeks after the host samples probed with rabbit anti-6728C IgG-HRP following becomes infected. the method of the present invention in the fourth Example. 15 The present invention therefore includes methods, devices, FIG. 26 shows Western blotting with different fecal and kits that use antibodies and/or fragments thereof to spe samples probed with rabbit anti-6728N IgG-HRP following cifically capture and detect and distinguish between round the method of the present invention in the fourth Example. worm, whipworm and hookworm antigens in a mammal. The ability of the present invention to detect and diagnose round DETAILED DESCRIPTION OF THE PREFERRED worm even when one or more other worm types are also EMBODIMENTS OF THE INVENTION present allows the mammals caregiver the opportunity to optimally select a treatment for ridding the roundworm, I. Introduction whipworm and/or hookworm from the mammal. Further, the The present invention is generally directed to methods, ability of the present invention to, in some cases, detect devices, and kits for detecting and distinguishing between 25 roundworm, whipworm and/or hookworm as early as 9 days roundworm, whipworm and hookworm in a fecal sample after the mammal is first infected provides the possibility that obtained from a mammal. The present invention relates to the caregiver may begin Such treatment before the mammal roundworm coproantigens from Toxocara. Such as Toxocara becomes severely sickened. An intervention prior to appear canis or Toxocara cati, whipworm coproantigens from Tri ance of ova in the feces would also greatly reduce or eliminate churis, such as Trichuris vulpis, and hookworm coproanti 30 the possibility that the infestation is spread to other animals or gens Ancylostoma Such as Anclostoma Caninum, for example. humans. In particular, the present invention relates methods, devices II. Definitions and Uses of Term and kits for detecting and distinguishing between round The term “compositions of the invention” refers to all of the worm, Such as Toxocara, Toxascaris, Baylisascaris, Ascar nucleic acids, polypeptides, antibodies, and mixtures that idia, Parascaris, Ascaris, Anisakis, or Pseudoterranova, 35 include one or more of those nucleic acids, polypeptides, and including T. canis, T. Cati, T. vitulorum, T. leonina, T. vitu antibodies and one or more other compounds, that can be used lorum, B. procyonis, A. galli, Pequorum, A. lumbricoides, to detect the presence or absence of roundworm, whipworm and A. suum, A. simplex, and P. decipiens, whipworm, Such as and/or hookworm in a sample obtained from a mammal by Trichuris vulpis, Trichuris Serrata, T. campanula, and Trichu carrying out the method of the present invention that are ris trichiura, and hookworm, Such as Ancylostoma caninum, 40 explicitly described, implicitly encompassed or otherwise Ancylostoma braziliense, Ancylostoma duodenal, Ancylos disclosed herein. toma ceylanicum, Ancylostoma tubaeforme and Ancylostoma “A sample from a mammal’ in which roundworm, whip pluridentatum, Necator americanus, and Uncinaria Steno worm and/or hookworm can be detected by the present inven cephala, for example. tion includes all bodily components and extracts thereof. Such The present invention provides a superior alternative to the 45 as any fluid, Solid, cell or tissue, that are capable of containing existing microscopic inspection techniques. This is true roundworm, whipworm and/or hookworm antigen. Exem because the present invention provides devices, kits and plary samples therefore include, but are not limited to being, methods for detecting the presence or absence of roundworm, feces, milk, whole blood and portions thereof, including whipworm and hookworm in a sample from a mammal that: serum, and further include tissue extracts, including tissue (1) are both easy to use and yield consistently reliable results: 50 from mammary gland, intestine, liver, heart, lung, esophagus, (2) allow for the absence or presence of whipworm in a brain, muscle, and eye, for example. The sample may be taken mammal to be confirmed regardless of whether that mammal directly from the mammal or the sample may be taken from is infected with hookworm, roundworm, and/or heartworm; anything that has contacted the mammal. For example, the (3) can detect roundworm, whipworm and hookworm prior to sample may be fresh or decaying fecal droppings from the the time that the ova first appear in the infected hosts feces: 55 mammal. As another example, the sample may include soil. and (4) can distinguish between roundworm, whipworm and dirt, sand, plant material, or any other material that may be hookworm infections. mixed with bodily components that may be left behind by a The present invention is based in part on the discovery of mammal, such as feces, for example. As such, the sample may unexpected properties of compositions specific to round be taken from an environmental source, including soil. worm, whipworm and hookworm infections. Specifically, it 60 decomposing material, or fecal matter from forests, farms, or was determined that antibodies raised against worm specific residential settings, including litter boxes, yards, gardens, polypeptides (or raised against an extract of whole worms, or sandboxes, playgrounds, parks, and beaches. No matter the extract of worm reproductive organs, or extract of worm origin or the content of the sample, this sample sometimes is intestines) can be used to capture, detect, and distinguish referred to herein as the “sample, the “mammalian sample, between roundworm antigens, whipworm antigens, and 65 the “test sample' or the “sample under test”. hookworm antigens in a mammal. The specificity for each As used herein, “nucleic acid is synonymous with, and type of worm is Surprising because roundworms, whip therefore is used interchangeably with, "gene”, “DNA’. US 7,951,547 B2 7 8 “cDNA”, “EST, “polynucleotide”, “oligonucleotide', includes the genera Toxocara, Toxascaris, Baylisascaris, “polynucleic acid”, “RNA” and “mRNA'. A nucleic acid Ascaridia, Parascaris, Ascaris, Anisakis, and Pseudoterra may be in double-stranded form or it may be in single nova. Thus, the term "roundworm', as used herein, does not stranded form. Further, a nucleic acid is either naturally iso refer to the entirety of the phylum Nematoda. Therefore, lated, such as from a whole roundworm, whipworm and/or “roundworm” does not include any member of the genera hookworm or a portion thereof, for example, or it is artifi Ancylostoma, Uncinaria, Necator, Trichuris, Wuchereria, cially synthesized, either in a recombinant host organism or Brugia or Dirofilaria. by any other artificial means knownto the skilled artisan, Such A“roundworm coproantigen' or a "coproantigen of round as by employing a PCR-based technique, by creating a trans worm' is any roundworm product that is present in the feces genic organism that synthesizes the nucleic acid, by using a 10 of a mammal having a roundworm infection and that may be DNA synthesizing machine, or by any another molecular specifically bound by one or more of the antibodies of the based technique, for example. invention. For example, a roundworm coproantigen may be, “Polypeptide', 'peptide' and “protein’ are synonymous but is not limited to being, one or more of the polypeptides of terms that are used interchangeably herein to refer to a poly the invention. The present inventors have determined that a mer of amino acid residues. A polypeptide, peptide and pro 15 novel C-terminal 7kD isoform of DIV6728, which is a excre tein of the present invention may be either naturally isolated, tory/secretory protein of T. canis, is present in feces of T. Such as from a whole roundworm, whipworm or hookworm canis—infected canines as early as 38 days after the canines or from a portion of roundworm, whipworm or hookworm for first became infected with the T. canis. Therefore, a “round example, or artificially synthesized, either in a recombinant worm coproantigen' may be this novel C-terminal 7 kD iso host organism or by any other artificial means known to the form of DIV6728 (which is referred to herein as skilled artisan. “Copro6728) that has been observed in canine feces by the The term “antibody' or “antibody of the present invention” present inventors. refers to any antibody that is able to specifically bind to one or The term “whipworm', as used herein, refers to helminths more antigens for the particular worm without binding to Such as intestinal whipworms of the genera Trichuris and antigens from the other worms. For example antibodies to the 25 Trichocephalus. Exemplary whipworms therefore include one or more roundworm antigens are able to specifically bind Trichuris vulpis, Trichuris campanula, Trichuris serrata, Tri to one or more roundworm antigens, but not to any antigens churis Suis, Trichuris trichiura, Trichuris discolor and Tri from hookworm or whipworm and antibodies to the one or chocephalus trichiuris. Further, the term “whipworm', as more whipworm antigens are able to specifically bind to one used herein, does not refer to the entirety of the phylum or more whipworm antigens, but not to any antigens from 30 Nematoda. For example, “whipworm” does not include any roundworm and hookworm. The antibodies of the present member of the genera Ancylostoma, Uncinaria, Necator, invention may be raised against one or more immunogenic Toxocara, Toxascaris, Ascaris, Wuchereria, Brugia or Dirofi polypeptides of the present invention. Unless otherwise laria. stated, it is to be understood that the antibody of the present A “whipworm coproantigen' or a "coproantigen of whip invention may include a mixture of two or more different 35 worm' is any whipworm product that is present in the feces of types of antibody. For example, the antibody may be a mix a mammal having a whipworm infection and that may be ture of two types of antibodies, wherein one of the two types specifically bound by one or more of the antibodies of the specifically binds to one particular antigen and the other of the invention. For example, a whipworm coproantigen may be, two types specifically binds to some other antigen. but is not limited to being, one or more of the polypeptides of The term “first antibody” as used herein means one or more 40 the invention. antibodies capable of specifically binding a roundworm The term “hookworm, as used herein, refers to helminthes coproantigen, but not a whipworm or hookworm coproanti Such as intestinal hookworm of the genera Ancylostoma, gen. Necator and Uncinaria. Exemplary hookworms therefore The term “second antibody” as used herein means one or include Ancylostoma caninum, Ancylostoma braziliense, more antibodies capable of specifically binding a whipworm 45 Ancylostoma duodenal, Ancylostoma ceylanicum, Ancylos coproantigen, but not a roundworm or hookworm coproanti toma tubaeforme and Ancylostoma pluridentatum, Necator gen. americanus, and Uncinaria Stenocephala. Further, the term The term “third antibody” as used herein means one or “hookworm,” as used herein, does not refer to the entirety of more antibodies capable of specifically binding a hookworm the phylum Nematoda. For example, “hookworm” does not coproantigen, but not a whipworm or roundworm coproanti 50 include any member of the genera Trichuris, Trichocephalus gen. Toxocara, Toxascaris, Ascaris, Wuchereria, Brugia or Dirofi The “immunogenic polypeptide of the present invention” laria. and, more simply, “the polypeptide of the present invention'. A "hookworm coproantigen' or a "coproantigen of hook is an immunogen against which the antibodies of the present worm' is any hookworm product that is present in the feces of invention may be raised. All “polypeptides of the present 55 a mammal having a hookworm infection and that may be invention” are immunogenic and therefore may be used to specifically bound by one or more of the antibodies of the elicit an immune response in a host animal to produce the invention. For example, a hookworm coproantigen may be, antibodies of the present invention. Unless otherwise stated, it but is not limited to being, one or more of the polypeptides of is to be understood that the polypeptide of the present inven the invention. The present inventors have determined that a tion may be one component of a mixed composition of a 60 novel N-terminal 28 kDa isoform of ASP5, which is a excre plurality of components. tory/secretory protein of Ancylostoma, is present in feces of An “immunogen' is any agent, Such as the immunogenic Ancylostoma-infected canines as early as 9 days after the polypeptide of the present invention, for example, that is canines first became infected with the Ancylostoma. There capable of eliciting an immune response in an animal that is fore, a “hookworm coproantigen' may be this novel N-ter exposed to that agent. 65 minal 28 kDa isoform of ASP5 (which is referred to hereinas The term “roundworm', as used herein, refers to helminths “CoproASP5') that has been observed in canine feces by the such as intestinal roundworms of the order Ascaridida, which present inventors. US 7,951,547 B2 10 “Specific for”, “specifically binds', and “stably binds” G or Gly: Histidine is Hor H is: Isoleucine is I or Ile; Leucine means that a particular composition of the invention, such as is L or Leu; Lysine is K or Lys; Methionine is Mor Met; an antibody, polypeptide, or oligonucleotide of the present Phenylalanine is For Phe; Proline is P or Pro; Serine is S or invention, for example, recognizes and binds to one or more Ser; Threonine is T or Thr; Tryptophan is W or Trp; Tyrosine other agents with greater affinity than to at least one other is Y or Tyr; and Valine is V or Val. Except where defined agent. As one example, an antibody of the present invention is otherwise herein, X or Xaa represents any amino acid. Other said to be “specific for', to “specifically bind', and to “stably relevant amino acids include, but are not limited to being, bind roundworm antigens whenever that antibody is able to 4-hydroxyproline and 5-hydroxylysine. In all cases, the recognize and bind to those roundworm antigens with greater amino acid sequence of a polypeptide described or otherwise affinity than to any other antigens from a non-roundworm 10 parasitic worm. Such binding specificity can be tested using referred to herein is presented in conventional form in that the methodology well known in the art, for example, ELISA or a left-most, or first, amino acid residue of the sequence is the radioimmunoassay (RIA). Based on information observed N-terminal residue and the right-most, or last, amino acid regarding the binding specificity of a particular composition residue of the sequence is the C-terminal residue. of the invention, the method of the present invention can be 15 A “conservative variant' of any particular nucleic acid carried out under conditions that allow that composition to sequence includes any sequence having one or more degen bind to (and therefore to allow the detection of such binding erate codon Substitutions to that particular nucleic acid to) a particular agent or agents, but not to significantly bind sequence, any sequence having one or more nucleotide Sub other agents, while those conditions are maintained. As one stitutions to, insertions to, and deletions from that particular example, the method of the present invention can be carried nucleic acid sequence, and the complementary sequence of out under conditions that allow an antibody of the present that particular nucleic acid and the conservative variants of invention to bind to (and therefore to allow the detection of that complementary sequence. Conservative variants of a par Such binding to) one or more roundworm antigens present in ticular nucleic acid sequence preferably have at least about a particular sample, but not significantly to any hookworm or 85% identity, more preferably have at least about 90% iden whipworm antigen that may be present in that sample, 25 tity, and even more preferably at least about 95-99% identity, thereby allowing for the distinction between roundworm, to that particular nucleic acid sequence. Conservative variants whipworm and hookworm. of a particular nucleic acid sequence may be artificially Syn "Detecting roundworm' means detecting one or more thesized or they may be isolated in their natural form from an roundworm-specific products, including one or more of the organism. polypeptides, antibodies and nucleic acids of the present 30 A “conservative variant of any particular polypeptide invention, or one or more roundworm antigens, or Copro6728, for example. The presence of one or more such sequence is any polypeptide having an amino acid sequence roundworm products in a sample from a mammal is indicative that varies from the amino acid sequence of that particular that the mammal has a roundworm infection, regardless of polypeptide but still retains the specific binding properties of whether any whole roundworm organism or ovum thereof is 35 that particular polypeptide. Such that an antibody of the also present in that sample. Conversely, the absence of one or present invention that is raised against the particular polypep more such roundworm products a sample from a mammal is tide is capable of specifically binding the variant polypeptide. indicative that the mammal does not have a roundworm infec Therefore, for example, a conservative variant of a particular tion. polypeptide may have one or more amino acid Substitutions, "Detecting whipworm' means detecting one or more 40 deletions, additions, and insertions to that particular polypep whipworm-specific products, including one or more of the tide. For example, a conserved variant of aparticular polypep polypeptides, antibodies and nucleic acids of the present tide may have 30 or fewer, 25 or fewer, 20 or fewer, 15 or invention, or one or more whipworm antigens, for example. fewer, 10 or fewer, or 5 or fewer, conserved amino acid The presence of one or more such whipworm products in a Substitutions to that particular polypeptide. Conservative sample from a mammal is indicative that the mammal has a 45 variants of a particular polypeptide preferably, but not essen whipworm infection, regardless of whether any whole whip tially, have at least about 80% identity, more preferably have worm organism or ovum thereof is also present in that sample. at least about 90% identity, and even more preferably at least Conversely, the absence of one or more such whipworm prod about 91-99% identity, to that particular polypeptide. A per ucts a sample from a mammal is indicative that the mammal cent identity for any Subject nucleic acid or amino acid does not have a whipworm infection. 50 sequence (e.g., any of polypeptides described herein) relative "Detecting hookworm' means detecting one or more hookworm-specific products, including one or more of the to another"target nucleic acid oramino acid sequence can be polypeptides, antibodies and nucleic acids of the present determined as follows. First, a target nucleic acid or amino invention, or one or more hookworm antigens, or acid sequence of the invention can be compared and aligned CoproASP5, for example. The presence of one or more such 55 to a Subject nucleic acid or amino acid sequence, using the hookworm products in a sample from a mammal is indicative BLAST 2 Sequences (B12seq) program from the stand-alone that the mammal has a hookworm infection, regardless of version of BLASTZ containing BLASTN and BLASTP (e.g., whether any whole hookworm organism or ovum thereof is version 2.0.14). The stand-alone version of BLASTZ can be also present in that sample. Conversely, the absence of one or obtained at www.ncbi.nlm.nih.gov. Instructions explaining more such hookworm products a sample from a mammal is 60 how to use BLASTZ, and specifically the B12seq program, indicative that the mammal does not have a hookworm infec can be found in the readme file accompanying BLASTZ. tion. The programs also are described in detail by Karlin et al. Amino acid refers to naturally occurring and synthetic (1990) Proc. Natl. Acad. Sci. 87:2264; Karlin et al. (1990) amino acids. Amino acid residues are abbreviated as follows: Proc. Natl. Acad. Sci. 90:5873; and Altschul et al. (1997) Alanine is A or Ala; Arginine is R or Arg, Asparagine is N or 65 Nucl. Acids Res. 25:3389. ASn; Aspartic Acid is D or Asp; Cysteine is C or Cys; “CoproASP5” refers to an N-terminal 28 kD fragment of Glutamic Acid is E or Glu: Glutamine is Q or Gln; Glycine is ASP5 found in mammalian feces. US 7,951,547 B2 11 12 “Copro6728” refers to a C-terminal 7 kD portion of that these variants include, but are not limited to, those have DIV6728 found in mammalian feces. In a specific embodi one or more Substitutions of basic amino acid residues, one or ment, coproG728 does not include the C-terminus of the full more Substitutions of acidic amino acid residues, one or more length DIV6728. Substitutions of polar amino acid residues, one or more Sub B12seq. performs a comparison between the Subject stitutions of hydrophobic amino acid residues, one or more sequence and a target sequence using either the BLASTN Substitutions of aromatic amino acid residues, and one or (used to compare nucleic acid sequences) or BLASTP (used more substitutions of small amino acid residues. (“Basic’ to compare amino acid sequences) algorithm. Typically, the amino acid residues are K, R and H. "Acidic' amino acid default parameters of a BLOSUM62 scoring matrix, gap residues are D and E. "Polar amino acid residues are N and existence cost of 11 and extension cost of 1, a word size of 3, 10 Q. “Hydrophobic' amino acids are I, L, and V. Aromatic' an expect value of 10, a per residue cost of 1 and a lambda amino acid residues are F.Y. and W. “Small amino acids are ratio of 0.85 are used when performing amino acid sequence G. S. A. T and M.) alignments. The output file contains aligned regions, of III. Nucleic Acids and Polypeptides of the Invention homology between the target sequence and the Subject The nucleic acids and polypeptides of the invention are sequence. Once aligned, a length is determined by counting 15 described in detail in Provisional Applications: “Methods, the number of consecutive nucleotides oramino acid residues Devices, Kits And Compositions For Detecting Whipworm.” (i.e., excluding gaps) from the target sequence that align with Application Ser. No. 61/128,077, filed May 19, 2008: “Meth sequence from the Subject sequence starting with any ods, Devices, Kits And Compositions For Detecting Round matched position and ending with any other matched posi worm.” Application Ser. No. 61/128,079, filed May 19, 2008: tion. A matched position is any position where an identical “Methods, Devices, Kits And Compositions For Detecting nucleotide or amino acid residue is present in both the target Roundworm. Application Ser. No. 61/128,076, files May 19, and Subject sequence. Gaps of one or more residues can be 2008; “Methods, Devices, Kits And Compositions For inserted into a target or subject sequence to maximize Detecting Roundworm. Application Ser. No. 61/128,099, sequence alignments between structurally conserved filed May 19, 2008; “Compositions, Devices, Kits and Meth domains (e.g., C.