RU2782463C1 - Methods, devices, kits, and compositions for detection of tapeworms - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular methods, devices, kits, and compositions for detection of the presence or absence of one or more coproantigens of tapeworms in a sample are disclosed. Methods, device, kits, and compositions, according to the invention, can be used for confirmation of the presence or absence of tapeworms in a sample of mammal faeces, as well as they allow for distinguishing of different types of tapeworms in the presence of one or more infections caused by helminths (such as roundworms, hookworms, trichocephals, and ascarids), Giardia, and parvovirus. The confirmation of the presence or absence of a tapeworm in a mammal can be performed, for example, in order to select an optimal course of treatment of a mammal and/or to confirm the destruction of an infection in a mammal after the treatment.

EFFECT: treatment of mammals from infections caused by helminths.

94 cl, 20 dwg, 5 ex

Description

cross reference

[001] This application claims priority to U.S. Provisional Application No. 62/740100, filed October 2, 2018, 62/741849, filed October 5, 2018; and 62/746805, filed October 17, 2018, which are incorporated herein by reference in their entirety.

Prerequisites for the invention

1. Scope of invention

[002] The present invention relates to compositions, devices, kits and methods for detecting and identifying species of tapeworms in mammals. More specifically, the present invention relates to antibodies and antibody compositions, devices, kits and methods for detecting the presence or absence of certain species of tapeworms in a sample taken from a mammal and for distinguishing between tapeworm antigens.

2. Description of the prior art

[003] Infections caused by parasitic worms are very common in animals and if not diagnosed and treated can lead to serious illness or death in animals. Current methods for diagnosing infections caused by parasitic worms mainly include the examination of faecal samples under a microscope, either directly in faecal smears or after concentration of eggs and parasites by solid media flotation or sedimentation. Despite the widespread use of this method, this method has significant drawbacks. Such methods of examination under a microscope require a lot of time and special equipment. In addition, the accuracy of the results of these methods is highly dependent on the skill and experience of the specialist. For example, the presence of tapeworms is determined by looking for eggs or proglottids, but they are excreted at intervals and in small quantities. Therefore, it is not surprising that tapeworm infection is often not diagnosed by routine faecal analysis.

[004] Dealing with faeces is unpleasant and dangerous. Sanitary procedures for handling faeces are quite inconvenient and often very difficult. Such procedures may involve weighing, centrifugation and storage and are quite difficult unless they are carried out in a clinical laboratory equipped with suitable instruments, protective equipment and experienced personnel. Therefore, any reduction in the number of steps is necessary to perform faecal analysis, and any reduction in contact between the qualified person performing the analysis and the material being tested is desirable. In clinical laboratories, immunoassay methods are used to detect various viruses, bacteria and non-helminthic parasites and organisms in feces. However, the need to develop a simple immunoassay method for detecting parasitic worm infection in faeces, whole blood or serum remains relevant.

The essence of the invention

[005] In one aspect, the present invention provides an isolated antibody that can specifically bind to a tapeworm coproantigen, such as a Taenia coproantigen, e.g., a Taenia pisiformis coproantigen, or a Taenia Taeniaeformis coproantigen , or a Dipylidium caninum coproantigen. The antibody may be detectably labeled or attached to a solid support.

[006] In one embodiment, the isolated antibody specifically binds to a Taenia pisiformis coproantigen. The antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia taeniaeformis tapeworm, Dipylidium tapeworm, hookworm (e.g. Ancylostoma ), roundworms ( Toxocara ), trichocephalus (e.g. Trichuris ), Giardia and parvovirus, where the Dipylidium tapeworm is Dipylidium caninum ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus.

[007] In another embodiment, the isolated antibody specifically binds to a Taenia taeniaeformis coproantigen . The antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia pisiformis tapeworm, Dipylidium tapeworm, hookworm (e.g. Ancylostoma ), roundworms ( Toxocara ), trichocephalus (e.g. Trichuris ), Giardia and parvovirus, where the Dipylidium tapeworm is Dipylidium caninum ; hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus.

[008] In other embodiments, the isolated antibody specifically binds to a Dipylidium caninum coproantigen. The antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia tapeworm, Dipylidium tapeworm, hookworm (e.g. Ancylostoma ), roundworms ( Toxocara ), trichocephalus (e.g. Trichuris ) Giardia , and parvovirus where the Taenia tapeworm is Taenia pisiformis or Taenia taeniaeformis ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus.

[009] In another aspect, the present invention provides an immunocomplex comprising a tapeworm coproantigen and one or more antibodies specifically associated with the tapeworm coproantigen. In one embodiment, the tapeworm coproantigen may be a coproantigen derived from Taenia pisiformis , Taenia taeniaeformis, Taenia crassiceps, or Dipylidium caninum . In another embodiment, the antibody is produced by immunization with whole tapeworm extract, tapeworm E/S product, tapeworm-derived wash, or tapeworm TCA solute. In another embodiment of the invention, the antibody specifically binds to a tapeworm coproantigen in a sample, such as a fecal sample obtained from a mammal infected with tapeworms. In some embodiments, the mammal is additionally infected with one or more worms, such as roundworms, trichocephali, and hookworms, and the antibody does not specifically bind to any antigen derived from one or more worms, such as roundworms, trichocephali, or hookworms, which may be present in the sample. In some embodiments of the invention, the antibody may be detectably labeled or associated with a solid carrier.

[010] In another aspect, the present invention provides an immunocomplex comprising a tapeworm coproantigen, an antibody specifically associated with a tapeworm coproantigen, and a lectin associated with a tapeworm coproantigen. A lectin may specifically bind to one particular type of carbohydrate or may bind to two or more carbohydrates on a tapeworm coproantigen.

[011] In another aspect, the present invention relates to a device for specific binding and isolation of helminth antigens from a sample, for example, coproantigens from a fecal sample, where such a device contains a solid carrier, where the specified solid carrier has one or more antibodies immobilized on it, selected from the group consisting of (a) a first antibody capable of specifically binding to a first tapeworm coproantigen but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen. Such a device may be, for example, but not limited to, an ELISA device such as a sidestream immunoassay device or a microtiter plate. Samples that can be tested for the presence of tapeworms using such a device include, but are not limited to, feces, gastrointestinal mucosa, urine, whole blood, serum, breast milk, and intact tissue such as, for example, breast tissue. gland, small intestine, liver, heart, lungs, esophagus, brain, muscles, and eyes. In addition, the device may also include, but not necessarily, one or more reagents for the detection of one or more groups, consisting of: one or more parasitic helminths; non-parasites, one or more protozoa, and one or more bacteria. In some embodiments, the solid support also has one or more antibodies selected from: (i) an antibody capable of specifically binding to a coproantigen of roundworms, but not to a coproantigen derived from the group consisting of trichocephalus, hookworm, tapeworm, immobilized thereon. worm, Giardia and parvovirus; (ii) an antibody capable of specifically binding to a trichocephalic coproantigen, but not to a coproantigen derived from the group consisting of roundworms, hookworm, tapeworms, Giardia , and parvovirus; (iii) an antibody capable of specifically binding to the hookworm coproantigen but not to the coproantigen derived from the group consisting of trichocephalus, roundworms, tapeworms, Giardia and parvovirus; (iv) an antibody capable of specifically binding to a Giardia copro antigen, but not to a copro antigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and parvovirus coproantigen; and (v) an antibody capable of specifically binding to a parvovirus coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and Giardia coproantigen. In some embodiments, the hookworm is Ancylostoma, the roundworm is Toxocara , and the trichocephalus is Trichuris . In other embodiments, the hookworms are Ancylostoma caninum or Ancylosloma tubaeforme ; roundworms are Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia ; and parvovirus is a feline or canine parvovirus.