-helices, B-sheets, and loops). 25 ods for Detecting Hookworm'. Application Ser. No. 61/122, The percent identity over a particular length is determined 254, filed Dec. 12, 2008; and Utility Applications: “Round by counting the number of matched positions over that par worm Coproantigen Detection, application Ser. No. 1 1/763, ticular length, dividing that number by the length and multi 592, filed Jun. 15, 2007 and “Device, Kit and Method for plying the resulting value by 100. For example, if (i) a 500 Hookworm Antigen Detection', application Ser. No. 1 1/763, amino acid target sequence is compared to a subject amino 30 583, filed Jun. 15, 2007, "Methods, Devices, Kits, Composi acid sequence, (ii) the B12seq program presents 200 amino tions for Detecting Roundworm” filed concurrently herewith, acids from the target sequence aligned with a region of the and are all incorporated by reference in their entirety. Subject sequence where the first and last amino acids of that In an attempt to identify compositions that may be used to 200 amino acid region are matches, and (iii) the number of confirm the presence or absence of roundworm in a fecal matches over those 200 aligned amino acids is 180, then the 35 sample and to distinguish roundworm from other parasitic 500 amino acid target sequence contains a length of 200 and worm infections, a plurality of oligonucleotide primers were a sequence identity over that length of 90% (i.e., 180/200x designed, synthesized and used in 5 RACE, 3RACE and 100–90). It will be appreciated that a nucleic acid or amino RT-PCR reactions that included total RNA isolated from acid target sequence that aligns with a subject sequence can either whole adult Toxocara Canis or whole adult Toxocara result in many different lengths with each length having its 40 cati. As a result of these efforts, an 469-nucleotide cDNA own percent identity. It is noted that the percent identity value sequence was deduced from Toxocara canis (identified herein can be rounded to the nearest tenth. For example, 78.11, as SEQ ID NO:25), and a 548-nucleotide cDNA sequence 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, was deduced from Toxocara Cati (identified hereinas SEQID 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It is also NO:26). (BLAST searches that were carried out using SEQ noted that the length value will always be an integer. 45 ID NO:25 and SEQID NO:26 indicated these sequences are Conservative variants of a particular polypeptide sequence likely to encode members of serine protease inhibitor families may be artificially synthesized or they may be isolated in their that were first identified in Ascaris, but that had not been natural form from an organism, including from a roundworm identified in either T. canis or T. cati until now.) organism, such as Toxocara canis, Toxocara cati, and In an attempt to identify compositions that may be used to Ascaris, from a whipworm organism, such as Trichuris and 50 confirm the presence or absence of whipworm in a fecal Trichocephalus, and hookworm organism, such as Ancylos sample and to distinguish whipworm from other parasitic toma, Necator and Uncinaria for example. In one specific, worm infections, a plurality of oligonucleotide primers were non-limiting example for roundworm, the polypeptide of the designed, synthesized and used in 5 RACE, 3RACE and invention having an amino acid sequence corresponding to RT-PCR reactions that included total RNA isolated from SEQID NO: 27 shown below is a conservative variant of the 55 whole adult Trichuris vulpis. As a result of these efforts, a polypeptide of the present invention having an amino acid 1210-nucleotide cDNA sequence and a 1059-nucleotide sequence corresponding to SEQ ID NO:26 in that SEQ ID cDNA sequence were deduced (identified herein as SEQID NO:27 is more than 95% identical to SEQID NO:26 over an NO: 1 and SEQID NO:2, respectively. (BLAST searches that alignment of 126 amino acids. More generally, each one of were carried out using SEQ ID NO: 1 and SEQ ID NO:2 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID 60 indicated these sequences are likely to encode porin, which is NO:29 and SEQ ID NO:30 are conserved variants of each a major whipworm excretory/secretory protein that has been other. It is also to be understood that other conserved variants described in the mouse parasite Trichuris muris and the of the SEQID NO:26, SEQID NO:27, SEQID NO:28, SEQ human parasite Trichuris trichiuria, but that has not been ID NO:29 and SEQ ID NO:30 are contemplated by the identified in Trichuris vulpis until now.) present invention as described herein, but the skilled artisan 65 Previously, Zhan and colleagues described the molecular would recognize that all of these contemplated variants are identification and partial characterization of ASP-5, which is too numerous to list. The skilled artisan will also recognize an excretory/secretory protein of Ancylostoma (See Zhan et US 7,951,547 B2 13 14 al., International Journal for Parasitology 33:897-907 (2003)). In their studies, the Zhan group described a single (SEQ ID NO: 32) form of the ASP-5 protein having a mass of about 56 kDa, ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCG secreted from in vitro cultured parasites. The ASP-5 protein including an N-terminal His6 tag (SEQ CGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGAT ID NO:33) may be encoded by the nucleic sequence of SEQ CCGAATTCGAGCTCACCACTTGTCCAGGAAATGATCTAACAGATGCTGAA ID NO:31: CGCACACTGCTAACTAGGGTGCACAATTCCATTCGACGGGAAATAGCGCA (SEQ ID NO: 31) AGGAGTTGCAAACAACTACCATGGTGGTAAACTGCCTGCTGGAAAGAACA ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCG 10 TATACAGGATGAGATACAGCTGTGAGCTGGAACAGGCTGCTATTGATGCT CGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGAT AGTCAAACCTTCTGTTCCGCATCATTGGAGGAACCACAGAAATATGGACA CCGAATTCGAGCTCACCACTTGTCCAGGAAATGATCTAACAGATGCTGAA AAACATCCAAGCATACGTCACACCATCTATAATCGCTCGCCCGAAAAACG CGCACACTGCTAACTAGGGTGCACAATTCCATTCGACGGGAAATAGCGCA 15 ACCTTCTTGAAGATGCAGTGAAACAATGGTATCTGCCTGTTATCTACTAC AGGAGTTGCAAACAACTACCATGGTGGTAAACTGCCTGCTGGAAAGAACA GGCCAGCGCGACGCGGCCAACAAGTTTACGGATCCGCGCTTGTACACATT TATACAGGATGAGATACAGCTGTGAGCTGGAACAGGCTGCTATTGATGCT TGCAAACCTCGCCTACGACAAGAACACTGCACTTGGCTGTCACTATGCGA AGTCAAACCTTCTGTTCCGCATCATTGGAGGAACCACAGAAATATGGACA AATGTCAAGGCCCTGACAGAATCGTCATTAGTTGCATGTACAACAACGTC AAACATCCAAGCATACGTCACACCATCTATAATCGCTCGCCCGAAAAACG GTTCCTGACAACGCAGTGATCTACGAGCCTGGAACTGCTTGCGTAAAAGA ACCTTCTTGAAGATGCAGTGAAACAATGGTATCTGCCTGTTATCTACTAC TGCGGACTGCACTACTTATCCTCAGTCCACATGCAAGGACAGCCTTTGCA GGCCAACGCGACGCGGCCAACAAGTTCACCGATCCGCGCTTGTACACATT 25 TTATTCCTACGCCACATCCACCAAATCCACCAAATCCACCACCAGCAATG TGCAAACCTCGCCTACGACAAGAACACTGCACTTGGCTGTCACTATGCGA AGTCCATGAGCGGCCGC AATGTCAAGGCCCTGACAGAATCGTCATTAGTTGCATGTACAACAACGTC A skilled artisan will appreciate that due to the degeneracy GTTCCTGACAACGCTGTGATCTACGAGCCAGGAACTGCTTGCGTAAAAGA of the genetic code, nucleic acid sequences other than SEQID 30 NO:32 can code for the polypeptide of SEQ ID NO:34 if TCAGGACTGCACTACTTATCCTCAGTCCACATGCAAGGACAGCCTTTGCA appropriate (silent) codon Substitutions are made. TTATTCCTACGCCACATCCACCAAATCCACCAAATCCACCACCTGCAATG Analysis of the roundworm sequences corresponding to SEQIDNO:24 and SEQID NO:25 indicated that each one of TGTCCAAACGCTGAAATGACTGATGCAGCACGAAAGAAGGTCCTCGACAT 35 these roundworm sequences contains a large open reading GCACAACTGGCGCAGATCGCAGCTCGCTCTGGGAAACGTTCAAAACGGGA frame (ORF). Specifically, the large ORF of SEQID NO:24 corresponds to nucleotides 21 through 446 of SEQID NO:24 AAAATGCTTACAACTGCCCCACTGCAACAGACATGTACAAGATGGAATAT and is predicted to encode a roundworm polypeptide having GATTGCGACCTCGAGAACAGCGCTCTAGCGTATGCAAAGCAATGTAGTCT the following amino acid sequence: 40 CGTTGGTTCAGCAGAAGGAACTCGTCCAGGAGAAGGCGAGAATGTCCACA (SEQ ID NO: 26) AAGGCGCTCTCGTAACCGATCCGGAGGCTGCAGTTCAGACCGCAGTTCAA MLPITFLLAIIWGAAVAHRKCGPNEEWTECTGCEIKCGOGEOPCPMMCRP GCATGGTGGAGTCAAATCTCACAAAATGGACT CAATGCACAGATGAAATT PSCECMAGKGLRRTADGRCWPEAOCPKRMVKRDEKCGPNEKFLKCRGCEG 45 CACTGCTTTCTTGAAGGACAAGCCTGACGCTCCGACAGCGTTTACACAGA TCKERLWPCPRMCKPPGCECPASEGFWRNDKGECIKFDDCPK. TGGCGTGGGCCAAATCCGTAAAGCTTGGATGTGCTGTCTCTAATTGTCAG Further, the large ORF of SEQ ID NO:25 corresponds to nucleotides 21 through 446 of SEQID NO:25 and is predicted GCAGATACCTTCACCGTCTGTAGATACAAAGCTGCCGGAAACATCGTGGG to encode a roundworm polypeptide having the following CGAATTCATCTATACCAAGGGAAATGTATGCGACGCCTGTAAAGCCACAT 50 amino acid sequence: GCATTACCGCGGAAGGTCTTTGCCCAACGCCTTGAGCGGCCGC In an effort to identify tools for capturing and detecting (SEO ID NO: 27) hookworm and/or hookworm antigen in hookworm-infected MLPLTFLLAFIWGAAWAHRKCGPNEEWTECTGCEMKCGEGETPCPMMCRP mammals, the present inventors have determined that a modi 55 fied protein of about 28 kDa, rather than the 56 kDa version, PSCECMAGKGLRRTPDGRCWPEAOCPKHMVKRDEKCGKNEKFLKCRGCEG is present in the feces of canines that are infected by Ancy TCKERLWPCPKMCKPPGCECPASEGFWRNDKHECIKFDDCPK. lostoma. (This 28 kDa version of ASP5 is referred to hereinas “CoproASP5': the detection of CoproASP5 in feces of Ancy SEQID NO:24, SEQID NO:25, SEQID NO:26, SEQID lostoma-infected canines is described in the Example section 60 NO:27, SEQIDNO: 28, SEQID NO:29 and SEQIDNO:30 included herein.) In one aspect, therefore, the present inven are outlined in Provisional Application Ser. No. 61/128,076 tion provides polypeptides that may be used to generate anti filed May 19, 2008 which is incorporated by reference in its bodies that may be used to specifically capture and detect entirety. CoproASP5. One such polypeptide that may be used to gen Analysis of the whipworm sequences corresponding to erate antibodies that may be used to bind Copro ASP5 is 65 SEQ ID NO: 1 and SEQID NO:2 indicated that each one of referred to as ASP5-1 polypeptide, which may be encoded by these whipworm sequences contains a large ORF. Specifi the following nucleic acid sequence: cally, the large ORF of SEQID NO: 1 corresponds to nucle US 7,951,547 B2 15 16 otides 32 through 1147 of SEQID NO: 1 and is predicted to encode a whipworm polypeptide having the following amino (SEQ ID NO: 13) acid sequence: IYGVAASRRRRHHFTLEKSLDTHLKWLSHEOKEELLKMKKDGKSKKELOD KVMHFYEHLEGDAKHEATEOLKGGCREILKHWVGEEKAAEIKALKDSGAS (SEQ ID NO : 3) KDELKAKVEDALHAVTDEEKKOHIAEFGPACKEIFGVPIDVRHKRDPYTN MRLVFHAVIYLTLGFLTDAVREKRGKCPPEPPIAGNTIYCRDDFDCGGRO MTPDEWAEGLRS KCCTIAEGRGCVPPYGEOHFEVVKPGHCPAIPAVTGMANFCNTDGDCDGP SEQID NO:10, SEQID NO:11, SEQID NO:12, SEQ ID KKCCLTSRGYDCTHPLHFPIOPOPPVGOCPPSKPRIPGKWVDICAKHANC 10 NO:13, SEQID NO:14, SEQID NO: 15, and SEQID NO: 16 PDPEKCCDTEYGNRCMDVGLVPGOGERPGNCPNEPRIRGTKYDCRRDDDC are outlined in Provisional Application Ser. No. 61/128,079 filed May 19, 2008 which is incorporated by reference in its DGVOKCCFTVEGRECVEPSRKPLDKPGHCPPIPADVGSARYCDTDRDCDG entirety. PRKCCLSSRGYECKHPWHYPDRWEPLWGECPPSRPRIPGKWWDICSKHAN Analysis of the roundworm sequences corresponding to 15 SEQID NO: 17 and SEQID NO: 18 indicated that each one CPDPEKCCDTEYGNRCMDVGLVPGOGEKPANCPKEPRIRGTKYDCRRDDD of these roundworm sequences contains a large open reading frame (ORF). Specifically, the large ORF of SEQID NO: 17 CDGKOKCCYTTEGRECWHGIWP. corresponds to nucleotides 28 through 456 of SEQID NO: 17 and is predicted to encode a roundworm polypeptide having Further, the large ORF of SEQ ID NO:2 corresponds to the following amino acid sequence: nucleotides 1 through 1059 of SEQID NO:2 and is predicted to encode a whipworm polypeptide having the following amino acid sequence: (SEQ ID NO: 19) MLSVLALFALITFAVAGPESCGPNEWWTECTGCELKCGODENTPCTLNCR 25 (SEQ ID NO : 4) PPSCECSPGRGMRRTNDGRCIPASOCPOHRAKREEOCKPNEOWSPCRGCE WREKRGKCPPEPPIAGNTIYCRDDFDCGGROKCCTIAEGRGCVPPYGEOD GTCAORFVPCTRNCRPPGCECVAGAGFVRDAEGNCIKFDDCPK. FEWWKPGHCPAIPAWTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFP Further, the large ORF of SEQ ID NO:2 corresponds to IOPOPPVGOCPPSKPRVPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVG 30 nucleotides 29through 457 of SEQID NO:18 and is predicted to encode a roundworm polypeptide having the following LVAGOGERPGNCPNEPRIRGTKYDCRRDDDCDGVOKCCFTVEGRECVEPS amino acid sequence: RKPLDKPGHCPPIPADWGSARYCDTDRDCDGPRKCCLSSRGYECKHPWHY PDRWEPLWGECPPSRPRIPGKWWDICSKHANCPDPEKCCDTEYGNRCMDW 35 (SEQ ID NO: 2O) MLSVLALFALITFAVADPKSCGPNEWWTECTGCELKCGODEDTPCTLNCR GLVPGOGEKPANCPKEPRIRGTKYDCRRDDDCDGKOKCCYTTEGRECWHG PPSCECSPGRGMRRTDDGRCIPASOCPOHRAKREEOCKPNEOWSPCRGCE IWP. GTCAORFVPCTRNCRPPGCECVAGAGFVRDAAGNCIKFDDCPK. SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID 40 SEQID NO:17, SEQID NO:18, SEQID NO:19, SEQ ID NO;4, SEQID NO:5, SEQID NO: 6, SEQIDNO: 7, SEQID NO:20, SEQID NO:21, SEQID NO:22, and SEQID NO. 23 NO: 8, and SEQID NO:9 are outlined in Provisional Appli are outlined in Provisional Application Ser. No. 61/128,099 cation Ser. No. 61/128,077 filed May 19, 2008 which is incor filed May 19, 2008 which is incorporated by reference in its porated by reference in its entirety. entirety Analysis of the roundworm sequences corresponding to 45 Analysis of the hookworm sequences corresponding to SEQID NO:31 and SEQID NO:32 indicated that each one SEQID NO: 10 and SEQID NO: 11 indicated that each one of these hookwormworm sequences contains a large open of these sequences contains a large ORF. Specifically, as reading frame (ORF). Specifically, the large ORF of SEQID shown in, the large ORF of SEQ ID NO:10 corresponds to NO:31 is predicted to encode a hookworm polypeptide hav nucleotides 2 through 616 of SEQID NO:11 and is predicted 50 to encode a roundworm polypeptide having the following ing the following amino acid sequence: amino acid sequence: (SEQ ID NO: 33) MGSSHHHHHHSSGLVPRGSHMASMTGGOOMGRGSEFELTTCPGNDLTDAE (SEQ ID NO: 12) 55 RTLLTRVHNSIRREIAOGVANNYHGGKLPAGKNIYRMRYSCELEOAAIDA KKIYGVAASRRRRHHFTLENSLDTHLKWLSHEOKEELLOMKKDGKSKKEL SOTFCSASLEEPOKYCONIOAYVTPSIIARPKNDLLEDAWKOWYLPWIYY QDKIMHYYEHLEGDAKHEATEOLKGGCREILKHWVGEEKAAEIKALKDSG GORDAANKFTDPRLYTFANLAYDKNTALGCHYAKCOGPDRIVISCMYNNW ASKDELKAKVEEALHAVTDEEKKOHIAEFGPACKKIYGVAASRRRRHHFT WPDNAVIYEPGTACVKDODCTTYPOSTCKDSLCIIPTPHPPNPPNPPPAM LENSLDTHLKWLSHEOKEELLOMKKDGKSKKELODKIMHYYEHLEGMLLA 60 CPNAEMTDAARKKVLDMHNWRRSOLALGNWONGKNAYNCPTATDMYKMEY LCILY. DCDLENSALAYAKQCSLVGSAEGTRPGEGENVHKGALVTDPEAAWOTAVO Further, the large ORF of SEQID NO: 11 corresponds to AWWSOISONGLNAOMKFTAFLKDKPDAPTAFTOMAWAKSWKLGCAVSNCO nucleotides 1 through 486 of SEQID NO: 11 and is predicted 65 to encode a roundworm polypeptide having the following ADTFTWCRYKAAGNIWGEFIYTKGNWCDACKATCITAEGLCPTP. amino acid sequence: US 7,951,547 B2 17 18 The first 38 amino acids of SEQID NO:33 are derived from - Continued a cloning vector, the skilled artisan will appreciate that this SRKPLDKPGHCPPIPADWGSARYCDTDRDCDGPRKCCLSSRGYECKHPWH portion may be omitted or substituted with other suitable fusion partners. YPDRWEPLWGECPPSRPRIPGKWWDICSKHANCPDPEKCCDTEYGNRCMD Further, the large ORF of SEQ ID NO:32 is predicted to WGLVPGOGEKPANCPKEPRIRGTKYDCRRDDDCDGKOKCCYTTEGRECWH encode a hookworm polypeptide having the following amino acid sequence: GIWP. In another specific example, the roundworm polypeptide of the present invention has the following amino acid sequence: (SEQ ID NO: 34) 10 MGSSHHHHHHSSGLVPRGSHMASMTGGOOMGRGSEFELTTCPGNDLTDAE RTLLTRVHNSIRREIAOGVANNYHGGKLPAGKNIYRMRYSCELEOAAIDA (SEQ ID NO: 14) MHHFTLENSLDTHLKWLSHEOKEELLOMKKDGKSKKELODKIMHYYEHLE SOTFCSASLEEPOKYCONIOAYVTPSIIARPKNDLLEDAWKOWYLPWIYY 15 GDAKHEATEOLKGGCREILKHWVGEEKAAEIKALKDSGASKDELKAKVEE GORDAANKFTDPRLYTFANLAYDKNTALGCHYAKCOGPDRIVISCMYNNW ALHAVTDEEKKOHIAEFGPACKKIYGVAAS. VPDNAVIYEPGTACVKDADCTTYPOSTCKDSLCIIPTPHPPNPPNPPPAM In another specific example, the roundworm polypeptide of SP. the present invention has the following amino acid sequence: The polypeptides of the present invention are encoded for by nucleic acids that have a nucleotide sequence that corre (SEQ ID NO: 21) sponds to all or portions of SEQID NO: 1, SEQID NO: 2, MGPESCGPNEVWTECTGCELKCGODENTPCTLNCRPPSCECSPGRGMRRT SEQID NO: 10, SEQ ID NO: 11, SEQID NO: 17, SEQ ID NDGRCIPASOCPOHRAKREEOCKPNEOWSPCRGCEGTCAORFVPCTRNCR NO: 18, SEQID NO: 24, SEQ ID NO: 25, SEQID NO:31 25 and SEQ ID NO: 32 and all conservative variants of those PPGCECWAGAGFWRDAEGNCIKFDDCPK. sequences. It is to be understood therefore that the amino acid sequence of the polypeptide of the present invention is vari In another specific example, the hookworm polypeptide of able. the invention includes an amino acid sequence that is identi For example, the polypeptide of the present invention may cal to or is homologous to a sequence represented by SEQID have an amino acid sequence that corresponds to all or a 30 NO: 34. portion of SEQID NO:3, SEQID NO:4, SEQID NO:5, SEQ The 125 amino acid residues that follow the N-terminal IDNO: 6, SEQID NO:7, SEQIDNO: 8, SEQID NO:9 SEQ methionine residue of the roundworm polypeptide corre ID NO:12, SEQID NO:13, SEQID NO: 14, SEQID NO:15, sponding to SEQID NO: 28 specifically represent the amino SEQID NO: 16, SEQ ID NO: 19, SEQID NO: 20, SEQ ID acid residues 18 through 142 of SEQID NO:26. The N-ter NO: 21, SEQID NO: 22, SEQID NO. 23, SEQID NO:26, 35 minal methionine was artificially added to the N-terminus of SEQID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID this polypeptide by carrying outa Standard cloning technique. NO:30, SEQID NO:33 and SEQID NO:34 or allora portion Antibody raised against the polypeptide corresponding to of a conservative variant of SEQ ID NO:3, SEQ ID NO: 4, SEQID NO: 28 was useful for detecting roundworm antigen. SEQID NO:5, SEQID NO: 6, SEQID NO: 7, SEQID NO: Because the N-terminal methionine was artificially added, 8, SEQID NO: 9 SEQID NO:12, SEQID NO:13, SEQID 40 and is not thought to naturally exist in Toxocara (the residue NO: 14, SEQID NO: 15, SEQID NO: 16, SEQID NO: 19, that is immediately prior to the histidine residue at position 18 SEQID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID in each one of SEQID NO:26 and SEQID NO:27 is alanine, NO: 23, SEQID NO: 26, SEQID NO: 27, SEQID NO: 28, and not methionine), it is therefore contemplated that the SEQID NO:29, SEQIDNO:30, SEQIDNO:33 and SEQID polypeptide of the present invention may have an amino acid NO: 34. 45 sequence that corresponds to amino acid residues 18 through In one specific example, the roundworm polypeptide of the 142 of SEQID NO:26, or, more specifically: present invention has the following amino acid sequence: (SEQ ID NO: 29) (SEQ ID NO: 28) 50 HRKCGPNEEWTECTGCEIKCGOGEOPCPMMCRPPSCECMAGKGLRRTADG MHRKCGPNEEWTECTGCEIKCGOGEOPCPMMCRPPSCECMAGKGLRRTAD RCVPEAOCPKRMVKRDEKCGPNEKFLKCRGCEGTCKERLVPCPRMCKPPG GRCVPEAOCPKRMVKRDEKCGPNEKFLKCRGCEGTCKERLVPCPRMCKPP CECPASEGFWRNDKGECIKFDDCPK. GCECPASEGFWRNDKGECIKFDDCPK. 