[012] In some embodiments, the solid support has two or more antibodies immobilized thereon, three or more antibodies, four or more antibodies, five or more antibodies, six or more antibodies, seven or more antibodies, or eight or more antibodies for multiplex reaction.

[013] In another aspect, the present invention relates to a device for detecting the presence or absence of one or more tapeworm coproantigens in a fecal sample; wherein said device comprises a solid carrier, a lectin, and one or more antibodies selected from the group consisting of (a) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen, wherein the solid support has one or more lectins or one or more antibodies immobilized thereon. In some embodiments of the invention, the lectin is immobilized on a solid support. In other embodiments of the invention, the first, second and third antibodies are immobilized on a solid support. In other embodiments, the device also comprises one or more species of tapeworm antigens, wherein the one or more species of tapeworm antigens are specifically associated with antibodies. In some embodiments, the first tapeworm is Taenia pisiformis, the second tapeworm is Taenia taeniaeformis , and/or the third tapeworm is Dipylidium caninum .

[014] In yet another aspect, the present invention relates to a method for detecting the presence or absence of one or more helminth antigens in a sample, for example, coproantigens in a fecal sample, where this method includes: (a) contacting a mammalian sample with one or more antibodies, selected from the group consisting of: (i) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen, or to a third tapeworm coproantigen; (ii) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (iii) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen; (b) formation of antibody-coproantigen complexes in the presence of coproantigens, if present in the sample; and (c) detecting the presence or absence of antibody-coproantigen complexes, if any. Tapeworm coproantigens may include coproantigens of Taenia species, eg Taenia pisiformis and Taenia taeniaeformis ; and Dipylidium species, such as Dipylidium caninum . In one embodiment of the invention, the step of detecting the presence or absence of complexes also includes the step of obtaining a lectin that binds to at least one of the complexes. In some embodiments of the invention, the lectin can be detectably labeled or immobilized on a solid support. In other embodiments, the first, second, and third antibodies are detectably labeled or immobilized on a solid support. In some embodiments, the first, second, and third antibodies may be immobilized on a solid support and the lectin may be detectably labeled. In other embodiments of the invention, the first, second and third antibodies can be detectably labeled and the lectin can be immobilized on a solid support.

[015] In another embodiment, the method, prior to the step of contacting the mammalian fecal sample with at least one antibody, further comprises the step of contacting the fecal sample with one or more lectins. One or more lectins may be detectably labeled or immobilized on a solid support. Alternatively, the first, second, and third antibodies may be detectably labeled or immobilized on a solid support. In one embodiment of the invention, the first, second, and third antibodies may be immobilized on a solid support, and one or more lectins may be detectably labeled. In another embodiment of the invention, the first, second and third antibodies can be detectably labeled and one or more lectins can be immobilized on a solid support.

[016] In one aspect of the invention, the method is carried out to test a mammalian fecal sample for tapeworm coproantigen. However, this method is not limited to analyzing a fecal sample. Therefore, in addition to faeces, the sample may be, but is not limited to, for example, whole blood, serum, breast milk, and intact tissue such as, for example, breast tissue, small intestine, liver, heart, lung, esophagus, brain brain, muscles and eyes.

[017] In another aspect, the present invention relates to a method for diagnosing infection of a mammal with one or more parasitic worms, where the method includes the steps of: (a) contacting a sample of a mammal with one or more antibodies selected from the group consisting of: (i a) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen, or to a third tapeworm coproantigen; (ii) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (iii) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen; (b) forming antibody-coproantigen complexes in the presence of coproantigens, if present in the sample; (c) detecting the presence or absence of antibody-coproantigen complexes, if any; and (d) diagnosing in the mammal (i) infection with a first tapeworm if an antibody-coproantigen complex against the first tapeworm is present, (ii) infection with a second tapeworm if an antibody-coproantigen complex against the second tapeworm is present, and (iii) infection with a third tapeworm if an anti-third tapeworm antibody-coproantigen complex is present. In one embodiment, two or more antibodies, three or more antibodies, four or more antibodies, five or more antibodies, six or more antibodies, seven or more antibodies, or eight or more antibodies are selected. In some embodiments of the invention, the step of detecting the presence or absence of complexes also includes the step of obtaining one or more lectins that bind to at least one of the complexes. In some embodiments of the invention, the lectin can be detectably labeled or immobilized on a solid support. In other embodiments, the first, second, and third antibodies are detectably labeled or immobilized on a solid support. In some embodiments, the first, second, and third antibodies may be immobilized on a solid support and the lectin may be detectably labeled. In other embodiments of the invention, the first, second and third antibodies can be detectably labeled and the lectin can be immobilized on a solid support.

[018] In another embodiment, the method, before the step of contacting the mammalian fecal sample with at least one antibody, further comprises the step of contacting the fecal sample with one or more lectins. The lectin may be detectably labeled or immobilized on a solid support. Alternatively, the first, second, and third antibodies may be detectably labeled or immobilized on a solid support. In one embodiment of the invention, the first, second and third antibodies can be immobilized on a solid support and the lectin can be detectably labeled. In another embodiment of the invention, the first, second and third antibodies can be detectably labeled and the lectin can be immobilized on a solid support.

[019] In yet another aspect, the present invention provides a method for diagnosing and treating a mammal infected with one or more parasitic worms, wherein the method comprises the steps of: (a) contacting a sample of the mammal with one or more antibodies selected from the group consisting of : (i) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (ii) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (iii) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen; (b) forming antibody-coproantigen complexes in the presence of coproantigens, if present in the sample; (c) detecting the presence or absence of antibody-coproantigen complexes, if any; and (d) diagnosing in the mammal (i) infection with a first tapeworm if an antibody-coproantigen complex against the first tapeworm is present, (ii) infection with a second tapeworm if an antibody-coproantigen complex against the second tapeworm is present, and (iii) infection with a third tapeworm if an anti-third tapeworm antibody-coproantigen complex is present; and (e) administering an effective amount of one or more therapeutic agents to treat a mammal infected with the first, second or third tapeworm, or a combination thereof. In one embodiment of the invention, two or more antibodies are selected. In other embodiments, step (e) further comprises administering one or more additional therapeutic agents for treating an infection caused by one or more parasitic helminths, one or more non-worm parasites, one or more viruses, one or more fungi, one or more protozoa or one or more bacteria. In other embodiments, step (e) further comprises administering one or more therapeutic agents to eradicate, repel, or kill the intermediate host of the flatworm parasite, parasitic helminth, non-worm parasites, virus, fungus, or bacteria. Representative examples of intermediate hosts include fleas and dog lice, which can carry tapeworm eggs.

[020] In other embodiments of the methods described above, the antibody group of step (a) further consists of: (i) an antibody capable of specifically binding to a coproantigen of roundworms, but not to a coproantigen derived from the group consisting of trichocephalus, hookworm, tapeworm, Giardia and parvovirus; (ii) an antibody capable of specifically binding to a trichocephalic coproantigen, but not to a coproantigen derived from the group consisting of roundworms, hookworm, tapeworms, Giardia , and parvovirus; (iii) an antibody capable of specifically binding to the hookworm coproantigen but not to the coproantigen derived from the group consisting of trichocephalus, roundworms, tapeworms, Giardia and parvovirus; (iv) an antibody capable of specifically binding to a Giardia ' coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and parvovirus coproantigen; and (v) an antibody capable of specifically binding to a parvovirus coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and Giardia coproantigen. In some embodiments, the hookworm is Ancylostoma, the roundworm is Toxocara , and the trichocephalus is Trichuris . In other embodiments, the hookworms are Ancylostoma caninum or Ancylosloma tubaeforme ; roundworms are Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia ; and parvovirus is feline parvovirus or canine parvovirus. Thus, a fecal sample is contacted with two or more antibodies, three or more antibodies, four or more antibodies, five or more antibodies, six or more antibodies, seven or more antibodies, or eight or more antibodies to provide a multiplex response.