55 The 353 amino acid residues that follow the N-terminal In one specific example, the whipworm polypeptide of the methionine residue of the whipworm polypeptide corre present invention has the following amino acid sequence: sponding to SEQ ID NO:5 specifically represent the amino acid residues 20 through 353 of SEQID NO:3. The N-termi nal methionine was artificially added to the N-terminus of this (SEO ID NO; 5) 60 polypeptide by carrying out a standard cloning technique. MVREKRGKCPPEPPIAGNTIYCRDDFDCGGROKCCTIAEGRGCVPPYGEO Antibody raised against the polypeptide corresponding to HFEWWKPGHCPAIPAWTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHF SEQ ID NO:5 was useful for detecting whipworm antigen. Because the N-terminal methionine was artificially added, PIOPOPPVGOCPPSKPRIPGKWVDICAKHANCPDPEKCCDTEYGNRCMDV and is not thought to naturally exist in Trichuris vulpis (the GLVPGOGERPGNCPNEPRIRGTKYDCRRDDDCDGWOKCCFTVEGRECVEP 65 residue that is immediately prior to the valine residue at position 20 in SEQ ID NO:3 is alanine), it is therefore con templated that the polypeptide of the present invention may US 7,951,547 B2 19 20 have an amino acid sequence that corresponds to amino acid sponding to SEQID NO: 14 specifically represent the amino residues 20 through 353 of SEQID NO:3, or, more specifi acid residues 14 through 142 of SEQID NO: 12. The N-ter cally: minal methionine was artificially added to the N-terminus of this polypeptide by carrying outa Standard cloning technique. Antibody raised against the polypeptide corresponding to (SEQ ID NO : 6) SEQID NO: 14 was useful for detecting roundworm antigen. WREKRGKCPPEPPIAGNTIYCRDDFDCGGROKCCTIAEGRGCVPPYGEOH Because the N-terminal methionine was artificially added, FEWWKPGHCPAIPAWTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFP and is not thought to naturally exist in Toxocara (the residue that is immediately prior to the histidine residue at position 14 IOPOPPVGOCPPSKPRIPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVG 10 in each one of SEQID NO: 12and SEQID NO: 13 is arginine, and not methionine), it is therefore contemplated that the LVPGOGERPGNCPNEPRIRGTKYDCRRDDDCDGVOKCCFTVEGRECVEPS polypeptide of the present invention may have an amino acid RKPLDKPGHCPPIPADWGSARYCDTDRDCDGPRKCCLSSRGYECKHPWHY sequence that corresponds to amino acid residues 14 through 142 of SEQID NO: 12, or, more specifically: PDRWEPLWGECPPSRPRIPGKWWDICSKHANCPDPEKCCDTEYGNRCMDW 15 GLVPGOGEKPANCPKEPRIRGTKYDCRRDDDCDGKOKCCYTTEGRECWHG (SEQ ID NO: 15) IWP. HHFTLENSLDTHLKWLSHEOKEELLOMKKDGKSKKELODKIMHYYEHLEG In another specific example, the whipworm polypeptide of DAKHEATEOLKGGCREILKHWVGEEKAAEIKALKDSGASKDELKAKVEEA the present invention has the following amino acid sequence: LHAVTDEEKKOHIAEFGPACKKIYGVAAS. With 128 amino acids, the roundworm protein DIV6728 (SEO ID NO: 7) (SEQID NO: 21) is about 14 kD in size and with a theoretical MVREKRGKCPPEPPIAGNTIYCRDDFDCGGROKCCTIAEGRGCVPPYGEO 25 pI is about 6.54. This protein belongs to TIL Superfamily, DFEWWKPGHCPAIPAWTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHF which is a group of serine protease inhibitors. In an effort to identify tools for capturing and detecting roundworm and/or PIOPOPPVGOCPPSKPRVPGKWVDICAKHANCPDPEKCCDTEYGNRCMDV roundworm antigen in roundworm-infected mammals, the GLVAGOGERPGNCPNEPRIRGTKYDCRRDDDCDGWOKCCFTVEGRECVEP present inventors have determined that only a truncated por 30 tion (about 7 kDa) of the full-length (14 kDa) protein, and SRKPLDKPGHCPPIPADWGSARYCDTDRDCDGPRKCCLSSRGYECKHPWH therefore not the 14 kDa version, is present in the feces of canines that are infected by T. canis. (This 7 kDa truncated YPDRWEPLWGECPPSRPRIPGKWWDICSKHANCPDPEKCCDTEYGNRCMD portion of DIV6728 is referred to herein as “Copro6728; the WGLVPGOGEKPANCPKEPRIRGTKYDCRRDDDCDGKOKCCYTTEGRECWH detection of Coproð728 in feces of T. canis-infected canines 35 is described in the Example section included herein.) In one GIWP. aspect, therefore, the present invention provides polypeptides that may be used to generate antibodies that may be used to The 353 amino acid residues that follow the N-terminal specifically capture and detect CoproG.728. methionine residue of the whipworm polypeptide corre The 127 amino acid residues that follow the N-terminal sponding to SEQ ID NO:7 specifically represent the amino methionine residue of the roundworm polypeptide corre acid residues 1 through 353 of SEQID NO:4. The N-terminal 40 sponding to SEQID NO: 21 specifically represent the amino methionine was artificially added to the N-terminus of this acid residues 17 through 143 of SEQID NO: 19. The N-ter polypeptide by carrying out a standard cloning technique. minal methionine was artificially added to the N-terminus of Antibody raised against the polypeptide corresponding to this polypeptide by carrying outa Standard cloning technique. SEQ ID NO:7 was useful for detecting whipworm antigen. Also as described throughout the Example section, antibody Because the N-terminal methionine was artificially added, it 45 raised against the polypeptide corresponding to SEQID NO: is therefore contemplated that the polypeptide of the present 21 was useful for detecting roundworm antigen. Because the invention may have an amino acid sequence that corresponds N-terminal methionine was artificially added, and is not to amino acid residues 1 through 353 of SEQ ID NO:4, or, thought to naturally exist in Toxocara (the residue that is more specifically: immediately prior to the glycine residue at position 17 in each 50 one of SEQID NO: 19 and SEQID NO:20 is alanine, and not methionine), it is therefore contemplated that the polypeptide (SEQ ID NO: 8) of the present invention may have an amino acid sequence WREKRGKCPPEPPIAGNTIYCRDDFDCGGROKCCTIAEGRGCVPPYGEOD that corresponds to amino acid residues 17 through 143 of FEWWKPGHCPAIPAWTGMANFCNTDGDCDGPKKCCLTSRGYDCTHPLHFP 55 SEQ ID NO:19, or, more specifically: IOPOPPVGOCPPSKPRVPGKWVDICAKHANCPDPEKCCDTEYGNRCMDVG (SEQ ID NO: 22) LVAGOGERPGNCPNEPRIRGTKYDCRRDDDCDGVOKCCFTVEGRECVEPS GPESCGPNEWWTECTGCELKCGODENTPCTLNCRPPSCECSPGRGMRRTN RKPLDKPGHCPPIPADWGSARYCDTDRDCDGPRKCCLSSRGYECKHPWHY 60 DGRCIPASOCPOHRAKREEOCKPNEOWSPCRGCEGTCAORFVPCTRNCRP PDRWEPLWGECPPSRPRIPGKWWDICSKHANCPDPEKCCDTEYGNRCMDW PGCECWAGAGFWRDAEGNCIKFDDCPK. GLVPGOGEKPANCPKEPRIRGTKYDCRRDDDCDGKOKCCYTTEGRECWHG Further, an alignment of SEQID NO: 28 (mostly Toxocara IWP. canis-derived sequence; with the only exception being the 65 N-terminal methionine residue) to SEQID NO: 27 (Toxocara The 129 amino acid residues that follow the N-terminal cati-derived sequence) is shown in FIG. 14. Because antibody methionine residue of the roundworm polypeptide corre raised against a polypeptide having sequence corresponding US 7,951,547 B2 21 22 to SEQID NO:28 was useful for detecting Toxocara cati, it is absent, the X at position 157 is A or absent, the X at position additionally contemplated that the polypeptide of the present 158 is E or absent, the X at position 159 is G or absent, the X invention may have the amino acid sequence corresponding at position 160 is L or absent, the X at position 161 is R or to SEQID NO:30, wherein the Xat position 1 is Mor absent, absent, and the X at position 162 is S or absent. the X at position 2 is L or absent, the X at position 3 is P or Further, an alignment of SEQID NO: 21 (mostly Toxocara absent, the X at position 4 is L or absent (or the X at position canis-derived sequence; with the only exception being the 4 is I, which occupies position 4 of SEQID NO:26), the X at N-terminal methionine residue) to SEQID NO:20 (Toxocara position 5 is T or absent, the X at position 6 is For absent, the cati-derived sequence) is shown in FIG. 11. Because antibody X at position 7 is L or absent, the Xat position 8 is L or absent, raised against a polypeptide having sequence corresponding the X at position 9 is A or absent, the X at position 10 is For 10 to SEQID NO: 21 was useful for detecting Toxocara Cati, it absent (or the Xat position 10 is I, which occupies position 10 is additionally contemplated that the polypeptide of the of SEQID NO:26), the X at position 11 is I or absent, the X present invention may have the amino acid sequence corre at position 12 is V or absent, the X at position 13 is G or sponding to SEQ ID NO:23, wherein the X at position 1 is absent, the Xat position 14 is A or absent, the Xat position 15 absent or M, the Xat position 2 is absent or L, the Xat position is A or absent, the X at position 16 is V or absent, the X at 15 3 is absent or S, the X at position 4 is absent or V, the X at position 16 is Mor A, the X at position 35 is I or M, the X at position 5 is absent or L, the X at position 6 is absent or A, the position 39 is Q or E, the X at position 42 is Q or T, the X at X at position 7 is absent or L, the X at position 8 is absent or position 65 is A or P, the X at position 78 is R or H, the X at F, the X at position 9 is absent or A, the X at position 10 is position 88 is P or K, the X at position 111 is R or K, and the absent or L, the Xat position 11 is absent or I, the Xat position X at position 132 is G or H. 12 is absent or T, the X at position 13 is absent or F, the X at Further, an alignment of SEQID NO:3 with respect to SEQ position 14 is absent or A, the X at position 15 is absent or V. ID NO:4 is shown in FIG. 5. It is additionally contemplated the X at position 16 is Mor A, the X at position 17 is G or D, that the polypeptide of the present invention may have the the X at position 19 is E or K, the X at position 42 is N or D, amino acid sequence corresponding to SEQ ID NO:9, the X at position 66 is N or D, and the X at position 132 is E wherein the X at position 1 is Mor absent, the X at position 2 25 or A. is R or absent, the X at position 3 is L or absent, the X at A polypeptide of the present invention may have the amino position 4 is V or absent, the X at position 5 is For absent, the acid sequence corresponding to SEQID NO:38, wherein the X at position 6 is Hor absent, the Xat position 7 is A or absent, amino acid at position 54 is E or A. Antibodies that specifi the X at position 8 is V or absent, the X at position 9 is I or cally bind to this polypeptide are included in the invention. absent, the Xat position 10 is Y or absent, the X at position 11 30 Because the first 38 amino acid residues of the polypeptide is L or absent, the X at position 12 is T or absent, the X at having the amino acid sequence that corresponds to SEQID position 13 is L or absent, the X at position 14 is G or absent, NO: 34 were not derived from Ancylostoma (i.e., they are the X at position 15 is For absent, the Xat position 16 is Lor vector sequence), it is further contemplated that the peptide of absent, the Xat position 17 is T or absent, the X at position 18 the present invention may include an amino acid sequence is D or absent, the X at position 19 is A or is absent (or the X 35 that is identical to or is homologous to a sequence represented at position 19 is M, which occupies position 19 of SEQ ID by SEQID NO:34, wherein the Xat position 1 is Mor absent, NO:5), the X at position 69 is Hor D, the X at position 136 is the X at position 2 is G or absent, the X at position 3 is S or I or V and the X at position 172 is P or A. absent, the X at position 4 is S or absent, the X at position 5 is Further, an alignment of SEQID NO:14 (mostly Toxocara Hor absent, the Xat position 6 is Hor absent, the Xat position canis-derived sequence; with the only exception being the 40 7 is H or absent, the X at position 8 is H or absent, the X at N-terminal methionine residue) to SEQID NO: 13 (Toxocara position 9 is H or absent, the X at position 10 is H or absent, cati-derived sequence) is shown in FIG. 8. Because antibody the X at position 11 is S or absent, the X at position 12 is S or raised against a polypeptide having sequence corresponding absent, the X at position 13 is G or absent, the Xat position 14 to SEQID NO: 14 was useful for detecting Toxocara Cati, it is L or absent, the X at position 15 is V or absent, the X at is additionally contemplated that the polypeptide of the 45 position 16 is P or absent, the X at position 17 is R or absent, present invention may have the amino acid sequence corre the X at position 18 is G or absent, the X at position 19 is S or sponding to SEQID NO:16, wherein the X at position 1 is I or absent, the X at position 20 is Horabsent, the Xat position 21 absent, the X at position 2 is Y or absent, the X at position 3 is M or absent, the X at position 22 is A or absent, the X at is G or absent, the X at position 4 is V or absent, the X at position 23 is S or absent, the X at position 24 is Mor absent, position 5 is A or absent, the X at position 6 is A or absent, the 50 the X at position 25 is T or absent, the X at position 26 is Gor X at position 7 is S or absent, the Xat position 8 is R or absent, absent, the X at position 27 is G or absent, the Xat position 28 the X at position 9 is R or absent, the X at position 10 is Ror is Q or absent, the X at position 29 is Q or absent, the X at absent, the X at position 11 is R or M, the X at position 18 is position 30 is Mor absent, the Xat position 31 is G or absent, N or K, the X at position 37 is Q or K, the X at position 52 is the X at position 32 is R or absent, the X at position 33 is Gor I or V. X at position 55 is Y or F, the X at position 110 is E or 55 absent, the Xat position 34 is S or absent, the Xat position 35 D, the X at position 133 is K or E, the X at position 135 is Y is E or absent, the X at position 36 is F or absent, the X at or F, the X at position 138 is A or P, the X at position 139 is A position 37 is E or absent, and the X at position 38 is L or or I, the X at position 140 is S or D, the X at position 141 is V absent. Furthermore, because the Sat position 251 in the SEQ or absent, the Xat position 142 is R or absent, the Xat position ID NO:33 was artificially substituted during the cloning pro 143 is H or absent, the X at position 144 is K or absent, the X 60 cess described in the Example section herein in that sequence at position 145 is R or absent, the X at position 146 is D or (the ASP5 protein of wild-type Ancylostoma includes a C absent, the X at position 147 is P or absent, the X at position residue at that position), it is contemplated that the X at 148 is Y or absent, the X at position 149 is T or absent, the X position 251 of SEQID NO:34 may be either S or C. at position 150 is N or absent, the X at position 151 is Mor It is also contemplated that any one or more of the SEQID absent, the X at position 152 is T or absent, the X at position 65 NO:3, SEQIDNO:4, SEQIDNO:5, SEQID NO: 6, SEQID 153 is P or absent, the X at position 154 is D or absent, the X NO:7, SEQID NO: 8, SEQID NO:9 SEQID NO: 12, SEQ at position 155 is E or absent, the X at position 156 is V or ID NO:13, SEQID NO:14, SEQID NO:15, SEQIDNO:16, US 7,951,547 B2 23 24 SEQID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID carried out to isolate naturally existing polypeptides having NO: 22, SEQID NO. 23, SEQID NO: 26, SEQID NO: 27, amino acid sequence corresponding to SEQ ID NO:3, SEQ SEQID NO: 28, SEQ ID NO: 29, SEQID NO:30, SEQ ID IDNO:4, SEQIDNO:5, SEQID NO: 6, SEQID NO:7, SEQ NO:33 and SEQID NO:34 may be only a portion of a larger ID NO: 8, SEQ ID NO: 9 SEQID NO:12, SEQ ID NO:13, polypeptide sequence, and therefore may represent partial 5 SEQID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID sequence of one or more proteins that normally are expressed NO: 19, SEQID NO: 20, SEQID NO: 21, SEQID NO:22, in roundworm, for example, or one or more polypeptide SEQID NO. 23, SEQ ID NO: 26, SEQID NO: 27, SEQ ID sequences that are artificially fused to SEQID NO:3, SEQID NO: 28, SEQID NO: 29, SEQID NO:30, SEQID NO:33, NO: 4, SEQID NO. 5, SEQID NO: 6, SEQID NO: 7, SEQ SEQ ID NO:34, SEQ ID NO:38, and Copro6728 and any ID NO: 8, SEQID NO: 9 SEQID NO: 12, SEQID NO:13, 10 naturally occurring variant of those polypeptides. These tech SEQID NO: 14, SEQ ID NO: 15, SEQID NO: 16, SEQ ID niques further include those that may be carried out to artifi NO: 19, SEQID NO: 20, SEQID NO: 21, SEQID NO:22, cially generate the polypeptides having amino acid sequence SEQID NO. 23, SEQ ID NO: 26, SEQID NO: 27, SEQ ID corresponding to SEQID NO:3, SEQID NO: 4, SEQID NO: NO: 28, SEQID NO: 29, SEQID NO:30, SEQID NO:33, 5, SEQID NO: 6, SEQID NO:7, SEQID NO:8, SEQIDNO: SEQ ID NO:34, or SEQ ID NO: 38, or Copro6728. The 15 9 SEQID NO:12, SEQID NO:13, SEQID NO: 14, SEQ ID skilled artisan will recognize that are a variety of techniques NO: 15, SEQID NO: 16, SEQID NO: 19, SEQID NO: 20, exist for artificially fusing two or more polypeptide fragments SEQID NO: 21, SEQ ID NO: 22, SEQID NO. 23, SEQ ID together. NO: 26, SEQID NO: 27, SEQID NO: 28, SEQID NO: 29, It is even further contemplated that the polypeptide of the SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQID present invention may include more than one of the SEQID NO:38, and Coproð728 and any conserved variant of those NO:3, SEQIDNO:4, SEQIDNO:5, SEQID NO: 6, SEQID polypeptides. Such variants may be generated, for example, NO: 7, SEQID NO: 8, SEQID NO:9 SEQID NO:12, SEQ by employing any one or more mutagenesis techniques or by IDNO:13, SEQID NO: 14, SEQID NO:15, SEQID NO:16, direct synthesis. SEQID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID The polypeptides of the present invention are capable of NO: 22, SEQID NO. 23, SEQID NO: 26, SEQID NO: 27, 25 eliciting an immune response in a host animal that is exposed SEQID NO: 28, SEQ ID NO: 29, SEQID NO:30, SEQ ID to these polypeptides to produce one or more of the antibodies NO:33 SEQID NO:34, SEQID NO:38, and Copro6728. For of the present invention. Regardless of the technique by example, the polypeptide of the present invention may which they are derived, the polypeptides of the present inven include the SEQID NO:3, SEQ ID NO: 4, SEQID NO: 5, tion are preferably prepared in substantially pure form when SEQID NO: 6, SEQID NO: 7, SEQID NO: 8, SEQID NO: 30 they are to be used for the purpose of raising antibody. Pref 9 SEQID NO:12, SEQID NO:13, SEQID NO: 14, SEQ ID erably, these polypeptides are at least about 80% pure, more NO: 15, SEQID NO: 16, SEQID NO: 19, SEQID NO:20, preferably are at least about 90-95% pure, and even more SEQID NO: 21, SEQ ID NO: 22, SEQID NO. 23, SEQ ID preferably are at least about 99% pure. Exemplary techniques NO: 26, SEQID NO: 27, SEQID NO: 28, SEQID NO: 29, for eliciting an immune response in a host organism and for SEQID NO:30, SEQID NO:33, SEQID NO:34, or SEQID 35 isolating antibodies therefrom are described herein, but it is to NO: 38, or Copro6728. Also, it is contemplated that the be understood that the present invention is not limited to those polypeptide of the present invention may include a plurality techniques. The skilled artisan will recognize that there are a of polypeptide fragments corresponding to SEQ ID NO:3, plurality of techniques for achieving this same goal without SEQID NO:4, SEQID NO:5, SEQID NO: 6, SEQID NO: deviating from the scope and spirit of the invention. 7, SEQ ID NO: 8, SEQID NO: 9 SEQ ID NO:12, SEQ ID 40 IV. Antibodies of the Invention NO:13, SEQ ID NO: 14, SEQID NO: 15, SEQID NO: 16, The present invention further includes antibodies and anti SEQID NO: 19, SEQ ID NO: 20, SEQID NO: 21, SEQ ID gen-binding fragments thereofthat are raised againstand that NO: 22, SEQID NO. 23, SEQID NO: 26, SEQID NO: 27, specifically bindall or part of one or more polypeptides of the SEQID NO: 28, SEQ ID NO: 29, SEQID NO:30, SEQ ID present invention, and also includes compositions that NO:33, SEQID NO:34, or SEQID NO:38, or Copro6728. 45 include said antibodies and antigen-binding fragments For example, the polypeptide of the present invention may be thereof. When contacted to a sample obtained from a mam formed by a plurality of polypeptide fragments correspond mal, these antibodies and antigen-binding fragments are able ing to SEQ ID NO:5, SEQ ID NO: 14, SEQ ID NO: 21, or to specifically bind to a particular helminthic worm antigen. SEQID NO: 28 that are fused together. In another example, For example the roundworm antibodies and antigen-binding the polypeptide of the present invention may be formed by a 50 fragments are able to specifically bind roundworm antigens plurality of polypeptide fragments corresponding to SEQID present in the sample, but are notable to specifically bind any NO:5 SEQID NO: 14, SEQID NO: 21, or SEQID NO: 28 antigen from hookworm or whipworm that may be present in and a plurality of polypeptide fragments corresponding to the sample. As a further example, the whipworm antibodies SEQID NO:7, SEQID NO: 16, SEQID NO. 23, or SEQ ID and antigen-binding fragments are able to specifically bind NO:30 that are fused together in any combination. 55 whipworm antigens present in the sample, but are notable to Whereas one particular polypeptide of the present inven specifically bind any antigen from hookworm or roundworm tion was expressed and isolated by a specific technique (in that may be present in the sample. The antibodies of the which is described in the Example section included herein), present invention are suitable for being used only to capture the skilled artisan will recognize that any of the polypeptides one or more roundworm antigens, whipworm antigens and/or of the present invention may be isolated by employing any 60 hookworm antigens, only to detect one or more roundworm one or more of a variety of techniques. (See, e.g., Sewald and antigens, whipworm antigens and/or hookworm antigens, or Jakubke, Peptides. Chemistry and Biology, Wiley Publishing more preferably, to both capture and detect one or more (2002); Peptide Synthesis and Applications (Methods in roundworm antigens, whipworm antigens and/or hookworm Molecular Biology) Howl, ed., Humana Press (2005); Jones, antigens. Amino Acid and Peptide Synthesis, Oxford University Press 65 The antibodies of the present invention may belong to any (2002), each one of which is incorporated herein by reference antibody class, including for example, IgG, IgM, IgA, Ig|D in its entirety.) These techniques include those that may be and IgE, and may be prepared by any of a variety of tech US 7,951,547 B2 25 26 niques known to the skilled artisan. (See, e.g., Dean, Methods “anti-DIV6901 or “anti-DIV6902.) The skilled artisan will Mol. Biol. 80:23-37 (1998); Dean, Methods Mol. Biol. recognize that the production and isolating of anti-DIV6901, 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88 anti-DIV6901 or any other antibody of the present invention, (1994); Gullick, Methods Mol. Biol. 32:389-99 (1994): is not limited to any specific technique. Drenckhahn et al Methods Cell Biol. 37:7-56 (1993); Morri In another embodiment, a hookworm antibody of the son, Ann. Rev. Immunol. 10:239-65 (1992); Wright etal. Crit. present invention is an antibody that is raised in rabbit by Rev. Immunol. 12:125-68 (1992); Harlow and Lane, Antibod immunizing that host animal with the polypeptide having the ies: A Laboratory Manual, Cold Spring Harbor Laboratory amino acid sequence corresponding to SEQ ID NO. 34. (1988); and Making and Using Antibodies: A Practical Hand (Hereinafter, this particular antibody is referred to as “anti book, Howard and Kaser, eds. CRC Press (2006), each one of 10 Asp5-1”.) The skilled artisan will recognize that the produc which is incorporated herein by reference in its entirety.) tion and isolating of anti-Asp5-1, or any other antibody of the In one technique, the polypeptide of the invention is intro present invention, is not limited to any specific technique. duced into a host animal. Such as into rabbit, mouse, rat, In an additional embodiment, the hookworm antibody of guinea pig, goat, pig, cow, sheep, donkey, dog, cat, chicken, or the present invention is an antibody that is raised in rabbit by horse, for example. An enhanced immune response may be 15 immunizing that host animal with the polypeptide having the elicited in the host animal by associating the polypeptide with amino acid sequence corresponding to SEQ ID NO:33, i.e. a carrier and/or by exposing the host to an adjuvant, but it is to with substantially the full-length ASP5 protein from hook be understood that the present invention does not require that worm, as described in U.S. application Ser. No. 1 1/763.583 the polypeptide be associated with a carrier or that the host be entitled “Device, Kit and Method for Hookworm Antigen exposed to the adjuvant. An exemplary carrier that may be Detection', filed Jun. 15, 2007. used for this purpose is bovine serum albumin, bovine thyro In other embodiments, the antibodies of the present inven globulin, and Soybean trypsin inhibitor. Exemplary adjuvants tion are raised in a host against one or more polypeptides include Freund's complete or incomplete adjuvant and MDL having an amino acid sequence that is a conservative variant TDM adjuvant. Regardless of whether the polypeptide is of the sequence corresponding to SEQIDNO:5, SEQID NO: associated with such a carrier or whether the host is exposed 25 14, SEQID NO: 21, SEQID NO: 28, and SEQID NO:34. In to an adjuvant, booster immunizations optionally may be some other embodiments, the antibodies of the present inven made with the host animal being bled one or more times tion are raised in a host against any one or more polypeptides thereafter. Polyclonal antibodies that specifically bind the having an amino acid sequence corresponding to the polypeptide may then be purified from antisera obtained from sequence of SEQID NO:3, SEQ ID NO: 4, SEQID NO: 6, the bleed or bleeds. Such purification may be achieved, for 30 SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 SEQ ID example, by employing affinity chromatography techniques NO:12, SEQ ID NO:13, SEQ ID NO: 15, SEQID NO: 16, that involve associating the polypeptide to a solid support. SEQID NO: 19, SEQ ID NO:20, SEQID NO:22, SEQID Such affinity chromatography techniques are well known by NO: 23, SEQID NO: 26, SEQID NO: 27, SEQID NO: 29, the skilled artisan. SEQID NO:30, SEQID NO:33, SEQID NO:38, Copro6728 In several embodiments, the roundworm antibody of the 35 and CoproAP5, or one or more polypeptides having an amino present invention is an antibody that is raised in rabbit by acid sequence that is a conservative variant of any of those immunizing that host animal with the polypeptide having the Sequences. amino acid sequence corresponding to SEQID NO: 28, SEQ In another embodiment, the antibodies of the present IDNO: 14, and SEQID NO:21, (Hereinafter, these particular invention are antibodies that specifically bind one or more the antibodies are referred to as “anti-DIV6744”, “anti 40 polypeptides having the amino acid sequence corresponding DIV6716,” and “anti-DIV6728 respectively) The skilled arti to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID san will recognize that the production and isolating of anti NO:6, SEQID NO:7, SEQID NO:8, SEQID NO:9 SEQID DIV6744, anti-DIV6716 and anti-DIV6728, or any other NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQID NO: 15, antibody of the present invention, is not limited to any specific SEQID NO: 16, SEQ ID NO: 19, SEQID NO: 20, SEQ ID technique. 45 NO: 21, SEQID NO: 22, SEQID NO. 23, SEQID NO: 26, In another embodiment, the roundworm antibody of the SEQID NO: 27, SEQ ID NO: 28, SEQID NO: 29, SEQ ID present invention is an antibody that is raised in rabbit by NO:30, SEQID NO:33, SEQID NO:34, SEQID NO:38, immunizing that host animal with a polypeptide having the Copro6728 or CoproAP5, or antigenic portions thereof. amino acid sequence corresponding to 6728C (SEQ ID In yet other embodiments, the antibodies of the present NO:38). (Hereinafter, this particular antibody is referred to an 50 invention specifically bind one or more polypeptides having “anti-CoproG728C.) A specific technique for producing and an amino acid sequence that is a conservative variant of the isolating this antibody is described in the Example section sequence corresponding to SEQ ID NO:5, SEQ ID NO: 14, herein, but the skilled artisan will recognize that the produc SEQID NO: 21, SEQID NO: 28, or SEQID NO:34. In some tion and isolating of anti-CoproG728C is not limited to that other embodiments, the antibodies of the present invention specific technique. 55 specifically bind one or more polypeptides having an amino In additional embodiments, the roundworm antibody of the acid sequence corresponding to the sequence of SEQ ID present invention is an antibody that is raised in rabbit by NO:3, SEQID NO:4, SEQID NO:6, SEQID NO:7, SEQID immunizing that host animal with extract of whole round NO: 8, SEQID NO:9 SEQID NO:12, SEQID NO:13, SEQ worm, extract of roundworm intestine, or extract of round IDNO:15, SEQID NO:16, SEQIDNO: 19, SEQID NO:20, worm reproductive organs, as described in U.S. application 60 SEQID NO: 22, SEQ ID NO. 23, SEQID NO: 26, SEQ ID Ser. No. 1 1/763,592 entitled “Roundworm Coproantigen NO: 27, SEQID NO: 29, SEQID NO:30, SEQID NO:33, Detection', filed Jun. 15, 2007. SEQ ID NO:38, Copro6728 or CoproAP5, or one or more In a further embodiment, the whipworm antibody of the polypeptides having an amino acid sequence that is a conser present invention is an antibody that is raised in rabbit by Vative variant of any of those sequences. immunizing that host animal with the polypeptide having the 65 It is also to be understood that the antibodies of the inven amino acid sequence corresponding to SEQID NO:5 or SEQ tion optionally may be polyclonal or monoclonal antibodies, IDNO:7 (Hereinafter, this particular antibody is referred to as single chain antibodies (scEv), chimericantibodies, and frag US 7,951,547 B2 27 28 ments thereof. Monoclonal antibodies that are specific for the hookworm coproantigen are specifically bound to the anti polypeptide of interest may be obtained and purified, for bodies. The device is arranged to aid specifically binding and example, by preparing cell lines that generate antibodies hav isolating helminthic coproantigens from roundworm, whip ing the desired specificity to the polypeptide of interest. Cell worm and hookworm in a sample from a mammal. lines of this kind may be derived from cells of aparticular type In one aspect, the device includes a Solid Support, wherein (e.g., spleen cells) that are isolated from a host animal that had one or more antibodies of the invention are immobilized on previously been immunized with the polypeptide as the solid support. The solid support may be, but is not limited described before. In such a case, these cells could then be to being, the inner, bottom surface of a well of a microtiter immortalized, for example, by fusing them with myeloma plate or a substrate that is included as part of a lateral flow cells by carrying out any one of a variety of fusion techniques 10 device, for example. An exemplary microtiter plate is an known to the skilled artisan. In one exemplary technique, the Immulon 1B 96-well plate (which is commercially available cells from the immunized host animal are co-incubated with from Thermo Scientific of Milford, Mass.), but it is to be their fusion partner, e.g., the myeloma cells, in the presence of understood that the skilled artisan will recognize that a large a detergent for a short period of time before being plated on a variety of other microtiter plates that are not the Immulon 1B medium that supports the growth of hybrid cells (but not the 15 96-well plate allow for the immobilization of antibodies myeloma fusion partner). Such selection may beachieved, for thereon, and therefore would be suitable for providing the example, by using hypoxanthine, aminopterin, and thymidine Solid Support of the present invention. (HAT). When hybrid cells emerge during selection, in per An exemplary lateral flow device is the lateral flow device haps one or two weeks after commencing the selection pro that is described in U.S. Pat. No. 5,726,010, which is incor cess, single hybridcolonies (and their Supernatants) are tested porated herein by reference in its entirety. The device for for their ability to bind the polypeptide or polypeptides performing a lateral flow assay may be a SNAPR) device, against which the host animal was immunized. Hybrid colo which is commercially available from IDEXX Laboratories, nies having the most optimal binding specificity would rep Inc. of Westbrook, Me. However, it is to be understood that resent the best candidates from which monoclonal antibodies the skilled artisan will recognize that a large variety of other may be isolated. These monoclonal antibodies, for example, 25 lateral flow devices that are not SNAPR devices or described may be isolated directly from the Supernatant (i.e., medium) by U.S. Pat. No. 5,726,010 allow for the immobilization of an in which these colonies are grown by employing any one of a antibody thereon, and therefore would be suitable for being variety techniques known to the skilled artisan. used as the device of the present invention. These devices can The antibodies of the invention also may be a single chain include, for example, lateral flow devices that use colloidal antibody (ScPV), or an antigen binding fragment of an anti 30 gold technology. body. Antigen-binding fragments of antibodies are a portion Antibodies used in the device of the invention may be of an intact antibody comprising the antigen binding site or immobilized on the solid support by any methodology known variable region of an intact antibody, wherein the portion is in the art, including, for example, covalently or non-co free of the constant heavy chain domains of the Fc region of valently, directly or indirectly, attaching the antibodies to the the intact antibody. Examples of antibody fragments include 35 solid support. Therefore, while these antibodies may be Fab, Fab', Fab'-SH, F(ab') and F fragments. In addition to attached to the Solid Support by physical adsorption (i.e., production and purification from animals or mammalian without the use of chemical linkers), it is also true that these cells, antibodies, antibody fragments, or non-antibody Scaf antibodies may be immobilized to the solid support by any folds can be selected based upon various in vitro technolo chemical binding (i.e., with the use of chemical linkers) gies, including phage display, ribosomal display, or bacterial 40 method readily known to one of skill in the art. display. It is also to be understood that the solid support may be any Antibodies, including secondary antibodies, may be suitable material for the immobilization of the antibodies of labeled with any type of label known in the art, including, for the invention. For example, the solid Support may be beads, example, fluorescent, chemiluminescent, radioactive, particles, tubes, wells, probes, dipsticks, pipette tips, slides, enzymes, colloidal particles, radioisotopes and biolumines 45 fibers, membranes, papers, natural and modified celluloses, cent labels. In various embodiments of the invention, the one polyacrylamides, agaroses, glass, polypropylene, polyethyl or more of the antibodies of the invention are labeled with an ene, polystyrene, dextran, nylon, amylases, plastics, magne enzyme, a colloidal particle, a radionuclide or a fluorophor. tite or any other suitable material readily known to one of skill The particulate label can be, for example, a colored latex in the art. particle, dye Sol, or gold Sol conjugated to an antibody. 50 The device optionally may include one or more labeled Methods, Devices and Kits of the Invention antigen capture reagents that may be mixed with a sample Devices and Kits of the Invention from a mammal prior to application to a device of the inven The present invention, in one aspect, is a detecting the tion. When the labeled capture antigen reagent is included, presence or absence of one or more helminthic antigens from the labeled antigen capture reagent may or may not be depos a sample, the device comprising a solid Support, wherein the 55 ited or dried on a solid surface of the device. “Antigen capture solid support has immobilized thereon at least two antibodies reagent” refers to any compound that is specific for the anti selected from the group consisting of (a) a first antibody gen or antigens of interest. The labeled antigen capture capable of specifically binding a roundworm coproantigen, reagent, whether added to the mammalian sample or pre but not a whipworm or hookworm coproantigen; (b) a second deposited on the device, may be, for example, a labeled anti antibody capable of specifically binding a whipworm 60 body specific for a roundworm antigen, including, but not coproantigen, but not a roundworm or hookworm coproanti limited to, the antibodies of the present invention. In one gen; and (c) a third antibody capable of specifically binding a example, anti-DIV6744 conjugated with horseradish peroxi hookworm coproantigen, but not a whipworm or roundworm dase may be used as a labeled antigen capture reagent. In coproantigen; and optionally, (d) one or more types of round another example, anti-DIV6901 or anti-DIV6902 conjugated worm coproantigen, whipworm coproantigen, and/or hook 65 with horseradish peroxidase may be used as a labeled antigen worm coproantigen, wherein the one or more types of round capture reagent. In a further example, anti-DIV6716 conju worm coproantigen, whipworm coproantigen, and gated with horseradish peroxidase may be used as a labeled US 7,951,547 B2 29 30 antigen capture reagent. In a further example, anti-DIV6728 The plate may be used in conjunction with a method of the conjugated with horseradish peroxidase may be used as a present invention to detecting the presence or absence of one labeled antigen capture reagent. In yet a further example, or more helminthic coproantigens in a sample. For example, anti-Copro6728 conjugated with horseradish peroxidase may a roundworm infection may be diagnosed in a mammal by be used as a labeled antigen capture reagent. detecting one or more roundworm antigens with the anti The device also may optionally include a liquid reagent DIV6744 pAB, one or more whipworm antigens with the that transports (such as when the device is a SNAPR) device, anti-DIV6902 p AB, and one or more hookworm antigens for example), or otherwise facilitates removal of (such as with the anti-Asp5-1 pAB, that is immobilized on the solid when the device includes a microtiter plate, for example), Support. In one embodiment, the antigens that are detected are 10 coproantigens. “Coproantigens' are any product or products unbound material (e.g., unreacted portions of the mammalian of roundworm, whipworm or hookworm that are present in a sample, Such as, for example, unreacted portions of fecal fecal sample and that can specifically bind to antibodies. extract, and unbound antigen capture reagent) away from the Coproantigens therefore may be whole worm, worm eggs, reaction Zone (solid phase). The liquid reagent may be a wash worm fragments, or products secreted, excreted or shed from reagent and serve only to remove unbound material from the 15 worm or a combination thereof. Coproantigens further reaction Zone, or it may include a detector reagent and serve include the polypeptides of the present invention, Such as the to both remove unbound material and facilitate antigen detec polypeptides having an amino acid sequence corresponding tion. For example, in the case of an antigen capture reagent to SEQID NO:3, SEQID NO:4, SEQID NO:5, SEQID NO: conjugated to an enzyme, the detector reagent includes a 6, SEQID NO:7, SEQID NO:8, SEQID NO:9 SEQID NO: Substrate that produces a detectable signal upon reaction with 12, SEQID NO: 13, SEQID NO: 14, SEQID NO: 15, SEQ the enzyme-antibody conjugate at the reaction Zone (Solid IDNO: 16, SEQID NO: 19, SEQIDNO:20, SEQID NO:21, phase). Alternatively, in the case of a labeled antigen capture SEQID NO: 22, SEQ ID NO. 23, SEQID NO: 26, SEQ ID reagent conjugated to a radioactive, fluorescent, or light-ab NO: 27, SEQID NO: 28, SEQID NO: 29, SEQID NO:30, Sorbing molecule, the liquid reagent acts merely as a wash SEQID NO:33, SEQID NO:34, or SEQID NO:38, polypep Solution facilitating detection of complex formation at the 25 tides having an amino acid sequence that is a conservative reactive Zone by washing away unbound labeled reagent. variant of those sequences, and/orantigenic fragments of any The liquid reagent may further include a limited quantity of such polypeptides, and CoproASP5 and Copro6728, for an “inhibitor', i.e., a substance that blocks the development of example. the detectable end product. A limited quantity is defined as The invention further includes assay kits (e.g., articles of being an amount of inhibitor sufficient to block end product 30 manufacture) for detecting and distinguishing between development until most or all excess, unbound material is roundworm, whipworm and/or hookworm in a mammalian transported away from the second region, at which time sample. A kit therefore may include one or more devices detectable end product is produced. and/or compositions of the present invention. For example, The device of the present invention may also include vari the kit may include anti-roundworm antibodies and means for ous binding reagents immobilized at locations distinct from 35 determining binding of the antibodies to roundworm anti the antigen capture reagent or reagents. For example, an gens, anti-whipworm antibodies and means for determining immunoreagent (an antibody, antigen or polypeptide) that binding of the antibodies to whipworm antigens, and anti recognizes a species-specific (e.g., roundworm-specific) anti hookworm antibodies and means for determining binding of body portion of a labeled antibody orantigen capture reagent, the antibodies to hookworm antigens in the sample. In one or an enzyme portion of an enzyme-labeled reagent, can be 40 particular example, such a kit includes the device having an included as a positive control to assess the viability of the immobilized anti-roundworm antibody. Such as anti reagents within the device. For example, a positive control DIV6744, an anti-whipworm antibody, such as anti may be an anti-horseradish peroxidase antibody that has been DIV6902, and an anti-hookworm antibody, such as anti raised in, for example, goat or mouse. Additionally, a reagent, Asp5-1, for example, one or more antigen capture reagents e.g., an antibody, isolated from a non-immune member of the 45 (e.g., a non-immobilized labeled antigen capture reagent and species from which the antibody portion of the antigen-anti an immobilized antigen capture reagent) and wash reagent, as body complex was derived can be included as a negative well as detector reagent and positive and negative control control to assess the specificity of immunocomplex (i.e., anti reagents, if desired or appropriate. Other components such as gen-antibody complex) formation. buffers, controls, and the like, knownto those of ordinary skill In addition to being designed to specifically binding and 50 in art, may be included in Such test kits. The relative amounts isolating helminthic coproantigens from roundworm, whip of the various reagents can be varied, to provide for concen worm and hookworm in a mammalian sample, the device of trations in Solution of the reagents that Substantially optimize the invention optionally may be designed to allow one or the sensitivity of the assay. Particularly, the reagents can be more other diagnostic tests to be performed. For example, the provided as dry powders, usually lyophilized, which on dis Solid Support may also include reagents for the detection of 55 Solution will provide for a reagent Solution having the appro one or more non-worm parasites, one or more viruses, one or priate concentrations for combining with a sample. The more fungi, or one or more bacteria. The reagents for the present kit may further include instructions for carrying out detection of one or more non-worm parasites, one or more one or more methods of the present invention, including viruses, one or more fungi, or one or more bacteria may be, for instructions for using any device and/or composition of the example, one or more antibodies or one or more antigens 60 present invention that is included with the kit. recognized by antibodies specific for one or more non-worm B. Methods of the Invention parasites, one or more viruses, one or more fungi, or one or The present invention further includes methods for using more bacteria. one or more of the devices, kits and/or compositions of the In one embodiment, the device of the present invention is a present invention to detect the presence or absence of one or microtiter plate that includes a plurality of wells, wherein 65 more helminthic antigens in a sample. The methods therefore each well includes a solid support having anti-DIV6744 pAB may be carried out to detect the presence or absence of round immobilized thereupon. worm, whipworm and/or hookworm in a sample, such as, for US 7,951,547 B2 31 32 example, a fecal sample, that is obtained from a mammal, complexes formed, that a specific helminth is present in the including, but not limited to, a canine, feline, porcine, bovine host mammal and which helminth is present (roundworm, or human. Further, the methods may be carried out to detect whipworm and/or hookworm). When this is true, it may be Toxocara. Such as T. Canis or T. Cati, or T. Vitulorum, Ascaris, concluded that the mammal from which the test sample was Such as A. lumbricoides or A. suum, Anisakis, Such as A. obtained harbors an intestinal helminth infection. The con simplex, Pseudoterranova, Such as P. decipiens, Trichuris clusions that the mammal being tested harbors an intestinal and/or Trichocephalus, such as Trichuris vulpis, Trichuris helminth infection may be made by a clinician at a diagnostic campanula, Trichuris serrata, Trichuris suis, Trichuristrichi service provider or by a caregiver of the mammal. Such as the ura, Trichuris discolor and Trichocephalus trichiuris, Ancy mammal’s veterinarian, for example. When a caregiver of a lostoma caninum, Ancylostoma braziliense, Ancylostoma 10 mammal determines (or is otherwise informed that) a mam duodenal, Ancylostoma ceylanicum, Ancylostoma tubae mal harbors a helminth infection and which helminth is forme and Ancylostoma pluridentatum, Necator americanus, present, the caregiver may then subject the mammal to a and Uncinaria Stenocephala, for example. course of treatment that is optimally designed to rid the mam In the methods of the present invention, detection of round mal of the helminth specifically, rather than of a parasitic worm, whipworm and/orhookworm may be accomplished by 15 nematode infection generally. Further, the present invention detecting the presence or absence of one or more roundworm, can be used to confirm that any animal that has received whipworm and/or hookworm antigens, such as Copro6728 treatment for the specific helminth infection has been rid of and Copro ASP5 or the polypeptides having an amino acid that infection. A caregiver who learns that a sample includes sequence corresponding to SEQ ID NO:3, SEQ ID NO: 4, both roundworm and whipworm, but not hookworm, for SEQID NO:5, SEQID NO: 6, SEQID NO: 7, SEQID NO: example, could use that knowledge to treat the mammal from 8, SEQID NO: 9 SEQID NO: 12, SEQID NO:13, SEQ ID which the sample was taken specifically for roundworm by NO: 14, SEQID NO: 15, SEQID NO: 16, SEQID NO: 19, administering to that mammal a drug optimally effective SEQID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID against roundworm and a second drug optimally effective NO: 23, SEQID NO: 26, SEQID NO: 27, SEQID NO: 28, against whipworm. Absent such knowledge, the caregiver SEQ ID NO: 29, SEQ ID NO:30, SEQID NO:33, SEQID 25 may, for example, otherwise treat the mammal with a drug NO: 34, or SEQ ID NO:38 as well as antigenic fragments that is optimally effective