[021] In another embodiment of the methods described above, step (d) also includes diagnosing roundworm infection by the presence of an anti-roundworm antibody-coproantigen complex; infection with trichocephalus by the presence of an antibody-coproantigen complex against trichocephalus; infection with hookworm by the presence of an antibody-coproantigen complex against hookworm; infection with Giardia by the presence of an anti- Giardia antibody-coproantigen complex; and infection with parvovirus by the presence of an anti-parvovirus antibody-coproantigen complex.

[022] In some embodiments of the invention, the step of detecting the presence or absence of complexes further includes the step of obtaining one or more lectins that bind to at least one of the complexes. In some embodiments of the invention, the lectin may be detectably labeled or immobilized on a solid support. In other embodiments, the first, second, and third antibodies are detectably labeled or immobilized on a solid support. In some embodiments, the first, second, and third antibodies are detectably labeled or immobilized on a solid support. In some embodiments, the first, second, and third antibodies may be immobilized on a solid support and the lectin may be detectably labeled. In other embodiments of the invention, the first, second and third antibodies can be detectably labeled and the lectin can be immobilized on a solid support.

[023] The method can also be used to analyze tapeworms in the environment and identify their species. Environmental samples that can be tested for tapeworms with the device include, but are not limited to, soil, decaying material, or faeces from areas where animals are present, including yards, gardens, sandboxes, playgrounds. Testing sites may also include parks, beaches, forests, farms, or other sites contaminated with the faeces of dogs, cats, or other mammalian hosts of tapeworms. Faeces in manure boxes inside and outside can also be tested.

[024] In yet another aspect, the present invention includes a kit for carrying out one or more steps of the method according to the invention. The kit may, but does not have to, include, for example, a device and one or more compositions according to the invention and instructions for carrying out the method according to the invention. The kit may additionally, but not necessarily, include, for example, one or more indicator reagents, one or more antibody labeling compounds, one or more antibodies, one or more antigen capture reagents, one or more inhibitors, and one or more wash reagents that are used as part of a device and/or to carry out such a method.

Brief description of the drawings

[025] In FIG. 1 shows the OD determinations for the ADX131 antibody against worm extract and dog fecal extract that are negative or positive for tapeworm infection according to the method of the invention as shown in Example 1A. Positive=fecal extract (FEX) from dogs infected with T. pisiformis ; negative=FEX of dogs not infected with T. pisiformis ; worm extract= T. pisiformis worm extract (WE).

[026] In FIG. 2 shows the OD determinations for the ADX132 antibody against worm extract and dog fecal extract that are negative or positive for tapeworm infection according to the method of the invention as shown in Example 1A. Positive=fecal extract (FEX) from dogs infected with T. pisiformis ; negative=FEX of dogs not infected with T. pisiformis ; worm extract= T. pisiformis worm extract (WE).

[027] In FIG. 3 shows a microtiter plate in which an ELISA assay is performed using faecal extracts from tapeworm-infected dogs according to the method of the invention as shown in Example 1B. H11 is the negative control. A5, B6, B8, E6 and E10 are fecal extracts from five T. pisiformis positive dogs. Each well contains a faecal extract from dogs infected with hookworm Ancylostoma caninum or dogs infected with roundworms Toxocara canis or dogs infected with Trichuris vulpis or dogs infected with D. caninum .