against only roundworm, only and/or conservative variants of those sequences, and whipworm, or neither roundworm nor whipworm (in Such CoproASP5, for example. When the sample under test for cases, the mammal would be at risk of receiving Suboptimal helminthic coproantigens is feces, the Soluble portion of the treatment). In addition, humans who may come in contact feces may be collected by any protocol known in art. For 30 with the infested animal or its excretions may be advised to example, in addition to the specific protocol described in the take precautions against acquiring the parasite or parasites. In Example section herein, the soluble portions of the sample this context, it is important to determine the worm species generally may be collected by using filtration, extraction, with high specificity, as some helminths, such as roundworms centrifugation, or simple mixing followed by gravimetric and hookworms, can cause significant disease (e.g., larval settling. The skilled artisan will recognize that there are a 35 migrans) in humans, while it is generally accepted that whip variety of ways of extracting and preparing non-fecal samples worm does not play a Zoonotic role of importance in humans. from a mammal as well. For example, the sample may be a The steps of the method of the present invention may bodily fluid that is naturally excreted or otherwise released by include applying a mammalian sample to a device of the the mammal or that is artificially obtained from the mammal. invention, which includes a first antibody capable of specifi Such artificial extraction may be carried out by milking the 40 cally binding a roundworm coproantigen, but not a whip mammal or by injecting a syringe into the mammal and draw worm or hookworm coproantigen; a second antibody capable ing the fluid into the syringe. Once obtained, the fluid option of specifically binding a whipworm coproantigen, but not a ally may be fractionated (for example, serum may be frac roundworm or hookworm coproantigen; and a third antibody tionated from whole blood as then used as the sample). As capable of specifically binding a hookworm coproantigen, another example, the sample may be obtained by Swabbing 45 but not a whipworm or roundworm coproantigen to form the mammal. Such as the oral cavity of the mammal, for antibody -coproantigen complexes in the presence of the example. As yet another example, tissue sections may be coproantigens, if any, in the sample; and detecting the pres obtained by biopsy. ence or absence of the antibody-coproantigen complexes, if The methods include contacting the mammalian sample any. Antibodies specific for antigens of roundworms may be with one or more antibodies specific for helminthic coproan 50 directly or indirectly attached to a solid support or a substrate tigens under conditions that allow an antigen/antibody com Such as a microtiter well, antibody-immobilizing portion of a plex, i.e., an immunocomplex, to form. That is, an antibody SNAPR device, magnetic bead, non-magnetic bead, column, specifically binds to a coproantigen present in the sample. matrix, membrane, fibrous mat composed of synthetic or The skilled artisan is familiar with assays and conditions that natural fibers (e.g., glass or cellulose-based materials or ther may be used to detect such antigen/antibody complex bind 55 moplastic polymers, such as, polyethylene, polypropylene, or ing. For example, the antigen/antibody complex may be polyester), sintered structure composed of particulate mate detected using a secondary antibody that binds to the antigen/ rials (e.g., glass or various thermoplastic polymers), or cast antibody complex. The formation of a complex between anti membrane film composed of nitrocellulose, nylon, polysul gen and antibodies in the sample may be detected using any fone or the like (generally synthetic in nature). All of these suitable method known in the art. 60 Substrate materials may be used in Suitable shapes, such as Further, the relative amount of antibody-antigen com films, sheets, or plates, or they may be coated onto or bonded plexes that are formed in one particular reaction may be or laminated to appropriate inert carriers, such as paper, glass, measured with respect to those formed in any other reaction plastic films, or fabrics. Suitable methods for immobilizing by any methodology known in the art for achieving that goal. peptides on Solid phases include ionic, hydrophobic, covalent When it is determined that a sample under test has a specific 65 interactions and the like. (roundworm, whipworm and/or hookworm) antibody-anti The methods of the present invention do not require the use gen complexes, it can be concluded, based upon the specific of solid phases or substrates, however. The skilled artisan will US 7,951,547 B2 33 34 recognize that there are a number of ways that the present In some embodiments of the method, antibodies specific method may be carried out to detect the presence or absence for a roundworm, whipworm and hookworm coproantigens of roundworm without involving the use of solid phases or are attached to a solid phase or Substrate. A sample potentially Substrates. In just one example, immunoprecipitation meth including an antigen from roundworm, whipworm and/or ods that do not require the use of Solid phases or Substrates hookworm are added to the substrate. Antibodies that specifi may be carried out. cally bind roundworm, whipworm and/or hookworm are In some embodiments of the invention, the antigen/anti added. The antibodies may be the same antibodies used on the body complex is detected when an indicator reagent, such as Solid phase or they may be from a different source or species. an enzyme conjugate, which is bound to the antibody, cata Further, these antibodies may be linked to an indicator lyzes a detectable reaction. Optionally, an indicator reagent 10 reagent, such as an enzyme conjugate. Wash steps may be including a signal generating compound may be applied to performed prior to each addition. A chromophore or enzyme substrate may be added and color may be allowed to develop. the antigen/antibody complex under conditions that allow The color reaction may be stopped and the color may be formation of a detectable antigen/antibody/indicator com quantified using, for example, a spectrophotometer, and/or plex. Optionally, the antibody may be labeled with an indica 15 the color may be subjectively assessed by the human eye. tor reagent prior to the formation of an antigen/antibody In other embodiments of the method, antibodies specific complex. for a roundworm, whipworm and hookworm coproantigens The formation of an antigen/antibody complex or an anti are attached to a solid phase or Substrate. A sample potentially gen/antibody/indicator complex in Some of the methods of including a roundworm, whipworm and/or hookworm anti the present invention specifically may be detected by radio gen is added to the Substrate. Second anti-species antibodies metric, calorimetric, fluorometric, photometric, size-separa that specifically bind the coproantigens are added. These tion, or precipitation methods. Detection of an antigen/anti second antibodies are from a different species than are the body complex also may be accomplished by the addition of a Solid phase antibodies. Third anti-species antibodies that spe secondary antibody that is coupled to an indicator reagent cifically bind the second antibodies and that do not specifi including a signal generating compound. Indicator reagents 25 cally bind the solid phase antibodies are added. The third including signal generating compounds (labels) associated antibodies may include an indicator reagent, such as an with a polypeptide?antibody complex may be detected using enzyme conjugate. Wash steps may be performed prior to the methods described above and may include chromogenic each addition. A chromophore or enzyme Substrate may agents, catalysts such as enzyme conjugates, fluorescent added and color may be allowed to develop. The color reac compounds such as fluorescein and rhodamine, chemilumi 30 tion may be stopped and the color may be quantified using, for nescent compounds Such as dioxetanes, acridiniums, phenan example, a spectrophotometer, and/or the color may be Sub thridiniums, ruthenium, and luminol, radioactive elements, jectively assessed by the human eye. direct visual labels, as well as cofactors, inhibitors, magnetic In a specific example, the method of the present invention particles, and the like. Examples of enzyme conjugates is performed in conjunction with a device that is a lateral flow include alkaline phosphatase, horseradish peroxidase, beta 35 assay device by adding a prepared mammalian sample to a galactosidase, and the like. The selection of a particular label flow matrix of the device at a first region (a sample application is not critical, but it will be capable of producing a signal Zone). The prepared sample is carried in a fluid flow path by either by itself or in conjunction with one or more additional capillary action to a second region of the flow matrix where a Substances. particulate label capable of binding and forming a first com Methods of the invention include, but are not limited to 40 plex with an antigen in the sample exists. The particulate label those based on competition, direct reaction or sandwich-type can be, e.g., a colored latex particle, dye Sol, or gold Sol assays, including, but not limited to ELISA, RIA, immuno conjugated to an antibody specific for a roundworm antigen. fluorescent assays (IFA), hemagglutination (HA), fluores The first complex is carried to a third region of the flow matrix cence polarization immunoassay (FPIA), and microtiter plate where an antibody that specifically binds a roundworm anti assays (i.e., any assay done in one or more wells of a micro 45 gen is immobilized at a distinct location. A second complex is titer plate). One assay of the invention includes a reversible formed between the immobilized antibody and the first com flow chromatographic binding assay, which may be per plex. The particulate label that is part of the second complex formed, for example, by using a SNAPR) device. See U.S. Pat. can be directly visualized by the human eye. No. 5,726,010. Each specific helminth antibody may be an immobilized In some embodiments, the method of the invention facili 50 antigen capture reagent in a reaction Zone (solid phase). A tates sandwich or competition-type specific binding assays. second antigen capture reagent, i.e., a second specific helm In a sandwich assay, antigen capture reagents are immobi inth antibody that has been conjugated to a label, either may lized in a reactive Zone. These antigen capture reagents may be added to the sample before the sample is added to the specifically bind to antigens in the sample being tested for device, or the second antigen capture reagent can be incorpo roundworm, whipworm and/or hookworm. Following bind 55 rated into the device. For example, the labeled antigen capture ing of the antigen from the sample, the antigen capture reagent may be deposited and dried on a fluid flow path that reagent/antigen complex is detected by any Suitable method. provides fluid communication between a sample application For example, the complex may be reacted with labeled spe Zone and the solid phase. Contact of the labeled antigen cific binding reagents (e.g., an enzyme-antibody conjugate) capture reagent with the test sample can result in dissolution and antigen detected (e.g., upon reaction with Substrate). 60 of the labeled antigen capture reagent. In other embodiments of the method of the present inven In one embodiment of the method of the present invention, tion, a competition assay is performed. In a competition specific helminthic coproantigen is detected by ELISA. Spe assay, antigen capture reagents are immobilized at the reac cific examples of the ELISA method of the present invention tive Zone and are contacted simultaneously with antigen from is described in the Example section included herein. Although a sample and labeled antigen (e.g., an antigen-enzyme con 65 the present invention is described with respect to those spe jugate). The amount of label detected at the reactive Zone is cific ELISA methods, however, it is to be understood that inversely proportional to the amount of antigen in the sample. those of ordinary skill in the art will recognize that alternative, US 7,951,547 B2 35 36 additional or substitute ELISA steps may be used without absence of helminths is further evaluated by using the anti deviating from the basic goal achieved through this method of bodies of the present invention. Detection of one or more the invention. helminthic nucleic acids may be carried out by using any In another embodiment of the present invention, helmin nucleic acid detection techniques knownto the skilled artisan. thic coproantigen is detected by using a lateral flow device, 5 For example, such detection may be carried out by perform such as a SNAP(R) device, for example. ing a PCR-based technique, such as, but limited to, for Further, the methods of the invention for detection of hel example, a real-time PCR-based technique. Exemplary PCR minth infection can be combined with other diagnostic assays based techniques are described in, e.g., PCR Protocols (Meth to detect the presence of other organisms or conditions. For ods in Molecular Biology), 2" ed., Bartlett and Stirling, eds., example, assays of the invention can be combined with 10 reagents that detect one or more non-worm fecal parasites, Humana Press (2003); and Sambrook and Russell, Molecular one or more viruses, one or more fungi, one or more bacteria, Cloning: A Laboratory Manual, Cold Spring Harbor Labo one or more blood-borne parasites or occult blood or a com ratory Press (2001); each one of which is incorporated herein bination thereof. By providing two or more unique binding by reference in its entirety. sites in a single assay device (such as, for example, two 15 The present invention is specifically described with refer unique spots on a SNAP(R) assay device), the present inven ence to five Examples; however, it is not to be construed as tion allows for detection of two or more organisms from a being limited thereto. single sample. In one embodiment, there are three unique spots for detection of past or present infection or infestation EXAMPLES from three organisms (the spots being either antigen or anti body binding reagents) from a single sample (i.e., the same Unless otherwise indicated, the following materials and individual sample is exposed to the three capture reagents on techniques were used to generate data described in one or a single device). In yet another embodiment, there are four more of Examples 1-4 as described below. unique spots for detection of past or present infection or Polyclonal roundworm antibody preparation. The poly infestation from four organisms (the spots being either anti 25 clonal antibodies “anti-DIV6728 p.AB. (IgG) was raised in gen or antibody binding reagents) from a single sample (i.e., rabbit against a polypeptide having amino acid sequence the same individual sample is exposed to the four capture corresponding to SEQ ID NO:21, respectively and purified reagents on a single device. It is to be understood, however, from serum by using standard methods. Briefly, for anti that the same device may include more than four unique spots DIV6728 pAB nucleotides 76 through 456 of SEQID NO: 17 and/or allow for the detection of more than four organisms. 30 were cloned in-frame into a vector (D8223, which is a deriva The reagents for the detection of one or more non-worm tive of puC19) to create the plasmid D8245. Specifically, the parasites, one or more viruses, one or more fungi, or one or 125 amino acids of SEQID NO: 21 that follow the methion more bacteria may be, for example, one or more antibodies or ine residue at the N-terminus of that sequence correspond to one or more antigens recognized by antibodies specific for a portion of SEQID NO: 19 and are encoded for by the cloned one or more non-worm parasites, one or more viruses, one of 35 portion of SEQID NO: 17. In the D8245 plasmid, the N-ter more fungi, or one or more bacteria. minal methionine residue was encoded for by vector The method further may optionally include using one or sequence at the junction of that plasmid where the vector was more nucleic acids from roundworm, whipworm and hook ligated to the cloned sequence from SEQID NO: 17. worm, including, but not limited to, the nucleic acids of the DNA sequence encoding SEQID NO:21 was then cleaved present invention, to determine the presence or absence of 40 from the D8245 plasmid by restriction exonuclease digestion roundworm, whipworm and/or hookworm in a mammalian (NdeI and BamHI) and purified. This purified sequence was sample. Such use of these nucleic acids for determining the then ligated to linearized expression vector, pFT28a, and the presence of the helminth may be carried out before, after or resulting circular construct (pTDX198:DIV6728) was trans concomitantly with the carrying out of any other aspects of formed into BL21 (DE3) E. coli cells. (The complete the method, including the detection of roundworm, whip- 45 sequence of the insert was confirmed by DNA sequence worm and hookworm by antibody. Therefore, in one aspect, analysis.) Expression of His-tagged fusion protein was after roundworm, whipworm and/or hookworm is detected or induced by addition of 1 mM IPTG to cultures of the trans not detected in a particular sample and the mammal from formed E. coli. Recombinant protein was solubilized in 6 M which the sample was obtained is diagnosed as either having urea and purified by nickel affinity and ion exchange chro or not having a roundworm, whipworm and/or hookworm 50 matography. (This recombinant protein is hereinafter is infection, the sample (or a later-obtained sample from the referred to as “rDIV6728.) diagnosed mammal) may be tested for the presence or After rIV6728 was introduced into rabbits, anti absence of any one or more of the nucleic acids, including any DIV6728 pAB was purified from the plasma of the immu one or more nucleic acids of the invention. Anyone failing to nized rabbits by isolating IgG antibody by protein G affinity detect a specific helminth in a particular mammal by using 55 chromatography. one or more nucleic acids (after the helminth had been Polyclonal whipworm antibody preparation. The poly detected by using one or more antibodies) would need to take clonal antibody “anti-DIV6901 p AB (IgG) was raised in into consideration the possibility that the antibodies had rabbit against a polypeptide having amino acid sequence detected helminthic coproantigen prior to the appearance of corresponding to SEQID NO:5 and purified from serum by detectable helminthic nucleic acid in the sample. In such an 60 using standard methods. Briefly, in the case of anti-DIV6901 instance, the mammals caregiver may elect to ignore the pAB, nucleotides 89 through 1147 of SEQ ID NO:1 were observation that the nucleic acid had failed to detect the cloned in-frame into an expression vector (D8223, which is a helminth and proceed with treating the mammal specifically derivative of puC19) to create the plasmid D9073. Specifi for helminth infection based on the observation that the anti cally, the 353 amino acids of SEQ ID NO:5 that follow the bodies had in fact detected helminth. In another aspect, the 65 methionine residue at the N-terminus of that sequence corre nucleic acids are used to determine the presence or absence of spond to a portion of SEQID NO:3 and are encoded for by the helminths in a particular mammal, and then the presence or cloned portion of SEQID NO: 1. US 7,951,547 B2 37 38 DNA sequence encoding SEQID NO:5 was then cleaved ELISA assays. Purified anti-DIV6728 pAB, anti-DIV6901 from the D9073 plasmid by restriction exonuclease digestion pAB and anti-Asp5-1 pAB (100 ul/well;3 ug/ml for Example (NdeI and BamHI) and purified. This purified sequence was 2) were immobilized by physical adsorption on Immulon 1B then ligated to linearized expression vector, pFT28a, and the 96-well plates overnight at 4°C. The plates were then blocked resulting circular construct (ptDX233:DIV6901) was trans with 1% BSA in 0.1M Tris pH 7.0 or 3 hours at room tem formed into E. coli cells. (The complete sequence of the insert perature, followed by 2.5% Sucrose in 0.1M Tris buffer, pH was confirmed by DNA sequence analysis.) Expression of 7.0 for 3 hours at room temperature, aspirating the liquid, His-tagged fusion protein was induced by addition of 1 mM drying at room temperature. Approximately 100 ul of fecal IPTG to cultures of the transformed E. coli. Recombinant extract was added to each well and allowed to incubate at protein was solubilized in 6 Murea and purified by nickel 10 room temperature for one hour. The wells were then washed affinity and ion exchange chromatography. (This recombi five times with a PBS-Tween-20 solution according to stan nant protein is hereinafter is referred to as “rDIV6901.) dard methods known to those of ordinary skill in the art. In a Anti-DIV6901 p AB was purified from the plasma of the separate reaction vessel, free anti-DIV6728 pAB, anti immunized rabbits by isolating IgG antibody by protein G DIV6901 pAB, and anti-Asp5-1 pAB was labeled with horse affinity chromatography. 15 radish peroxidase (HRP) by using the crosslinker succinim Polyclonal hookworm antibody preparation and isolation. idyl 4-N-maleimidomethylcyclohexane-1-carboxylate The polyclonal antibody anti-ASP5-1 (IgG) was raised in (SMCC) to create a conjugate, and this conjugate (3 ug/ml for rabbit against a polypeptide having amino acid sequence Examples 2) was added to each well having immobilized corresponding to SEQID NO:34 and purified from serum by anti-DIV6728 p.AB, anti-DIV6901 p.AB, and anti-Asp5-1 using standard methods. Briefly, nucleotides 50 through 427 pAB. Following a 30-minute incubation period at room tem of SEQ ID NO: 32 were cloned in-frame into a plasmid. perature, unbound conjugate was washed from the wells by Specifically, the 129 amino acids of SEQ ID NO: 34 that using PBS-Tween-20 solution according to standard methods follow the methionine residue at the N-terminus of that known to those of ordinary skill in the art. 50 ul of sequence correspond to a portion of SEQID NO:33 and are TMBLUER) peroxidase substrate (SeraCare Life Sciences, encoded for by the cloned portion of SEQID NO:32. In the 25 West Bridgewater, Mass.) was then added to each well and the plasmid, the N-terminal methionine residue was encoded for plates were incubated for 10 minutes at room temperature. by vector sequence at the junction of that plasmid where the After stopping each enzymatic reaction with 0.1% sodium vector was ligated to the cloned sequence from SEQID NO: dodecyl sulfate (SDS) following the 10-minute incubation 32. period, the optical density (OD) value of each well of the DNA sequence encoding SEQID NO:32 was then cleaved 30 96-well plate was measured at A650 by standard spectropho from the plasmid by restriction exonuclease digestion (NotI tometric techniques by using an ELISA plate reader to gen and SacI) and purified. This purified sequence was then erate an “OD650 value” (or, more simply, an “OD value') for ligated to linearized expression vector, pET28a, and the each well. In this arrangement, the OD value obtained for any resulting circular construct was transformed into BL21 (DE3) particular well of the 96-well plate was directly proportional E. coli cells. (The complete sequence of the insert was con 35 to the amount of specifically boundantigen present in the well firmed by DNA sequence analysis.) Expression of His-tagged (if the IgG is not saturated). fusion protein was induced by addition of 1 mM IPTG to cultures of the transformed E. coli. Recombinant protein was Example 1 solubilized in 6 Murea and purified by nickel affinity and ion exchange chromatography. (This recombinant protein is 40 When tested by ELISA in a lateral flow format, anti hereinafter is referred to as “r ASP5-1”.) DIV6728 p AB specifically binds roundworm coproantigen, After raSP5-1 was introduced into rabbits, anti-ASP5-1 anti-DIV6901 pAB specifically binds whipworm coproanti pAB was purified from the plasma of the immunized rabbits gen, and anti-Asp5-1 pAB specifically binds hookworm by isolating IgG antibody by protein Gaffinity chromatogra coproantigen. There was no cross coproantigen binding phy. 45 between the helminthic antibodies and their non-specific hel Infection of canine and feline animals. Parasitic nematode minthics. The specific binding of roundworm coproantigen infection was effected by orally administering about 150-300 by anti-DIV6728 pAB, whipworm coproantigen by anti larvated eggs of roundworm (Toxocara canis), 150-300 infec DIV6901 pAB, and hookworm coproantigen by anti-Asp5-1 tive larvae of hookworm (Ancylostoma canium), or 700 lar pAB produces a calorimetric change that is readily observ vated eggs of whipworm (Trichuris vulpis), or any combina 50 able to the human eye. tion of the three to a healthy canine or feline. Infection was It was a goal of Example 1 to determine whether anti confirmed by microscopic observation of worm ova in fecal DIV6728 p.AB, anti-DIV6901 p.AB, and anti-Asp 5-1 pAB samples obtained from these host animals. can be used to capture and specifically bind their respective Canine and feline fecal sample preparation. Canine and helminthic coproantigens without non-specific binding in a feline animals known to be free of parasitic worm infection or 55 lateral flow ELISA. The lateral flow format that was used was to be infected with one, two or all three of roundworm, hook a SNAPR) assay device, similar to that which is described in worm, or whipworm provided the Source of fecal samples. U.S. Pat. No. 5,726,010. Further, the assay was performed Samples (approximately 1 gram) from frozen, unpreserved generally as described in that same patent. Briefly, among canine or feline fecal samples were suspended in 4 ml of other components, the SNAPR) assay device included a diluent solution (“diluent solution' is 0.05 MTris base; 1 mM 60 sample entry cup, a flow matrix, a sample prefilter pad for EDTA; 0.45% Kathon; 16 mg/l gentamicin sulfate; 0.05% removing interfering particulate matter, a specific binding Tween-20; 40% fetal bovine serum; 10% rabbit serum; and reagent pad, a reactive Zone, and an absorbent reservoir. Anti 5% mouse serum). The Suspension was centrifuged in a table DIV6728 p.AB, anti-DIV6901 p.AB, and anti-Asp 5-1 pAB top centrifuge at 4000 rpm for 20 minutes to produce a first were immobilized in the form of small, round spots at the supernatant. The first supernatant was centrifuged at 10,000 g 65 reactive Zone by drying. The reactive Zone was then blocked for 5 minutes to produce a second Supernatant, which is with BSA. A pooled fecal extract (150 ul) from roundworm referred to herein as "fecal extract’. infected canines was mixed with 200 ul (1.0 g/ml) conju US 7,951,547 B2 39 40 gated anti-DIV6728 pAB, anti-DIV6901 p.AB, or anti-Asp surfaces of wells G1-G12 and H1-H12, and anti-Asp5-1 pAB 5-1 pAB (the antibodies were affinity-purified before being was immobilized on the bottom surfaces of wells A1-A12 and labeled with HRP as described above. This mixture added to B1-B12 of a microtiter plate as described before. Following the sample cup and then was allowed to flow along the flow such immobilization, the A3, B3, D3, E3, G3 and H3 wells matrix. While in the flow matrix, the HRP labeled antibodies were exposed to fecal extract from a heartworm-infected specifically bound to their respective helminthic coproanti canine (indicated by “HW in FIG. 2). The A4, B4, D4, E4, gens present in the fecal extract. The resulting complexes G4 and H4 wells were exposed to fecal extract from a first (i.e., those that included the HRP labeled antibodies and their hookworm-infected canine, the A5, B5, D5, E5, G5, and H5 respective helminthic coproantigens) were allowed to specifi wells were exposed to fecal extract from a second hookworm cally bind to the immobilized anti-DIV6728 p.AB, anti 10 DIV6901 pAB, or anti-Asp 5-1 pAB at the reaction Zone. infected canine, and the A6, B6, D6, E6, G6, and H6 wells Flow along the flow matrix was reversed by contacting the were exposed to fecal extract from a third hookworm-infected absorbent reservoir with the flow matrix. At this time, detec canine. The A7, B7, D7, E7, G7 and H7 wells were exposed tor and wash Solution migrated into the flow matrix to remove to fecal extract from a first roundworm-infected canine, the any unbound components and to allow detection of any ana 15 A8, B8, D8, E8, G8, and H8 wells were exposed to fecal lyte complexes that were present where the capture reagent extract from a second roundworm-infected canine, and the was immobilized onto the reaction Zone. (This detection step A9, B9, D9, E9, G9, and H9 wells were exposed to fecal lasted about eight minutes.) Stopping of the detection of the extract from a third roundworm-infected canine. The A1, analyte complexes occurred by exposing the analyte com B10, D10, E0, G10, and H10 wells were exposed to fecal plexes to 0.1% sodium azide. extract from a first whipworm-infected canine, the A11, B11, As shown in FIG. 1C-E, detection of helminthic specific D11, E11, G11, and H11 wells were exposed to fecal extract analyte complexes where the roundworm, whipworm, and from a second whipworm-infected canine, and the A12, B12, hookworm specific antibodies were immobilized onto sepa D12, E12, G12, and H12 wells were exposed to fecal extract rate reaction Zones was visibly apparent for a roundworm from a third whipworm-infected canine. The A1. B1, D1, E1, infection (1D), a whipworm infection (1E) and a hookworm 25 G1. and H1 wells were exposed to rDIV6728, rDIV6901, and infection (1C). In the negative control sample shown in FIG. rAsp5-1 (1 lug/ml), and therefore those wells served as posi 1A (diluent solution only), no analyte complexes were tive controls. The A2, B2, D2, E2, G2, and H2 wells were not detected. In a negative control sample shown in FIG. 1B, no exposed to any fecal extract or to rDIV6728, rDIV6901, and analyte complexes were detected where the antibody was rAsp5-1 and therefore those wells served as negative controls. 30 After washing, wells D1-12 and E1-12 were exposed to HRP immobilized onto the reaction Zone of a separate device (the labeled rDIV6728 pAB; wells G1-G12 and H1-H12 were negative control sample was a pool of fecal extracts obtained exposed to HRP-labeled rDIV6901 pAB; wells A1-A12 and from canines that did not harbor a helminth infection). These B1-B12 were exposed to HRP-labeled rasp 5-1 pAB at 3 data therefore indicate that anti-DIV6728 pAB, anti ug/ml as described above. DIV6901 pAB, and anti-Asp 5-1 pAB can be used in a lateral 35 flow ELISA format to separately and specifically bind their Following incubation of all of these wells with TMBLUE(R) respective helminthic coproantigen. This specific binding is peroxidase substrate and the subsequent addition of the SDS, calorimetric change was visually observed in the anti readily visible to the human eye. DIV6728 pAB wells that had been exposed to fecal extract Example 2 40 from roundworm-infected canines (D7-D9 and E7-E9), but no calorimetric change was observed in any of the anti When tested by ELISA in a microtiter dish format, anti DIV6728 pAB wells that had been exposed to fecal extract DIV6728 pAB specifically binds roundworm coproantigen, from canines infected with either hookworm, whipworm or but does not specifically bind coproantigen from either hook heartworm. Colorimetric change was visually observed in the 45 anti-DIV6901 pAB wells that had been exposed to fecal worm, whipworm or heartworm; anti-DIV6901 p AB specifi extract from whipworm-infected canines (G10-G12 and cally binds whipworm coproantigen, but does not specifically H10-H12), but no calorimetric change was observed in any of bind coproantigen from either hookworm, roundworm or the anti-DIV6901 pAB wells that had been exposed to fecal heartworm; and anti-Asp5-1 pAB specifically binds hook extract from canines infected with either hookworm, round worm coproantigen, but does not specifically bind coproan 50 worm or heartworm. Colorimetric change was visually tigen from either roundworm, whipworm or heartworm. The observed in the anti-Asp5-1 pAB wells that had been exposed specific binding of roundworm coproantigen by anti to fecal extract from hookworm-infected canines (A4-A6 and DIV6728 p.AB, whipworm coproantigen by anti-DIV 6901 B4-B6), but no calorimetric change was observed in any of pAB, and hookworm coproantigen by anti-Asp5-1 pAB pro 55 the anti-Asp5-1 pAB wells that had been exposed to fecal duces a colorimetric change that is readily observable to the extract from canines infected with either roundworm, whip human eye. worm or heartworm. It was a goal of Example 2 to determine whether specific These data indicate that anti-DIV6728 p.AB detects round binding between anti-DIV6728 p.AB and roundworm worm; anti-DIV6901 pAB detects whipworm; and anti coproantigen; anti-DIV6901 pAB and whipworm coproanti 60 gen; and anti-Asp5-1 pAB and hookworm coproantigen, Asp5-1 pAB detects hookworm in an ELISA format suffi while the anti-DIV6728 pAB, anti-DIV6901 pAB, and anti ciently enough to produce a calorimetric change that is robust Asp5-1 pAB are immobilized on a solid Support can produce and readily visible to the human eye. Further, these data a colorimetric change that is observable to the human eye. indicate that Such calorimetric change allows the human eye Referring to FIG. 2, anti-DIV6728 p.AB (3 g/ml) was 65 to readily distinguish the between the helminthic specific immobilized onto the bottom surfaces of wells D1-D12 and fecal samples containing roundworm, hookworm and whip El-E12, anti-DIV6901 pAB was immobilized on the bottom WO US 7,951,547 B2 41 42 Example 3 of the full-length DIV6728, confirming that the 7 kb band identified by Western blot is derived from DIV6728. The A truncated version of DIV6728, Copro6728, is present in location of the two peptide sequences indicates that a C-ter T. canis infected canine feces. minal portion of DIV6728 (Copro6728) was present in the T. A. Canine Fecal Sample Preparation canis positive fecal samples. FIG. 18 shows the full-length of Canine animals known to harbor a roundworm (T. canis) DIV6728 (SEQID NO: 21) with the two peptides identified infection or to not have a parasitic worm infection provided by MS analysis highlighted in the shaded boxes. the Source of fecal samples. A sample (approximately 1 gram) of frozen, unpreserved canine feces pooled from five round Example 4 worm-infected or uninfected canines was Suspended in 4 ml 10 of extraction buffer (“extraction buffer is 1X phosphate Two recombinant proteins were generated that correspond buffered saline (PBS), pH 7.0–7.5 with 0.05% Tween-20). to 64 amino acids within the N-terminal portion of DIV6728 This suspension was Vortexed for 2 minutes and then was and 65 amino acids within the C-terminal portion of centrifuged at 13,000 rpm for 25 minutes to produce a first DIV6728. Supernatant. This first Supernatant was then centrifuged at 15 Based on the MS analysis, western blotting and SDS 10,000 rpm for 5 minutes to produce a second supernatant. PAGE data, two new expression constructs encoding trunca This second supernatant hereinafter is referred to as "fecal tions of DIV6728 were made. They were named 6728N (SEQ extract. ID NO:37) and 6728C (SEQID NO:38) for the N-terminus B. Ion Exchange and C-terminus of full-length DIV6728, respectively. FIG. 19 Ion exchange chromatography can enrich Copro6728 from shows an alignment of the 6728N (SEQ ID NO: 37) and a fecal sample. PLRS samples were used for this study. Fecal 6728C (SEQID NO: 38) amino acid sequences encoded by sample was extracted first with PBST (0.05% Tween 20), pH the constructs. 7.3. Sample was diluted with sodium citrate buffer, pH 3.0 A. Synthetic Genes for Expressing Recombinant 6728N first and then the pH was adjusted to 3 with HC1. Finally, and 6728C sample was centrifuged and the Supernatant was loaded onto 25 The genes for expressing 6728N and 6728C polypeptides a sulfopropyl (SP) column. The SP column was eluted with 20 were codon optimized for expression in E. coli. Synthesized mM sodium citrate buffer, pH 3 with 1 M NaCl, and the and cloned into vector pET28(a) with six His(6) tags at the elution fractions were evaluated by ELISA. The ELISA plate N-terminus of each recombinant protein by GeneArt, (Josef was coated with rabbitanti-6728 IgG at 3 ug/ml. Based on the Engert-Str. 11 D-93053 Regensburg, Germany). results shown in FIG. 15, it is clear that Coproð728 can be 30 B. Recombinant Protein 6728N and 6728C Expression partially purified and enriched by eluting the SP column with Recombinant proteins 6728N and 6728C were expressed sodium citrate buffer with 1 MNaCl and Copro6728 is in the in E. coli BL21 (DE3) and purified with a single nickel col fraction between A11 and C9 (FIG. 15) umn. Plasmid pET28(a) 6728N was transformed into BL21 C. Western Blotting and SDS-Page (DE3), grown to an OD-0.8 and induced with 1 mM IPTG Western blotting and SDS-PAGE gel showed that the 35 (Isopropyl-1-thio-f-D-galactopyranoside) at 37° C. for 2 molecular weight of Copro6728 is about 7 kD. Elution frac hour. Cells were lysed with Microfluidizer(R) Processor, tions from the SP column were mixed and buffer pH was M-11 EH. Recombinant 6728N was soluble in the 20 mM Tris adjusted to 7 with NaOH before loading onto an affinity buffer, pH 8.0, with 500 mM. NaCl and was purified by step column, which was prepared by linking the rabbit anti-6728 eluting the nickel column with different concentration of IgG with AminoLink resin (Pierce. Thermo Scientific). The 40 imidazole in the 20 mM Tris buffer, pH 8.0, with 500 mM column was washed and eluted according to manufacturers NaCl. The recombinant 6728N was eluted from the Nickel instructions. Elution fractions were loaded to a 10 well 4-12% column by the same buffer with 500 mM imidazole. FIG. 20 Bis-Tris gradient gel and transferred to nitrocellulose mem is a SDS-PAGEgel loaded with different samples to check the brane for western blotting. Probed with rabbit anti-6728IgG purification of the recombinant 6728N. Recombinant 6728N HRP, western blotting showed that the major band (Co 45 is about 12 kD in size (lane 9) on the gel. pro6728) is about 7 kD (red arrow on FIG. 16). After further Plasmid pET28(a) 6728C was transformed into BL21 concentration, the same samples were visualized on an SDS (DE3), grown to an OD-0.8 and induced with 1 mM IPTG PAGE gel with Imperial Protein Stain (Pierce. Thermo Sci (Isopropyl-1-thio-f-D-galactopyranoside) at 37° C. for 2 entific). A 7 kD band corresponding to the size indicated by hour. Cells were lysed with Microfluidizer(R) Processor, anti-6728IgG-HRP is visible (red arrow on FIG. 17). 50 M-11 EH. Recombinant 6728C was soluble in the mM Tris D. Mass Spectrometry Analysis buffer, pH 8.0, with 500 mM. NaCl and was purified by step Mass spectrometry analysis on the band cut from SDS eluting the nickel column with different concentration of PAGE gel (pointed by a red arrow on FIG. 17) indicated that imidazole in the 20 mM Tris buffer, pH 8.0, with 500 mM this band contains Coproð728, and that the C-terminal por NaCl. The recombinant 6728C was eluted from the Nickel tion of DIV6728 contains Copro6728. 55 column by the same buffer with 500 mM imidazole. FIG. 21 The 7 kD band that corresponds to the 7 kD band on the is a SDS-PAGE gel loaded with different samples to confirm Western blotting was cut out from the SDS-PAGEgeland sent the purification of the recombinant 6728C. Recombinant to the Keck Center at Yale University for Mass spectrometry 6728C is about 12 kD in size (lane 9) on this gel. analysis. The sample in the gel was first trypsin digested and C. Rabbit Polyclonal Antibodies then analyzed by LC-MS/MS using the Q-Tofof Ultima Mass 60 Rabbit polyclonal antibody raised against 6728C detects spectrometer (Waters). Two specific peptides were found in antigen in fecal ELISA, whereas polyclonal antibody against the sample by Mass Spectrometry analysis: Peptide 1: 6728N does not detect antigen in fecal ELISA R.FVPCTR.N (SEQIS NO:35) and Peptide 2: R.DAEGN Recombinant proteins 6728N and 6728C, purified with a CIK.F (SEQID NO:36). single Nickel column, were used to immunize rabbits for Alignment analysis on the sequences of DIV6728 (SEQID 65 polyclonal antibody production. Polyclonal antibodies from NO: 21) and the two peptides identified by MS analysis the immunized rabbit sera were affinity purified with Protein indicated that both peptides are located in the C terminal end G resin and used to coat Immulon I plates at 2 Lig/ml. Four US 7,951,547 B2 43 44 different canine samples were tested with different antibody Blotting, but not the polyclonal antibody against recombinant coated plates. Antibodies from the two rabbits immunized 6728N. In addition to T. canis whole worm extract (lane 1), with recombinant 6728C could differentiate the T. canis posi nematode negative (lanes 2-4) and T. canis positive (lanes tive fecal samples from hookworm, whipworm positive 5-7) fecal samples were fractionated by SP column with high samples and nematode negative samples. However, antibod- 5 salt elution buffer (1 MNaCl in 20 mM sodium citrate buffer, iessamp from the two rabbitsIe negativeimmunized withp recombinant~. 6728N pH 3). The T. canis worm extract (lane 1), samples loaded ld not differentiate the T. itive fecal 1 onto the column (lane 2 and 5), column flow-through (lane 3 E. isk 1 t e une 1. canis posi Samp i. and 6), and column elution (lane 4 and 7) were loaded to 10 rom nookworm, whipworm positive samples and nematode well, 4-12% Bis-Tris gradient gel and then further transferred negative samples (FIG.22). This ELISA data further demon- to nitrocellulose membrane, probed with different conjugates strates that CoproG728 is a C-terminal portion of full-length as indicated in FIGS. 24-26. Both the anti-full-length 6728 DIV6728. Further experiments showed that antibodies raised IgG-HRP and anti-6728C-IgG-HRP could differentiate the T. against 6728N and 6728C only recognize their cognate canis positive fecal sample from the nematode negative fecal recombinant proteins without cross reactivity. (FIG. 23). samples (FIGS. 24 and 25). However, the anti-6728N IgG Both of these polyclonal antibodies react with full-length is HRP could not differentiate these two different fecal samples recombinant DIV6728 as expected (FIG. 23). (FIG. 26). These data further confirmed that Coproð728 is D. Western Blotting only about half the size of full-length DIV6728, in agreement Rabbit polyclonal antibody against recombinant 6728C with the data obtained from Mass spectrometry analysis and can recognize the T. canis positive fecal samples in Western fecal ELISA. SEQUENCE LISTING <16 Os NUMBER OF SEO ID NOS: 38 <21 Os SEQ ID NO 1 &211s LENGTH: 1210 &212s. TYPE: DNA <213> ORGANISM: Trichuris vulpis <4 OOs SEQUENCE: 1 catt cactgc ggttgtaaaa gCagtgcaga aatgaggct g g tott C catg cggittattta 60 Cct cacattggggttcCtca cc.gacgc.cgt aagagaaaaa cqtggcaaat gtCctic ctga 12O accaccgatC go aggaalaca Catctact.g. cc.gc.gatgat tttgattgttg gaggaagaca 18O gaagtgctgt acaattgcag aaggacgtgg atgcgtgcc.g. ccct atggtgaacaac attt 24 O cgaagtggtg aalaccgggit C attgcc.ca.gc tatt coagcg gttacgggca tdgcgaactt 3 OO Ctgtaac act gatggcgact gtgatggacc gaaaaaatgt ttct cacat cqcgcggcta 360 cgattgcaca catcc attac actt.cccaat coagccacaa cct coagtag gacagtgc cc 42O to Cttcaaag ccc.cgitatcc caggaaaatggg tagaCat C togctaagc atgccaactg 48O Cccagaccca gagaagtgtt gcgacacgga gtatggcaac catgitatgg atgttggatt 54 O agtgc.cagga caaggagaaa gaccaggcaa ttgc.ccgaac galaccacgala talagaggaac 6 OO taaatacgat ticcgacgag acgatgactg. cacggtgtg cagaaatgct gctt cactgt 660 tgagggacgt gagtgcgtgg aaccalagtag aaaaccact g gacaa.gc.ccg gacattgtc.c 72O accaattico C gotgatgtgg gcticago Cag gtactgcgac actgat cqgg attgttgatgg 78O accaagaaaa togctgcct ct ctitcgcgtgg ctatgaatgt aaa.cat coag tacactatico 84 O cgatcgagtg gagcc actag taggagaatg cccaccatca cacct cqca tt CCtgggaa 9 OO atgggttgac atctgct cta agcatgccala Ctgcc.ca.gaC ccagagaaat gttgcgacac 96.O ggagtatggc aaccgatgta tigacgttgg attagtgcct ggacaaggag aaaaac Ctgc 102O caactgcc.ca aaggalaccac gaataagagg aactaagtac gactgtcgac gggacgatga 108O ctg.cgatggg aaacaaaagt gctgctacac aactgaaggc cgcgaatgcg to catggitat 114 O atggc cittaa atggttgctt ct tcc tataa taaaagcaaa cqaatcaaaa aaaaaaaaaa 12 OO aaaaaaaaaa. 