[028] In FIG. 4 shows the specificity of a coproantigen sandwich ELISA performed using an ADX131 antibody coated on a microtiter plate and an ADX132-HRP conjugate applied after addition of a patient sample. The analysis was performed on WE D. caninum , WE T. pisiformis , WE T. crassiceps , WE T. taeniaeformis , E/S T. taeniaeformis , as well as on FEX dogs positive for D. caninum , dogs negative for D. caninum , D. caninum positive cats D. caninum negative cats T. pisiformis positive dogs T. pisiformis negative dogs Group of three T. taeniaeformis positive cats Group of three T. taeniaeformis negative cats T. taeniaeformis , hookworm WE Ancylostoma caninum , roundworm WE Toxocara canis , and trichocephalus WE Trichuris vulpis , as discussed in Example 1B.

[029] In FIG. 5 shows the sensitivity of a coproantigen sandwich ELISA performed using the ADX185 antibody coated on a microtiter plate, followed by the addition of a patient sample, followed by the addition of T. taeniaeformis rabbit anti-WE pAb -HRP conjugate. The analysis was performed on FEX 7 cats positive for T. taeniaeformis and 3 cats negative for T. taeniaeformis, as discussed in Example 2B (part B).

[030] In FIG. 6 shows the specificity of the coproantigen sandwich ELISA performed using the ADX184 antibody coated on a microtiter plate and the ADX193-HRP conjugate applied after the addition of a patient sample. ADX193 is a mouse anti-E/S T. taeniaeformis mAb described below. The analysis was performed on WE D. caninum , WE T. pisiformis , WE T. crassiceps , WE T. taeniaeformis , E/S T. taeniaeformis , as well as on faecal extracts of dogs positive for D. caninum , dogs negative for D. caninum , D. caninum positive cats, D. caninum negative cats, T. pisiformis positive dogs, Taenia pisiformis negative dogs, group of three cats positive for T. taeniaeformis and group of three cats negative for T. taeniaeformis as discussed in Example 2B (Part B).

[031] In FIG. 7 shows the results of an ADX184/ADX193-HRP ELISA performed on fecal extracts from six T. taeniaeformis positive cats; fecal extracts from four cats negative for T. taeniaeformis ; and one sample of T. taeniaeformis E/S protein as discussed in Example 2C (Part A).

[032] In FIG. 8 shows the results of a coproantigen ELISA using ADX191 antibody coated on microtiter plates and ADX194 conjugated to HRP. Both mouse mAbs were used at a concentration of 3 μg/ml. This ELISA was performed on faecal extracts from six T. taeniaeformis positive cats; fecal extracts from four cats negative for T. taeniaeformis ; and one sample of T. taeniaeformis E/S protein as discussed in Example 2D.

[033] In FIG. 9 shows the results of an ELISA assay using rabbit anti-WE D. caninum pAb coated on plates and then contacted with FEX at 5 μg/mL, contacted with rabbit anti-WE D. caninum pAb-HRP at 3 μg/mL, and contact with the color substrate. ELISA analysis using a rabbit pAb against WE D. caninum was performed on worm extracts isolated from several helminth species: WE D. caninum , WE Taenia pisiformis , WE T. crassiceps , WE hookworm Ancylostoma caninum , WE roundworm Toxocara canis and WE trichocephalus Trichuris vulpis as discussed in Example 3B (Part A).

[034] In FIG. 10 shows the results of a coproantigen ELISA where mouse anti-WE D. caninum pAb was plated and then subjected to FEX contact, mouse anti-WE D. caninum pAb-HRP conjugate and color substrate contact as discussed in Example 3E. (part A). The assay was performed on fecal extracts from 4 D. caninum positive dogs and 3 D. caninum positive cats; one D. caninum negative cat infected with Giardia and T. taeniaeformis, one D. caninum negative cat infected with T. taeniaeformis, and one D. caninum negative cat infected with Toxocara canis and Taenia pisiformis , as discussed in Example 3E (part A).