1210 <21 Os SEQ ID NO 2 US 7,951,547 B2 47 48 - Continued Tyr Gly Asn Arg Cys Met Asp Val Gly Lieu Val Pro Gly Glin Gly Glu 1.65 17O 17s Arg Pro Gly Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly. Thir Lys Tyr 18O 185 19 O Asp Cys Arg Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Cys Phe 195 2OO 2O5 Thr Val Glu Gly Arg Glu. Cys Val Glu Pro Ser Arg Llys Pro Lieu. Asp 21 O 215 22O Llys Pro Gly. His Cys Pro Pro Ile Pro Ala Asp Val Gly Ser Ala Arg 225 23 O 235 24 O Tyr Cys Asp Thir Asp Arg Asp Cys Asp Gly Pro Arg Llys Cys Cys Lieu. 245 250 255 Ser Ser Arg Gly Tyr Glu. Cys Llys His Pro Val His Tyr Pro Asp Arg 26 O 265 27 O Val Glu Pro Leu Val Gly Glu. Cys Pro Pro Ser Arg Pro Arg Ile Pro 27s 28O 285 Gly Lys Trp Val Asp Ile Cys Ser Llys His Ala Asn. Cys Pro Asp Pro 29 O 295 3 OO Glu Lys Cys Cys Asp Thr Glu Tyr Gly Asn Arg Cys Met Asp Val Gly 3. OS 310 315 32O Lieu Val Pro Gly Glin Gly Glu Lys Pro Ala Asn. Cys Pro Lys Glu Pro 3.25 330 335 Arg Ile Arg Gly Thr Llys Tyr Asp Cys Arg Arg Asp Asp Asp Cys Asp 34 O 345 35. O Gly Lys Gln Lys Cys Cys Tyr Thr Thr Glu Gly Arg Glu. CyS Val His 355 360 365 Gly Ile Trp Pro 37 O <210s, SEQ ID NO 4 &211s LENGTH: 353 212. TYPE: PRT <213> ORGANISM: Trichuris vulpis <4 OOs, SEQUENCE: 4 Val Arg Glu Lys Arg Gly Lys Cys Pro Pro Glu Pro Pro Ile Ala Gly 1. 5 1O 15 Asn. Thir Ile Tyr Cys Arg Asp Asp Phe Asp Cys Gly Gly Arg Gln Lys 2O 25 3O Cys Cys Thr Ile Ala Glu Gly Arg Gly Cys Val Pro Pro Tyr Gly Glu 35 4 O 45 Gln Asp Phe Glu Val Val Llys Pro Gly His Cys Pro Ala Ile Pro Ala SO 55 6 O Val Thr Gly Met Ala Asn. Phe Cys Asn. Thir Asp Gly Asp Cys Asp Gly 65 70 7s 8O Pro Llys Lys Cys Cys Lieu. Thir Ser Arg Gly Tyr Asp Cys Thr His Pro 85 90 95 Lieu. His Phe Pro Ile Gln Pro Gln Pro Pro Val Gly Glin Cys Pro Pro 1OO 105 11 O Ser Llys Pro Arg Val Pro Gly Lys Trp Val Asp Ile Cys Ala Lys His 115 12 O 125 Ala Asn. Cys Pro Asp Pro Glu Lys Cys Cys Asp Thr Glu Tyr Gly Asn 13 O 135 14 O Arg Cys Met Asp Val Gly Lieu Val Ala Gly Glin Gly Glu Arg Pro Gly 145 150 155 160 US 7,951,547 B2 49 - Continued Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly Thr Llys Tyr Asp Cys Arg 1.65 17O 17s Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Cys Phe Thr Val Glu 18O 185 19 O Gly Arg Glu. CyS Val Glu Pro Ser Arg Llys Pro Lieu. Asp Llys Pro Gly 195 2OO 2O5 His Cys Pro Pro Ile Pro Ala Asp Val Gly Ser Ala Arg Tyr Cys Asp 21 O 215 22O Thir Asp Arg Asp Cys Asp Gly Pro Arg Lys Cys Cys Lieu. Ser Ser Arg 225 23 O 235 24 O Gly Tyr Glu. Cys Llys His Pro Val His Tyr Pro Asp Arg Val Glu Pro 245 250 255 Lieu Val Gly Glu. Cys Pro Pro Ser Arg Pro Arg Ile Pro Gly Lys Trp 26 O 265 27 O Val Asp Ile Cys Ser Llys His Ala Asn. Cys Pro Asp Pro Glu Lys Cys 27s 28O 285 Cys Asp Thr Glu Tyr Gly Asn Arg Cys Met Asp Val Gly Lieu Val Pro 29 O 295 3 OO Gly Glin Gly Glu Lys Pro Ala Asn. Cys Pro Lys Glu Pro Arg Ile Arg 3. OS 310 315 32O Gly. Thir Lys Tyr Asp Cys Arg Arg Asp Asp Asp Cys Asp Gly Lys Glin 3.25 330 335 Lys Cys Cys Tyr Thr Thr Glu Gly Arg Glu. Cys Val His Gly Ile Trp 34 O 345 35. O Pro <210s, SEQ ID NO 5 &211s LENGTH: 354 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 5 Met Val Arg Glu Lys Arg Gly Lys Cys Pro Pro Glu Pro Pro Ile Ala 1. 5 1O 15 Gly Asn. Thir Ile Tyr Cys Arg Asp Asp Phe Asp Cys Gly Gly Arg Glin 2O 25 3O Lys Cys Cys Thr Ile Ala Glu Gly Arg Gly Cys Val Pro Pro Tyr Gly 35 4 O 45 Glu Gln His Phe Glu Val Val Llys Pro Gly His Cys Pro Ala Ile Pro SO 55 6 O Ala Val Thr Gly Met Ala Asn. Phe Cys Asn. Thir Asp Gly Asp Cys Asp 65 70 7s 8O Gly Pro Llys Lys Cys Cys Lieu. Thir Ser Arg Gly Tyr Asp Cys Thr His 85 90 95 Pro Leu. His Phe Pro Ile Gln Pro Gln Pro Pro Val Gly Glin Cys Pro 1OO 105 11 O Pro Ser Llys Pro Arg Ile Pro Gly Lys Trp Val Asp Ile Cys Ala Lys 115 12 O 125 His Ala Asn. Cys Pro Asp Pro Glu Lys Cys Cys Asp Thr Glu Tyr Gly 13 O 135 14 O Asn Arg Cys Met Asp Val Gly Lieu Val Pro Gly Glin Gly Glu Arg Pro 145 150 155 160 Gly Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly Thr Llys Tyr Asp Cys 1.65 17O 17s US 7,951,547 B2 51 - Continued Arg Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Cys Phe Thr Val 18O 185 19 O Glu Gly Arg Glu. CyS Val Glu Pro Ser Arg Llys Pro Lieu. Asp Llys Pro 195 2OO 2O5 Gly His Cys Pro Pro Ile Pro Ala Asp Val Gly Ser Ala Arg Tyr Cys 21 O 215 22O Asp Thr Asp Arg Asp Cys Asp Gly Pro Arg Lys Cys Cys Lieu. Ser Ser 225 23 O 235 24 O Arg Gly Tyr Glu. Cys Llys His Pro Wal His Tyr Pro Asp Arg Val Glu 245 250 255 Pro Leu Val Gly Glu. Cys Pro Pro Ser Arg Pro Arg Ile Pro Gly Lys 26 O 265 27 O Trp Val Asp Ile Cys Ser Llys His Ala Asn. Cys Pro Asp Pro Glu Lys 27s 28O 285 Cys Cys Asp Thr Glu Tyr Gly Asn Arg Cys Met Asp Val Gly Lieu Val 29 O 295 3 OO Pro Gly Glin Gly Glu Lys Pro Ala Asn. Cys Pro Llys Glu Pro Arg Ile 3. OS 310 315 32O Arg Gly. Thir Lys Tyr Asp Cys Arg Arg Asp Asp Asp Cys Asp Gly Lys 3.25 330 335 Gln Lys Cys Cys Tyr Thr Thr Glu Gly Arg Glu. Cys Val His Gly Ile 34 O 345 35. O Trp Pro <210s, SEQ ID NO 6 &211s LENGTH: 353 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 6 Val Arg Glu Lys Arg Gly Lys Cys Pro Pro Glu Pro Pro Ile Ala Gly 1. 5 1O 15 Asn. Thir Ile Tyr Cys Arg Asp Asp Phe Asp Cys Gly Gly Arg Gln Lys 2O 25 3O Cys Cys Thr Ile Ala Glu Gly Arg Gly Cys Val Pro Pro Tyr Gly Glu 35 4 O 45 Gln His Phe Glu Val Val Llys Pro Gly His Cys Pro Ala Ile Pro Ala SO 55 6 O Val Thr Gly Met Ala Asn. Phe Cys Asn. Thir Asp Gly Asp Cys Asp Gly 65 70 7s 8O Pro Llys Lys Cys Cys Lieu. Thir Ser Arg Gly Tyr Asp Cys Thr His Pro 85 90 95 Lieu. His Phe Pro Ile Gln Pro Gln Pro Pro Val Gly Glin Cys Pro Pro 1OO 105 11 O Ser Llys Pro Arg Ile Pro Gly Lys Trp Val Asp Ile Cys Ala Lys His 115 12 O 125 Ala Asn. Cys Pro Asp Pro Glu Lys Cys Cys Asp Thr Glu Tyr Gly Asn 13 O 135 14 O Arg Cys Met Asp Val Gly Lieu Val Pro Gly Glin Gly Glu Arg Pro Gly 145 150 155 160 Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly Thr Llys Tyr Asp Cys Arg 1.65 17O 17s Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Cys Phe Thr Val Glu US 7,951,547 B2 53 - Continued 18O 185 19 O Gly Arg Glu. CyS Val Glu Pro Ser Arg Llys Pro Lieu. Asp Llys Pro Gly 195 2OO 2O5 His Cys Pro Pro Ile Pro Ala Asp Val Gly Ser Ala Arg Tyr Cys Asp 21 O 215 22O Thir Asp Arg Asp Cys Asp Gly Pro Arg Lys Cys Cys Lieu. Ser Ser Arg 225 23 O 235 24 O Gly Tyr Glu. Cys Llys His Pro Val His Tyr Pro Asp Arg Val Glu Pro 245 250 255 Lieu Val Gly Glu. Cys Pro Pro Ser Arg Pro Arg Ile Pro Gly Lys Trp 26 O 265 27 O Val Asp Ile Cys Ser Llys His Ala Asn. Cys Pro Asp Pro Glu Lys Cys 27s 28O 285 Cys Asp Thr Glu Tyr Gly Asn Arg Cys Met Asp Val Gly Lieu Val Pro 29 O 295 3 OO Gly Glin Gly Glu Lys Pro Ala Asn. Cys Pro Lys Glu Pro Arg Ile Arg 3. OS 310 315 32O Gly. Thir Lys Tyr Asp Cys Arg Arg Asp Asp Asp Cys Asp Gly Lys Glin 3.25 330 335 Lys Cys Cys Tyr Thr Thr Glu Gly Arg Glu. Cys Val His Gly Ile Trp 34 O 345 35. O Pro <210s, SEQ ID NO 7 &211s LENGTH: 354 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO > SEQUENCE: 7 Met Val Arg Glu Lys Arg Gly Lys Cys Pro Pro Glu Pro Pro Ile Ala 1. 5 1O 15 Gly Asn. Thir Ile Tyr Cys Arg Asp Asp Phe Asp Cys Gly Gly Arg Glin 2O 25 3O Lys Cys Cys Thr Ile Ala Glu Gly Arg Gly Cys Val Pro Pro Tyr Gly 35 4 O 45 Glu Glin Asp Phe Glu Val Val Llys Pro Gly His Cys Pro Ala Ile Pro SO 55 6 O Ala Val Thr Gly Met Ala Asn. Phe Cys Asn. Thir Asp Gly Asp Cys Asp 65 70 7s 8O Gly Pro Llys Lys Cys Cys Lieu. Thir Ser Arg Gly Tyr Asp Cys Thr His 85 90 95 Pro Leu. His Phe Pro Ile Gln Pro Gln Pro Pro Val Gly Glin Cys Pro 1OO 105 11 O Pro Ser Llys Pro Arg Val Pro Gly Lys Trp Val Asp Ile Cys Ala Lys 115 12 O 125 His Ala Asn. Cys Pro Asp Pro Glu Lys Cys Cys Asp Thr Glu Tyr Gly 13 O 135 14 O Asn Arg Cys Met Asp Val Gly Lieu Val Ala Gly Glin Gly Glu Arg Pro 145 150 155 160 Gly Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly Thr Llys Tyr Asp Cys 1.65 17O 17s Arg Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Cys Phe Thr Val 18O 185 19 O US 7,951,547 B2 55 - Continued Glu Gly Arg Glu. CyS Val Glu Pro Ser Arg Llys Pro Lieu. Asp Llys Pro 195 2OO 2O5 Gly His Cys Pro Pro Ile Pro Ala Asp Val Gly Ser Ala Arg Tyr Cys 21 O 215 22O Asp Thr Asp Arg Asp Cys Asp Gly Pro Arg Lys Cys Cys Lieu. Ser Ser 225 23 O 235 24 O Arg Gly Tyr Glu. Cys Llys His Pro Wal His Tyr Pro Asp Arg Val Glu 245 250 255 Pro Leu Val Gly Glu. Cys Pro Pro Ser Arg Pro Arg Ile Pro Gly Lys 26 O 265 27 O Trp Val Asp Ile Cys Ser Llys His Ala Asn. Cys Pro Asp Pro Glu Lys 27s 28O 285 Cys Cys Asp Thr Glu Tyr Gly Asn Arg Cys Met Asp Val Gly Lieu Val 29 O 295 3 OO Pro Gly Glin Gly Glu Lys Pro Ala Asn. Cys Pro Llys Glu Pro Arg Ile 3. OS 310 315 32O Arg Gly. Thir Lys Tyr Asp Cys Arg Arg Asp Asp Asp Cys Asp Gly Lys 3.25 330 335 Gln Lys Cys Cys Tyr Thr Thr Glu Gly Arg Glu. Cys Val His Gly Ile 34 O 345 35. O Trp Pro <210s, SEQ ID NO 8 &211s LENGTH: 353 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 8 Val Arg Glu Lys Arg Gly Lys Cys Pro Pro Glu Pro Pro Ile Ala Gly 1. 5 1O 15 Asn. Thir Ile Tyr Cys Arg Asp Asp Phe Asp Cys Gly Gly Arg Gln Lys 2O 25 3O Cys Cys Thr Ile Ala Glu Gly Arg Gly Cys Val Pro Pro Tyr Gly Glu 35 4 O 45 Gln Asp Phe Glu Val Val Llys Pro Gly His Cys Pro Ala Ile Pro Ala SO 55 6 O Val Thr Gly Met Ala Asn. Phe Cys Asn. Thir Asp Gly Asp Cys Asp Gly 65 70 7s 8O Pro Llys Lys Cys Cys Lieu. Thir Ser Arg Gly Tyr Asp Cys Thr His Pro 85 90 95 Lieu. His Phe Pro Ile Gln Pro Gln Pro Pro Val Gly Glin Cys Pro Pro 1OO 105 11 O Ser Llys Pro Arg Val Pro Gly Lys Trp Val Asp Ile Cys Ala Lys His 115 12 O 125 Ala Asn. Cys Pro Asp Pro Glu Lys Cys Cys Asp Thr Glu Tyr Gly Asn 13 O 135 14 O Arg Cys Met Asp Val Gly Lieu Val Ala Gly Glin Gly Glu Arg Pro Gly 145 150 155 160 Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly Thr Llys Tyr Asp Cys Arg 1.65 17O 17s Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Cys Phe Thr Val Glu 18O 185 19 O Gly Arg Glu. CyS Val Glu Pro Ser Arg Llys Pro Lieu. Asp Llys Pro Gly 195 2OO 2O5 US 7,951,547 B2 57 58 - Continued His Cys Pro Pro Ile Pro Ala Asp Wall Gly Ser Ala Arg Tyr Cys Asp 21 O 215 Thir Asp Arg Asp Cys Asp Gly Pro Arg Cys Lell Ser Ser Arg 225 23 O 235 24 O Gly Tyr Glu Cys Llys His Pro Wall His Tyr Pro Asp Arg Wall Glu Pro 245 250 255 Lieu Wall Gly Glu Cys Pro Pro Ser Arg Pro Arg Ile Pro Gly Trp 26 O 265 27 O Val Asp Ile Cys Ser Llys His Ala Asn Pro Asp Pro Glu Cys 285 Thir Glu Tyr Gly Asn Arg Met Asp Wall Gly Luell Wall Pro 29 O 295 3 OO Gly Glin Gly Glu Lys Pro Ala Asn Pro Lys Glu Pro Arg Ile Arg 3. OS 310 315 Gly Thr Tyr Asp Arg Asp Asp Asp Asp Gly Lys Glin 3.25 330 335 Tyr Thr Thir Glu Gly Arg Glu Wall His Gly Ile Trp 34 O 345 35. O Pro &21. Os SE Q I D NO 9 <211s LE NGT H: 372 &212s. TY PE: PRT <213> ORGANISM: Artificial ATU RE: HER INFORMAT ON: Synthetic ATU RE: <221 > NAMEA KEY: misc feature <222s. LOCATION: (1) . . (19) HER INFORMAT ON: Xaa Ca loe any naturally occurring amino acid ATU RE: <221 > NAMEA KEY: misc feature <222s. LOCATION: (69). . (69) HER INFORMAT ON: Xaa Ca loe any naturally occurring amino acid ATU RE: <221 > NAMEA KEY: misc feature <222s. LOCATION: (136 ) ... (136) HER INFORMAT ON: Xaa Ca loe any naturally occurring amino acid ATU RE: <221 > NAMEA KEY: misc feature <222s. LOCATION: (172 ) ... (172) HER INFORMAT ON: Xaa Ca loe any naturally occurring amino acid <4 OOs, SEQUENCE: 9 Xaa Xala Xala Xala Xala Xaa Xala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1. 5 15 Xaa Xala Xaa Val Arg Glu Lys Arg Gly Pro Pro Glu Pro Pro 2O 25 Ile Ala Gly Asn. Thir Ile Tyr Cys Arg Asp Asp Phe Asp Gly Gly 35 4 O 45 Arg Glin Thir Ile Ala Glu Gly Arg Gly Wall Pro Pro SO 55 6 O Tyr Gly Glu Glin Xaa Phe Glu Wall Wall Pro Gly His Pro Ala 65 70 Ile Pro Ala Wall. Thir Gly Met Ala Asn Phe Asn Thir Asp Gly Asp 85 90 95 Gly Pro Llys Luell Thir Ser Arg Gly Tyr Asp Cys 1OO 105 11 O Thir His Pro Lieu. His Phe Pro Ile Glin Pro Glin Pro Pro Wall Gly Glin 115 12 O 125 US 7,951,547 B2 59 60 - Continued Pro Pro Ser Llys Pro Arg Xaa Pro Gly Lys Trp Val Asp Ile Cys 13 O 135 14 O Ala His Ala Asn. Cys Pro Asp Pro Glu Lys Cys Cys Asp Thr Glu 145 150 155 160 Gly Asn Arg Cys Met Asp Val Gly Lieu Val Xaa Gly Glin Gly Glu 1.65 17O 17s Arg Pro Gly Asn. Cys Pro Asn. Glu Pro Arg Ile Arg Gly Thir 18O 185 19 O Asp Arg Arg Asp Asp Asp Cys Asp Gly Val Glin Lys Cys Phe 195 2O5 Thir Wall Glu Gly Arg Glu. Cys Val Glu Pro Ser Arg Pro Lieu. Asp 21 O 215 Lys Pro Gly His Cys Pro Pro Ile Pro Ala Asp Wall Ser Ala Arg 225 23 O 235 24 O Asp Thir Asp Arg Asp Cys Asp Gly Pro Arg Cys Lieu. 245 250 255 Ser Ser Arg Gly Tyr Glu. Cys Llys His Pro Wall His Pro Asp Arg 26 O 265 27 O Wall Glu Pro Lieu Val Gly Glu. Cys Pro Pro Ser Arg Pro Arg Ile Pro 285 Gly Lys Trp Val Asp Ile Cys Ser Llys His Ala Asn Pro Asp Pro 29 O 295 3 OO Glu Cys Asp Thr Glu Tyr Gly Asn Arg Met Asp Val Gly 3. OS 310 315 32O Lell Wall Pro Gly Glin Gly Glu Lys Pro Ala Asn Pro Glu Pro 3.25 330 335 Arg Ile Arg Gly. Thir Lys Tyr Asp Cys Arg Arg Asp Asp Asp 34 O 345 35. O Gly Glin Lys Cys Cys Tyr Thr Thr Glu Gly Arg Glu Wall His 355 360 365 Gly Ile Trp Pro 37 O SEQ ID NO 10 LENGTH: 86.5 TYPE: DNA ORGANISM: Toxocara canis < 4 OOs SEQUENCE: 1.O Caagaagatt tatggtgtgg Cagct tcgag acgaaggagg Cat Cact tca cgcticgaaaa 6 O Cagt ctggac acccacctga aatggcttag ccacgagcaa. aaggaggaac tgctgcaaat 12 O gaagaaggac ggcaaatcga agaaggagct cCaggataag at catgcact attacgagca 18O Ccticgaaggc gatgcgaaac atgaa.gcaac agagcaactg aaggg.cggat gcc.gc.gagat 24 O t cittaa.gcat gttgttggcg aggagaaagc agctgagatc aaagc actga aagattctgg 3OO agcaa.gcaaa gatgagctta aagccaaggt cgalagaggca ctic cacgcag t caccgacga 360 agaaaagaag caa.catat cq ccgaatt.cgg tcc.cgcatgc aagaagattt atggtgttggc agct tcgaga cgaaggaggc at Cacttcac gct caaaac agt ctggaca cccacctgaa atggcttagc cacgagcaaa aggaggaact gctgcaaatg aagaaggacg gcaaatcgaa 54 O gaaggagctic Caggataaga t catgcacta ttacgagcac Ctcgaaggga tgctic ct cqc gctatotatc citg tattgac ggcct tccaa CCt at Cacac Ctgtcagtgc ggcct tacat 660 tcgacgagcg tagaaagacic tgtct tcc ta agcagctggit aaagtactgc ggaatcc cag 72 O US 7,951,547 B2 61 - Continued aatctggaga ggcgt.cggcg gaagttggtg agt cqtacta acacagcacg Ctctcgttgg tgcagatgtt gtgttgaaata ctitttgtcag ttitt cogtgt gttittaaata aataaaaaat 84 O tcc.gtaaaaa aaaaaaaaaa aaaaa. 865 <210s, SEQ ID NO 11 &211s LENGTH: 632 &212s. TYPE: DNA <213> ORGANISM: Toxocara cati <4 OOs, SEQUENCE: 11 attt atggtg tdgcagct tc gaga.cgalagg aggcatcact t cacgct cqa aaaaagttctg 6 O gacacccacc tgaaatggct tagcc acgag caaaaggagg aactgctgaa aatgaagaaa 12 O gatgggaaat cgaagaagga gct coaggat alaggtgatgc actitctacga gcaccitcgaa 18O ggcgatgcga alacatgaagc alacagagcaa. Ctgaagggcg gatgcc.gcga gatcc ttaag 24 O Catgttgttg gtgaggagaa agcagctgag atcaaag cac tgaaagattic tggagcaa.gc 3OO aaagatgagc ttaaagccaa ggtcgaagat gcact coacg cggtcaccga agaagaaaag 360 aagcaa.cata tcqc.cgaatt tggtc.ca.gca tgcaaggaaa tttitcggggt gcc.ggttgat gttcgt caca aacgcgaccc ttatactaat atgacgc.ccg atgaagttgc tgaaggact a agaagttaac ggtgat Cag Ctttittgcaa aaactggttg atgcttittaa att Cttittaa. 54 O gcctttittct tgttgttattt cggaattgta ccacacgaac agittagttcc gaataaagaa ctgtaattat gtaaaaaaaa aaaaaaaaaa. a.a. 632 SEQ ID NO 12 LENGTH: 2O5 TYPE : PRT ORGANISM: Toxocara canis < 4 OOs SEQUENCE: 12 Lys Ile Tyr Gly Val Ala Ala Ser Arg Arg Arg Arg His His Phe 5 1O 15 Lel Glu Asn Ser Lieu. Asp Thr His Luell Lys Trp Lell Ser His Glu 25 Glin Glu Glu Lieu. Lieu. Glin Met Asp Gly Lys Ser 35 4 O 45 Glu Glin Asp Llys Ile Met His Glu His Lell Glu Gly Asp 55 6 O Ala His Glu Ala Thr Glu Glin Luell Gly Gly Arg Glu Ile 65 70 8O Lell His Val Val Gly Glu Glu Ala Ala Glu Ile Ala Lieu 85 90 95 Asp Ser Gly Ala Ser Lys Asp Glu Luell Lys Ala Wall Glu Glu 105 11 O Ala Luell His Ala Val Thr Asp Glu Glu Glin His Ile Ala Glu 115 12 O 125 Phe Gly Pro Ala Cys Llys Lys Ile Gly Wall Ala Ala Ser Arg Arg 13 O 135 14 O Arg Arg His His Phe Thir Lieu. Glu. Asn Ser Luell Asp Thir His Lieu Lys 145 150 155 160 Trp Luell Ser His Glu Gln Lys Glu Glu Luell Luell Glin Met Lys Asp 1.65 17O 17s Gly Ser Llys Lys Glu Lieu. Glin Asp Ile Met His Tyr Tyr Glu 18O 185 19 O US 7,951,547 B2 63 - Continued His Lieu. Glu Gly Met Lieu. Lieu Ala Lieu. Cys Ile Lieu. Tyr 195 2OO 2O5 <210s, SEQ ID NO 13 &211s LENGTH: 162 212. TYPE: PRT <213> ORGANISM: Toxocara cati <4 OOs, SEQUENCE: 13 Ile Tyr Gly Val Ala Ala Ser Arg Arg Arg Arg His His Phe Thr Lieu. 1. 5 1O 15 Glu Lys Ser Lieu. Asp Thr His Lieu Lys Trp Lieu. Ser His Glu Gln Lys 2O 25 3O Glu Glu Lieu. Lieu Lys Met Lys Lys Asp Gly Lys Ser Llys Lys Glu Lieu. 35 4 O 45 Glin Asp Llys Val Met His Phe Tyr Glu. His Lieu. Glu Gly Asp Ala Lys SO 55 6 O His Glu Ala Thr Glu Gln Lieu Lys Gly Gly Cys Arg Glu Ile Lieu Lys 65 70 7s 8O His Val Val Gly Glu Glu Lys Ala Ala Glu Ile Lys Ala Lieu Lys Asp 85 90 95 Ser Gly Ala Ser Lys Asp Glu Lieu Lys Ala Lys Val Glu Asp Ala Lieu. 1OO 105 11 O His Ala Val Thr Asp Glu Glu Lys Lys Gln His Ile Ala Glu Phe Gly 115 12 O 125 Pro Ala Cys Lys Glu Ile Phe Gly Val Pro Ile Asp Val Arg His Lys 13 O 135 14 O Arg Asp Pro Tyr Thr Asn Met Thr Pro Asp Glu Val Ala Glu Gly Lieu. 145 150 155 160 Arg Ser <210s, SEQ ID NO 14 &211s LENGTH: 130 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 14 Met His His Phe Thr Lieu. Glu Asn Ser Lieu. Asp Thr His Leu Lys Trp 1. 5 1O 15 Lieu. Ser His Glu Glin Lys Glu Glu Lieu. Lieu Gln Met Llys Lys Asp Gly 2O 25 3O Llys Ser Lys Lys Glu Lieu. Glin Asp Llys Ile Met His Tyr Tyr Glu. His 35 4 O 45 Lieu. Glu Gly Asp Ala Lys His Glu Ala Thr Glu Glin Lieu Lys Gly Gly SO 55 6 O Cys Arg Glu Ile Lieu Lys His Val Val Gly Glu Glu Lys Ala Ala Glu 65 70 7s 8O Ile Lys Ala Lieu Lys Asp Ser Gly Ala Ser Lys Asp Glu Lieu Lys Ala 85 90 95 Llys Val Glu Glu Ala Lieu. His Ala Val Thir Asp Glu Glu Lys Lys Glin 1OO 105 11 O His Ile Ala Glu Phe Gly Pro Ala Cys Llys Lys Ile Tyr Gly Val Ala 115 12 O 125 Ala Ser 13 O US 7,951,547 B2 65 - Continued > SEQ ID NO 15 is LENGTH: 12 9 TYPE : PRT > ORGANISM: Artificial FEATURE: OTHER INFO RMAT ION: Synthetic <4 OOs, SEQUENCE: 15 His His Phe Thr Lel Glu Asn Ser Luell Asp Thir His Lell Trp Luell 1. 1O 15 Ser His Glu Glin Lys Glu Glu Luell Luell Glin Met Asp Gly 2O 25 3O Ser Lys Glu Lel Glin Asp Lys Ile Met His Glu His Luell 35 4 O Glu Gly Asp Ala His Glu Ala Thir Glu Glin Lell Gly Gly SO 55 6 O Arg Glu Ile Luell Lys His Wall Wall Gly Glu Glu Lys Ala Ala Glu Ile 65 70 8O Ala Luell Asp Ser Gly Ala Ser Lys Asp Glu Lell Ala Lys 85 90 95 Wall Glu Glu Ala Lell His Ala Wall Thir Asp Glu Glu Lys Glin His 1OO 105 11 O Ile Ala Glu Phe Gly Pro Ala Cys Ile Gly Wall Ala Ala 115 12 O 125 Ser <210s, SEQ I D NO 16 NGT H: 15 5 TYPE : PRT > ORGANISM: Artificial FEATU RE: OT HER INFO RMAT ON: Synthetic FEATU RE: > NAMEA KEY: misc feature LOCATION: (1) . . (11) INFO RMAT ON: Xaa Ca loe any natura y occurring amino acid E A. NA. misc feature O C f (18). . (18) ON: Xaa Ca loe any natura y occurring amino acid feature ... (37) ON: Xaa Ca loe any natura y occurring amino acid feature (52). . (52) RMAT ON: Xaa Ca loe any natura y occurring amino acid misc feature (55). . (55) INFO RMAT ON: Xaa Ca loe any natura y occurring amino acid misc feature C f (110 ) . . (110) INFO RMAT ON: Xaa Ca loe any natura y occurring amino acid misc feature (133 ) ... (133) INFO RMAT ON: Xaa Ca loe any natura y occurring amino acid misc feature C f (135 ) ... (135) INFO RMAT ON: Xaa Ca loe any natura y occurring amino acid A. : misc feature > LO (138 ) ... (155) INFO RMAT ON: Xaa Ca loe any natura y occurring amino acid US 7,951,547 B2 69 - Continued tgcaggaaat tdcatcaagt togacgattg ccc.gaagitaa ataataacca tactaattgc 48O tgattacaat taaaataata aatgagtcca gctgttaaaa aaaaaaaaaa aaaaaa 536 <210s, SEQ ID NO 19 &211s LENGTH: 143 212. TYPE: PRT <213> ORGANISM: Toxocara canis <4 OOs, SEQUENCE: 19 Met Lieu. Ser Wall Lieu Ala Lieu. Phe Ala Lieu. Ile Thr Phe Ala Wall Ala 1. 5 1O 15 Gly Pro Glu Ser Cys Gly Pro Asn Glu Val Trp Thr Glu. Cys Thr Gly 2O 25 3O Cys Glu Lieu Lys Cys Gly Glin Asp Glu Asn Thr Pro Cys Thir Lieu. Asn 35 4 O 45 Cys Arg Pro Pro Ser Cys Glu. Cys Ser Pro Gly Arg Gly Met Arg Arg SO 55 6 O Thir Asn Asp Gly Arg Cys Ile Pro Ala Ser Glin Cys Pro Gln His Arg 65 70 7s 8O Ala Lys Arg Glu Glu Gln Cys Llys Pro Asn. Glu Gln Trp Ser Pro Cys 85 90 95 Arg Gly Cys Glu Gly Thr Cys Ala Glin Arg Phe Val Pro Cys Thr Arg 1OO 105 11 O Asn. Cys Arg Pro Pro Gly Cys Glu. Cys Val Ala Gly Ala Gly Phe Val 115 12 O 125 Arg Asp Ala Glu Gly ASn Cys Ile Llys Phe Asp Asp Cys Pro Llys 13 O 135 14 O <210s, SEQ ID NO 2 O &211s LENGTH: 143 212. TYPE: PRT <213> ORGANISM: Toxocara cati <4 OOs, SEQUENCE: 2O Met Lieu. Ser Wall Lieu Ala Lieu. Phe Ala Lieu. Ile Thr Phe Ala Wall Ala 1. 5 1O 15 Asp Pro Llys Ser Cys Gly Pro Asn Glu Val Trp Thr Glu. Cys Thr Gly 2O 25 3O Cys Glu Lieu Lys Cys Gly Glin Asp Glu Asp Thr Pro Cys Thir Lieu. Asn 35 4 O 45 Cys Arg Pro Pro Ser Cys Glu. Cys Ser Pro Gly Arg Gly Met Arg Arg SO 55 6 O Thir Asp Asp Gly Arg Cys Ile Pro Ala Ser Glin Cys Pro Gln His Arg 65 70 7s 8O Ala Lys Arg Glu Glu Gln Cys Llys Pro Asn. Glu Gln Trp Ser Pro Cys 85 90 95 Arg Gly Cys Glu Gly Thr Cys Ala Glin Arg Phe Val Pro Cys Thr Arg 1OO 105 11 O Asn. Cys Arg Pro Pro Gly Cys Glu. Cys Val Ala Gly Ala Gly Phe Val 115 12 O 125 Arg Asp Ala Ala Gly Asn. Cys Ile Llys Phe Asp Asp Cys Pro Llys 13 O 135 14 O <210s, SEQ ID NO 21 &211s LENGTH: 128 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: US 7,951,547 B2 71 - Continued <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 21 Met Gly Pro Glu Ser Cys Gly Pro Asn Glu Val Trp Thr Glu. Cys Thr 1. 5 1O 15 Gly Cys Glu Lieu Lys Cys Gly Glin Asp Glu Asn Thr Pro Cys Thr Lieu. 2O 25 3O Asn Cys Arg Pro Pro Ser Cys Glu. Cys Ser Pro Gly Arg Gly Met Arg 35 4 O 45 Arg Thr Asn Asp Gly Arg Cys Ile Pro Ala Ser Glin Cys Pro Gln His SO 55 6 O Arg Ala Lys Arg Glu Glu Glin Cys Llys Pro Asn. Glu Gln Trp Ser Pro 65 70 7s 8O Cys Arg Gly Cys Glu Gly Thr Cys Ala Glin Arg Phe Val Pro Cys Thr 85 90 95 Arg Asn. Cys Arg Pro Pro Gly Cys Glu. CyS Val Ala Gly Ala Gly Phe 1OO 105 11 O Val Arg Asp Ala Glu Gly Asn. Cys Ile Llys Phe Asp Asp Cys Pro Llys 115 12 O 125 <210s, SEQ ID NO 22 &211s LENGTH: 127 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 22 Gly Pro Glu Ser Cys Gly Pro Asn Glu Val Trp Thr Glu. Cys Thr Gly 1. 5 1O 15 Cys Glu Lieu Lys Cys Gly Glin Asp Glu Asn Thr Pro Cys Thir Lieu. Asn 2O 25 3O Cys Arg Pro Pro Ser Cys Glu. Cys Ser Pro Gly Arg Gly Met Arg Arg 35 4 O 45 Thir Asn Asp Gly Arg Cys Ile Pro Ala Ser Glin Cys Pro Gln His Arg SO 55 6 O Ala Lys Arg Glu Glu Gln Cys Llys Pro Asn. Glu Gln Trp Ser Pro Cys 65 70 7s 8O Arg Gly Cys Glu Gly Thr Cys Ala Glin Arg Phe Val Pro Cys Thr Arg 85 90 95 Asn. Cys Arg Pro Pro Gly Cys Glu. Cys Val Ala Gly Ala Gly Phe Val 1OO 105 11 O Arg Asp Ala Glu Gly Asn. Cys Ile Llys Phe Asp Asp Cys Pro Llys 115 12 O 125 <210s, SEQ ID NO 23 &211s LENGTH: 143 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (17 <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (19) . . (19) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (42) ... (42) US 7,951,547 B2 73 - Continued <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (66) . . (66) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (132) ... (132) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 23 Xaa Xala Xala Xala Xala Xala Xala Xala Xala Xala Xala Xaa Xaa Xaa Xaa Xala 1. 5 1O 15 Xaa Pro Xaa Ser Cys Gly Pro Asn Glu Val Trp Thir Glu Cys Thr Gly 2O 25 Cys Glu Lieu Lys Cys Gly Glin Asp Glu Xaa Thr Pro Cys Thir Luell Asn 35 4 O 45 Cys Arg Pro Pro Ser Cys Glu. Cys Ser Pro Gly Arg Gly Met Arg Arg SO 55 6 O Thr Xaa Asp Gly Arg Cys Ile Pro Ala Ser Glin Pro Glin His Arg 65 70 7s Ala Lys Arg Glu Glu Gln Cys Llys Pro Asn. Glu Glin Trp Ser Pro Cys 85 90 95 Arg Gly Cys Glu Gly. Thir Cys Ala Glin Arg Phe Wall Pro Cys Thr Arg 1OO 105 11 O Asn. Cys Arg Pro Pro Gly Cys Glu. Cys Val Ala Gly Ala Gly Phe Wall 115 12 O 125 Arg Asp Ala Xaa Gly ASn Cys Ile Llys Phe Asp Asp Pro 13 O 135 14 O <210s, SEQ ID NO 24 &211s LENGTH: 469 &212s. TYPE: DNA <213> ORGANISM: Toxocara canis <4 OOs, SEQUENCE: 24 atcago aggt titcgcttcaa atgct tcc.ga taacttittitt gctggcaatt attgtcggtg 6 O Cagcagtagc ticaccgtaaa ttggtc.caa acgaagagtg gaccgaatgc actggttgcg 12 O aaattalagtg cggtcaagga gag calaccat gcc ct atgat c catcgtgtg 18O aatgcatggc cqgcaaagga ttacgaagaa cagcggacgg aagatgcgtg ccggaggcac 24 O aatgcc caaa aagaatggta aag.cgagacg aaaaatgtgg gccaaacgag aaatticcitga 3OO agtgcagagg ttgtgagggit acctgcaaag aacgt.ct cqt tocctg.ccct agaatgtgca 360 alaccaccagg ttgcgaatgc ccc.gcttcag aaggatt.cgt tcqcaatgac aaaggcgaat gtat caagtt coacgactgc ccgaaataaa ttcaataaat caattttgt 469 <210s, SEQ ID NO 25 &211s LENGTH: 548 &212s. TYPE: DNA <213> ORGANISM: Toxocara cati <4 OOs, SEQUENCE: 25 atcago aggt titcgcttcaa atgct tcc.gt taacttittitt gctggcattt attgttgggtg 6 O Cagcgg tagc ticaccgtaaa ttggtc.caa atgaagagtg gacggaatgc actggctg.cg 12 O aaatgaagtg cggtgaagga gagacaccat gcc ct atgat c catcgtgtg 18O aatgcatggc cqgcaaagga ttacgaagaa caccggacgg aagatgtgtg ccggaggcac 24 O aatgc.ccgaa acatatggta aag.cgagatgaaaaatgtgg gaaaaacgag aaatticcitga US 7,951,547 B2 75 76 - Continued agtgcagagg atgttgagggit acgtgcaaag aagatgtgca 360 alaccaccagg ttgcgaatgc ccggctt.cgg aaggatt.cgt. tcqcaatgac aaacacgaat gtat caagtt cgacgactgc CCCaaataaa. ttcaataaat cagtc.ttgtt gataaataca atcgtgatgc t cacgtttitt tttitcttgcc ataaaat Cta tact tcc cala aaaaaaaaaa. 54 O aaaaaaaa. 548 SEQ ID NO 26 LENGTH: 142 TYPE : PRT ORGANISM: Toxocara canis < 4 OOs SEQUENCE: 26 Met Lieu. Pro Ile Thir Phe Leu Lleu. Ala Ile Wall Gly Ala Ala Wall 1. 5 15 Ala His Arg Lys Cys Gly Pro Asn Glu Trp Thir Glu Cys Thr Gly 25 Glu Ile Lys Cys Gly Glin Gly Glu Pro Pro Met Met Cys 35 4 O 45 Arg Pro Pro Ser Cys Glu. Cys Met Ala Gly Lell Arg Arg Thr SO 55 6 O Ala Asp Gly Arg Cys Val Pro Glu Ala Pro Arg Met Wall 65 70 8O Arg Asp Glu Lys Cys Gly Pro Asn Phe Lell Cys Arg 85 95 Glu Gly Thr Cys Lys Glu Arg Luell Wall Pro Pro Arg Met 105 11 O Pro Pro Gly Cys Glu. Cys Pro Ala Ser Glu Gly Phe Val Arg 115 12 O 125 Asn Asp Gly Glu. Cys Ile Llys Phe Asp Asp Cys Pro 13 O 135 14 O <210s, SEQ ID NO 27 &211s LENGTH: 142 212. TYPE : PRT &213s ORGANISM: Toxocara cati <4 OOs, SEQUENCE: 27 Met Lieu. Pro Lieu. Thir Phe Leu Lleu. Ala Phe Ile Wall Gly Ala Ala Wall 1. 5 15 Ala His Arg Lys Cys Gly Pro Asn Glu Glu Trp Thir Glu Cys Thr Gly 25 Glu Met Lys Cys Gly Glu Gly Glu Thir Pro Pro Met Met Cys 35 4 O 45 Arg Pro Pro Ser Cys Glu. Cys Met Ala Gly Lys Gly Lell Arg Arg Thr SO 55 6 O Pro Asp Gly Arg Cys Val Pro Glu Ala Glin Cys Pro His Met Wall 65 70 Arg Asp Glu Lys Cys Gly Lys Asn Glu Lys Phe Lell Cys Arg 85 90 95 Glu Gly Thr Cys Lys Glu Arg Luell Wall Pro Pro Lys Met 105 11 O Pro Pro Gly Cys Glu. Cys Pro Ala Ser Glu Gly Phe Val Arg 115 12 O 125 Asn Asp His Glu. Cys Ile Llys Phe Asp Asp Cys Pro 13 O 135 14 O US 7,951,547 B2 77 - Continued <210s, SEQ ID NO 28 &211s LENGTH: 126 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 28 Met His Arg Lys Cys Gly Pro Asn Glu Glu Trp Thr Glu. Cys Thr Gly 1. 5 1O 15 Cys Glu Ile Lys Cys Gly Glin Gly Glu Glin Pro Cys Pro Met Met Cys 2O 25 3O Arg Pro Pro Ser Cys Glu. Cys Met Ala Gly Lys Gly Lieu. Arg Arg Thr 35 4 O 45 Ala Asp Gly Arg Cys Val Pro Glu Ala Glin Cys Pro Lys Arg Met Val SO 55 6 O Lys Arg Asp Glu Lys Cys Gly Pro Asn. Glu Lys Phe Lieu Lys Cys Arg 65 70 7s 8O Gly Cys Glu Gly Thr Cys Lys Glu Arg Lieu Val Pro Cys Pro Arg Met 85 90 95 Cys Llys Pro Pro Gly Cys Glu. Cys Pro Ala Ser Glu Gly Phe Val Arg 1OO 105 11 O Asn Asp Llys Gly Glu. Cys Ile Llys Phe Asp Asp Cys Pro Llys 115 12 O 125 <210s, SEQ ID NO 29 &211s LENGTH: 125 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 29 His Arg Lys Cys Gly Pro Asn Glu Glu Trp Thr Glu. Cys Thr Gly Cys 1. 5 1O 15 Glu Ile Lys Cys Gly Glin Gly Glu Gln Pro Cys Pro Met Met Cys Arg 2O 25 3O Pro Pro Ser Cys Glu. Cys Met Ala Gly Lys Gly Lieu. Arg Arg Thr Ala 35 4 O 45 Asp Gly Arg Cys Val Pro Glu Ala Glin Cys Pro Lys Arg Met Val Lys SO 55 6 O Arg Asp Glu Lys Cys Gly Pro Asn. Glu Lys Phe Lieu Lys Cys Arg Gly 65 70 7s 8O Cys Glu Gly Thr Cys Lys Glu Arg Lieu Val Pro Cys Pro Arg Met Cys 85 90 95 Llys Pro Pro Gly Cys Glu. Cys Pro Ala Ser Glu Gly Phe Val Arg Asn 1OO 105 11 O Asp Llys Gly Glu. Cys Ile Llys Phe Asp Asp Cys Pro Llys 115 12 O 125 <210s, SEQ ID NO 3 O &211s LENGTH: 142 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (17 <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid US 7,951,547 B2 79 80 - Continued 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (35) ... (35) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (39) . . (39) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (42) ... (42) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (65) . . (65) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (78) . . (78) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (88) . . (88) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (111) ... (111) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (132) ... (132) &223s OTHER INFORMATION: Xala can loe any natura y occurring amino acid <4 OOs, SEQUENCE: 30 Xaa Xala Xala Xala Xala Xala Xala Xala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xala 1. 5 15 Xaa His Arg Lys Cys Gly Pro Asn Glu Glu Trp Thir Glu Cys Thr Gly 2O 25 Cys Glu Xaa Lys Cys Gly Xaa Gly Glu Xaa Pro Pro Met Met Cys 35 4 O 45 Arg Pro Pro Ser Cys Glu. Cys Met Ala Gly Lys Gly Lell Arg Arg Thr SO 55 6 O Xaa Asp Gly Arg Cys Val Pro Glu Ala Glin Cys Pro Xaa Met Wall 65 70 8O Lys Arg Asp Glu Lys Cys Gly Xaa Asn Glu Lys Phe Lell Cys Arg 85 90 95 Gly Cys Glu Gly Thr Cys Lys Glu Arg Luell Wall Pro Pro Xaa Met 1OO 105 11 O Cys Llys Pro Pro Gly Cys Glu. Cys Pro Ala Ser Glu Gly Phe Val Arg 115 12 O 125 Asn Asp Llys Xaa Glu. Cys Ile Llys Phe Asp Asp Cys Pro 13 O 135 14 O <210s, SEQ ID NO 31 &211s LENGTH: 1343 &212s. TYPE: DNA <213> ORGANISM: Ancylostoma <4 OOs, SEQUENCE: 31 atgggcagca gccatcatca toat catcac agcagcggcc cggcagc cat 6 O atggctag Ca tgactggtgg acagdaaatg ccgaatt cqa gct caccact 12 O tgtcCagg a.a. atgatctaa C agatgctgaa cgcacactgc taact agggit gcacaatticc 18O atticgacg 99 aaatagcgc a aggagttgca aacaact acc atggtggtaa actgcctgct 24 O ggaaagaa Ca tatacagga t gagatacagc tgttgagctgg aac aggctgc tattgatgct 3OO US 7,951,547 B2 83 84 - Continued Arg Gly Ser His Met Ala Ser Met Thir Gly Gly Gln Gln Met Gly Arg 25 Ser Glu Phe Glu Lell Thir Thir Pro Gly Asn Asp Luell Thir Asp 35 4 O 45 Glu Arg Thir Lell Lell Thir Arg Wall His ASn Ser Ile Arg Arg Glu SO 55 6 O Ala Glin Gly Wall Ala Asn Asn His Gly Gly Luell Pro Ala 70 Asn Ile Tyr Arg Met Arg Ser Glu Lell Glu Glin Ala 85 90 95 Ile Asp Ala Ser Glin Thir Phe Cys Ser Ala Ser Lell Glu Glu Pro 105 11 O Tyr Gly Glin Asn Ile Glin Ala Tyr Wall Thir Pro Ser Ile Ile 115 12 O 125 Arg Pro Asn Asp Lell Luell Glu Asp Ala Wall Glin Trp 13 O 135 14 O Lell Pro Wall Ile Tyr Tyr Gly Glin Arg Asp Ala Ala Asn Phe Thir 145 150 155 160 Asp Pro Arg Luell Tyr Thir Phe Ala Asn Luell Ala Asp Asn Thir 1.65 17O 17s Ala Luell Gly Cys His Ala Cys Glin Gly Pro Asp Arg Ile Wall 185 19 O Ile Ser Cys Met Tyr Asn Asn Wall Wall Pro Asp Asn Ala Wall Ile 195 Glu Pro Gly Thir Ala Wall Asp Glin Asp Cys Thir Thir Pro 21 O 215 22O Glin Ser Thir Lys Asp Ser Luell Ile Ile Pro Thir Pro His Pro 225 23 O 235 24 O Pro Asn Pro Pro Asn Pro Pro Pro Ala Met Pro Asn Ala Glu Met 245 250 255 Thir Asp Ala Ala Arg Wall Luell Asp Met His Asn Trp Arg Arg 26 O 265 27 O Ser Glin Luell Ala Lell Gly Asn Wall Glin Asn Gly Asn Ala Asn 285 Pro Thir Ala Thir Asp Met Met Glu Tyr Asp Asp Luell 29 O 295 3 OO Glu Asn Ser Ala Lell Ala Ala Glin Cys Ser Lell Wall Gly Ser 3. OS 310 315 Ala Glu Gly Thir Arg Pro Gly Glu Gly Glu ASn Wall His Gly Ala 3.25 330 335 Lell Wall Thir Asp Pro Glu Ala Ala Wall Glin Thir Ala Wall Glin Ala Trp 34 O 345 35. O Trp Ser Glin Ile Ser Glin Asn Gly Luell Asn Ala Glin Met Phe Thir 355 360 365 Ala Phe Luell Asp Pro Asp Ala Pro Thir Ala Phe Thir Glin Met 37 O 375 Ala Trp Ala Ser Wall Luell Gly Ala Wall Ser Asn Glin 385 390 395 4 OO Ala Asp Thir Phe Thir Wall Arg Lys Ala Ala Gly Asn Ile Wall 4 OS 41O 415 Gly Glu Phe Ile Tyr Thir Gly Asn Wall Cys Asp Ala Cys Ala 42O 425 43 O Thir Ile Thir Ala Glu Gly Luell Pro Thir Pro US 7,951,547 B2 85 86 - Continued 435 44 O <210s, SEQ ID NO 34 &211s LENGTH: 252 212. TYPE: PRT <213> ORGANISM: Ancylostoma <4 OOs, SEQUENCE: 34 Met Gly Ser Ser His His His His His His Ser Ser Gly Lieu Val Pro 1. 5 15 Arg Gly Ser His Met Ala Ser Met Thir Gly Gly Gln Gln Met Gly Arg 2O 25 3O Gly Ser Glu Phe Glu Lieu. Thr Thir Pro Gly Asn Asp Lieu. Thir Asp 35 4 O 45 Ala Glu Arg Thr Lieu. Lieu. Thr Arg Wall His ASn Ser Ile Arg Arg Glu SO 55 6 O Ile Ala Glin Gly Val Ala Asn Asn His Gly Gly Llys Lieu Pro Ala 65 70 8O Gly Lys Asn. Ile Tyr Arg Met Arg Ser Glu Lieu. Glu Glin Ala 85 90 95 Ala Ile Asp Ala Ser Glin Thr Phe Cys Ser Ala Ser Lieu. Glu Glu Pro 1OO 105 11 O Glin Llys Tyr Gly Glin Asn. Ile Glin Ala Wall Thr Pro Ser Ile Ile 115 12 O 125 Ala Arg Pro Lys Asn Asp Lieu Luell Glu Asp Ala Val Lys Gln Trp Tyr 13 O 135 14 O Lieu Pro Val Ile Tyr Tyr Gly Glin Arg Asp Ala Ala Asn Llys Phe Thr 145 150 155 160 Asp Pro Arg Lieu. Tyr Thr Phe Ala Asn Luell Ala Tyr Asp Lys Asn Thr 1.65 17O 17s Ala Lieu. Gly Cys His Tyr Ala Cys Glin Gly Pro Asp Arg Ile Val 18O 185 19 O Ile Ser Cys Met Tyr Asn Asn Wall Wall Pro Asp Asn Ala Val Ile Tyr 195 2OO 2O5 Glu Pro Gly Thr Ala Cys Val Asp Ala Asp Cys Thr Thr Tyr Pro 21 O 215 22O Gln Ser Thr Cys Lys Asp Ser Luell Ile Ile Pro Thr Pro His Pro 225 23 O 235 24 O Pro Asn. Pro Pro Asn. Pro Pro Pro Ala Met Ser Pro 245 250 <210s, SEQ ID NO 35 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 35 Arg Phe Val Pro Cys Thr Arg Asn 1. 5 <210s, SEQ ID NO 36 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 36 US 7,951,547 B2 - Continued Arg Asp Ala Glu Gly Asn. Cys Ile Llys Phe 1. 5 1O <210s, SEQ ID NO 37 &211s LENGTH: 64 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OO > SEQUENCE: 37 Met Gly Pro Glu Ser Cys Gly Pro Asn Glu Val Trp Thr Glu. Cys Thr 1. 5 1O 15 Gly Cys Glu Lieu Lys Cys Gly Glin Asp Glu Asn Thr Pro Cys Thr Lieu. 2O 25 3O Asn Cys Arg Pro Pro Ser Cys Glu. Cys Ser Pro Gly Arg Gly Met Arg 35 4 O 45 Arg Thr Asn Asp Gly Arg Cys Ile Pro Ala Ser Glin Cys Pro Gln His SO 55 6 O <210s, SEQ ID NO 38 &211s LENGTH: 65 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 38 Met Arg Ala Lys Arg Glu Glu Gln Cys Llys Pro ASn Glu Gln Trp Ser 1. 5 1O 15 Pro Cys Arg Gly Cys Glu Gly Thr Cys Ala Glin Arg Phe Val Pro Cys 2O 25 3O Thir Arg Asn. Cys Arg Pro Pro Gly Cys Glu. Cys Val Ala Gly Ala Gly 35 4 O 45 Phe Val Arg Asp Ala Glu Gly Asn. Cys Ile Llys Phe Asp Asp Cys Pro SO 55 6 O Lys 65 We claim: 45 ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID 1. A method of detecting the presence or absence of one or NO:15, SEQID NO: 16, SEQID NO: 19, SEQID NO: more helminthic antigens in a sample, the method compris 20, SEQID NO: 21, SEQID NO:22, SEQID NO. 23, 1ng: SEQID NO:26, SEQID NO:27, SEQID NO:28, SEQ (a) contacting a sample from a mammal with at least two ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 38, or antibodies selected from the group consisting of: 50 Copro6728; or the first antibody was raised against an (i) a first antibody capable of specifically binding a round extract of whole roundworms, or extract of roundworm worm coproantigen, but not a whipworm or hookworm reproductive organs, or extract of roundworm intestines; coproantigen; (b) the second antibody is capable of specifically binding a (ii) a second antibody capable of specifically binding a 55 polypeptide comprising an amino acid sequence of SEQ whipworm coproantigen, but not a roundworm or hook ID NO:3, SEQID NO: 4, SEQID NO. 5, SEQID NO: worm coproantigen; and 6, SEQID NO: 7, SEQID NO: 8, or SEQID NO: 9; and (iii) a third antibody capable of specifically binding a hook (c) the third antibody is capable of specifically binding a worm coproantigen, but not a whipworm or roundworm polypeptide comprising an amino acid sequence of SEQ coproantigen; 60 ID NO:33 or SEQID NO:34, or CoproASP5. (b) forming antibody-coproantigen complexes in the pres 3. The method of claim 1 wherein the roundworm coproan ence of the coproantigens, if any, in the sample; and tigen, hookworm coproantigen, and/or whipworm coproan (c) detecting the presence or absence of the antibody-co tigen is from a fecal sample. proantigen complexes, if any. 4. The method of claim 1 wherein the first, second and third 2. The method of claim 1 wherein: 65 antibodies are immobilized on a solid Support forming part of (a) the first antibody is capable of specifically binding a an enzyme-linked immunosorbent assay device comprising a polypeptide comprising an amino acid sequence of SEQ lateral flow immunoassay device. US 7,951,547 B2 89 90 5. The method of claim 1 further comprising the step of 11. The method of claim 1 wherein the first antibody is contacting the sample with one or more reagents to detect one capable of specifically binding a polypeptide comprising an or more of the group consisting of one or more non-worm amino acid sequence of SEQID NO:12, SEQIDNO:13, SEQ parasites, heartworm, one or more viruses, one or more fungi, IDNO: 14, SEQID NO:15, SEQIDNO:16, SEQID NO:19, and one or more bacteria. SEQID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID 6. The method of claim 5 wherein the reagents for the NO: 23, SEQID NO: 26, SEQID NO: 27, SEQID NO: 28, detection of any one or all of the one or more non-worm SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 38, or parasites, one or more viruses, one or more fungi and one or Copro6728; or the first antibody was raised againstan extract more bacteria are one or more antibodies or one or more of whole roundworms, or extract of roundworm reproductive antigens recognized by antibodies specific for the one or more 10 non-worm parasites, one or more viruses, one or more fungi organs, or extract of roundworm intestines. or one or more bacteria. 12. The method of claim 1 wherein the second antibody is 7. A method of diagnosing whether a mammal is infected capable of specifically binding a polypeptide comprising an with one or more parasitic worms, the method comprising the amino acid sequence of SEQID NO:3, SEQID NO: 4, SEQ steps of: 15 ID NO:5, SEQID NO: 6, SEQID NO:7, SEQID NO: 8, or (a) contacting a sample from a mammal with at least two SEQID NO:9. antibodies selected from the group consisting of: 13. The method of claim 1 wherein the third antibody is (i) a first antibody capable of specifically binding a capable of specifically binding a polypeptide comprising an roundworm coproantigen, but not a whipworm or amino acid sequence of SEQID NO:33 or SEQID NO:34, or hookworm coproantigen; CoproASP5. (ii) a second antibody capable of specifically binding a 14. The method of claim 1 wherein the sample is obtained whipworm coproantigen, but not a roundworm or from a mammal that is a canine or a feline. hookworm coproantigen; and 15. The method of claim 1 wherein the roundworm is (iii) a third antibody capable of specifically binding a Toxocara Canis or Toxocara Cati, Toxocara vitulorum, Toxas hookworm coproantigen, but not a whipworm or 25 caris leonina, Baylisascaris procyonis, Ascaridia galli, roundworm coproantigen; Parascaris equorum, Ascaris suum, Ascaris lumbricoides, (b) forming antibody-coproantigen complexes in the pres Anisakis simplex, or Pseudoterranova decipiens. ence of the coproantigens, if any, in the sample: 16. The method of claim 1 wherein the whipworm is Tri (c) detecting the presence or absence of the antibody-co churis vulpis, Trichuris campanula, Trichuris Serrata, Tri proantigen complexes, if any; and 30 churis Suis, Trichuris trichiura, Trichuris discolor and Tri (d) diagnosing the mammal as having: chocephalus trichiuris. (i) a roundworm infection if a roundworm antibody 17. The method of claim 1 wherein the hookworm is Ancy coproantigen complex is present; lostoma caninum, Ancylostoma braziliense, Ancylostoma (ii) a whipworm infection if a whipworm antibody-co duodenal, Ancylostoma ceylanicum, Ancylostoma tubae proantigen complex is present; and 35 forme and Ancylostoma pluridentatum, Necator americanus, (iii) a hookworm infection if a hookworm antibody and Uncinaria Stenocephala. coproantigen complex is present. 18. The method of claim 2 wherein the first, second and 8. The method of claim 7 wherein: third antibodies do not specifically bind any coproantigen (a) the first antibody is capable of specifically binding a derived from heartworm. polypeptide comprising an amino acid sequence of SEQ 40 19. The method of claim 1 wherein the step of detecting the ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID presence or absence of the complexes further includes the NO:15, SEQID NO: 16, SEQID NO: 19, SEQID NO: step of providing at least one secondary antibody that binds to 20, SEQID NO: 21, SEQID NO: 22, SEQID NO. 23, at least one of the complexes. SEQID NO:26, SEQID NO:27, SEQID NO:28, SEQ 20. The method of claim 19 wherein the at least one sec ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 38, or 45 ondary antibody is labeled. Copro6728; or the first antibody was raised against an 21. The method of claim 1 wherein one or more of the first, extract of whole roundworms, or extract of roundworm second and third antibodies are labeled. reproductive organs, or extract of roundworm intestines; 22. The method of claim 1 wherein the first, second and (b) the second antibody is capable of specifically binding a third antibodies are immobilized on a solid support. polypeptide comprising an amino acid sequence of SEQ 50 23. The method of claim 22 wherein the solid support ID NO:3, SEQID NO: 4, SEQID NO. 5, SEQID NO: forms part of an enzyme-linked immunosorbent assay device. 6, SEQID NO: 7, SEQID NO: 8, or SEQID NO: 9; and 24. The method of claim 1 further comprising the step of (c) the third antibody is capable of specifically binding a determining the presence or absence of a nucleic acid from polypeptide comprising an amino acid sequence of SEQ the roundworm, whipworm or hookworm. ID NO:33 or SEQID NO:34, or CoproASP5. 55 25. The method of claim 24 wherein the step of determin 9. The method of claim 7 further comprising the step of ing the presence or absence of the nucleic acid is carried out contacting the sample with one or more reagents to detect one by using a polymerase chain reaction (PCR)-based assay. or more of the group consisting of one or more non-worm 26. The method of claim 7 wherein the first antibody is parasites, heartworm, one or more viruses, one or more fungi, capable of specifically binding a polypeptide comprising an and one or more bacteria. 60 amino acid sequence of SEQID NO:12, SEQIDNO:13, SEQ 10. The method of claim 9 wherein the reagents for the IDNO: 14, SEQID NO:15, SEQIDNO:16, SEQID NO:19, detection of any one or all of the one or more non-worm SEQID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID parasites, one or more viruses, one or more fungi and the one NO: 23, SEQID NO: 26, SEQID NO: 27, SEQID NO: 28, or more bacteria are one or more antibodies or one or more SEQ ID NO: 29, SEQ ID NO:30, SEQ ID NO: 38, or antigens recognized by antibodies specific for one or more 65 Copro6728; or the first antibody was raised againstan extract non-worm parasites, one or more viruses, one or more fungi of whole roundworms, or extract of roundworm reproductive or one or more bacteria. organs, or extract of roundworm intestines. US 7,951,547 B2 91 92 27. The method of claim 7 wherein the second antibody is duodenal, Ancylostoma ceylanicum, Ancylostoma tubae capable of specifically binding a polypeptide comprising an forme and Ancylostoma pluridentatum, Necator americanus, amino acid sequence of SEQID NO:3, SEQID NO: 4, SEQ and Uncinaria Stenocephala. ID NO:5, SEQID NO: 6, SEQID NO:7, SEQID NO: 8, or 33. The method of claim 7 wherein the step of detecting the SEQID NO:9. 5 presence or absence of the complexes further includes the 28. The method of claim 7 wherein the third antibody is step of providing at least one secondary antibody that binds to capable of specifically binding a polypeptide comprising an the one or more complexes. amino acid sequence of SEQID NO:33 or SEQID NO:34, or 34. The method of claim 33 wherein the secondary anti CoproASP5. body is labeled. 10 35. The method of claim 7 wherein the first, second and 29. The method of claim 7 wherein the mammal is a canine third antibodies are immobilized on a solid support. or a feline. 36. The method of claim 35 wherein the solid support 30. The method of claim 7 wherein the roundworm is forms part of an enzyme-linked immunosorbent assay device. Toxocara Canis Or Toxocara Cati, Toxocara vitulorum, Toxas 37. The method of claim 36 wherein the enzyme-linked caris leonina, Baylisascaris procyonis, Ascaridia galli, 15 immunosorbent assay device is a lateral flow immunoassay Parascaris equorum, Ascaris suum, Ascaris lumbricoides, device. Anisakis simplex, or Pseudoterranova decipiens. 38. The method of claim 7 further comprising the step of 31. The method of claim 7 wherein the whipworm is Tri determining the presence or absence of a nucleic acid from churis vulpis, Trichuris campanula, Trichuris Serrata, Tri the roundworm, whipworm and hookworm. churis Suis, Trichuris trichiura, Trichuris discolor and Tri 39. The method of claim 38 wherein the step of determin chocephalus trichiuris. ing presence or absence of the nucleic acid is carried out by 32. The method of claim 7 wherein the hookworm is Ancy using a polymerase chain reaction (PCR)-based assay. lostoma caninum, Ancylostoma braziliense, Ancylostoma k k k k k