[035] In FIG. 11 shows the results of the first coproantigen ELISA using a mouse ADX226 mAb plated and then contacted with FEX, with a mouse ADX251-HRP mAb conjugate, and with a color substrate. The assay was performed on fecal extracts from 3 D. caninum positive dogs; 5 dogs negative for D. caninum ; 3 cats positive for D. caninum and 1 cat negative for D. caninum as discussed in Example 3F (Part A).

[036] In FIG. 12 shows the results of a second coproantigen ELISA using mouse ADX251 mAb plated and then contacted with FEX, mouse mAb ADX227-HRP conjugate, and color substrate. The assay was performed on fecal extracts from 3 D. caninum positive dogs; 5 dogs negative for D. caninum ; 3 D. caninum positive cats and 1 D. caninum negative cat, as discussed in Example 3F (Part A).

[037] In FIG. 13 shows the results of a third coproantigen ELISA using mouse mAb ADX251 plated and then contacted with FEX with rabbit anti-WE D. caninum pAb-HRP conjugate and color substrate. The assay was performed on fecal extracts from 3 D. caninum positive dogs; 5 dogs negative for D. caninum ; 3 D. caninum positive cats and 1 D. caninum negative cat, as discussed in Example 3F (Part A).

[038] In FIG. 14 shows the results of the first series of coproantigen ELISAs where mouse anti-WE D. caninum mAbs ADX224, ADX225, ADX226, and ADX227 were individually plated and then contacted with FEX, with a rabbit anti-WE D. caninum pAb-HRP conjugate. and with a color substrate as discussed in Example 3G (Part A).

[039] In FIG. 15 shows the results of a second series of coproantigen ELISA assays where mouse anti-WE D. caninum mAbs ADX226 and ADX227 were separately plated and then contacted with FEX, mouse anti-WE D. caninum mAb ADX227-HRP conjugate and color substrate as discussed in Example 3G (Part A).

[040] In FIG. 16 shows the results of glycosylation analysis of coproantigen associated with mouse anti-WE D. caninum mAb ADX226 by evaluating the ability of 21 different lectins to bind to coproantigen using a commercially available kit (biotinylated lectin kits I, II and III from Vector Laboratories, Burlingame, CA). ) as discussed in Example 3H (Part 1).

[041] In FIG. 17 shows the results of glycosylation analysis of coproantigen associated with D. caninum rabbit anti-WE pAb by evaluating the ability of 21 different lectins to bind to coproantigen using a commercially available kit (biotinylated lectin kits I, II and III from Vector Laboratories, Burlingame, CA) , as discussed in Example 3H (Part 2).

[042] In FIG. 18 shows the results of a glycosylation assay of coproantigen(s) associated with rabbit anti-WE D. caninum and ADX187 pAb (with mouse anti-WE D. caninum mAb) by evaluating the ability of 21 different lectins to bind to coproantigen using a commercially available kit ( biotinylated lectin kits I, II and III from Vector Laboratories, Burlingame, CA) as discussed in Example 3H (part 3).

[043] In FIG. 19 shows microtiter plates in which a series of ELISA assays for T. pisiformis , T. taeniaeformis , D. caninum , hookworms, roundworms, trichocephalus, and Giardia were performed using faecal extracts from a number of infected dogs and cats as discussed in Example 4. B Fecal extracts from the following sources were tested in ELISA assays: dogs positive for T. pisiformis (Fig. 19, column 1), cats infected with T. taeniaeformis (Fig. 19, column 2), dogs infected with D. caninum ( Fig. 19, column 3), cats infected with D. caninum (Fig. 19, column 4), dogs infected with hookworm A. caninum (Fig. 19, column 5), cats infected with hookworm A. tubaeforme (Fig. 19, column 6), dogs infected with T. canis roundworms (Fig. 19, column 7), cats infected with T. cati roundworms (Fig. 19, column 8), dogs infected with T. vulpis trichocephalus (Fig. 19, column 9), cats infected with trichocephalus T. felis (Fig. 19, hundred panel 10), Giardia -infected dogs (Fig. 19, column 11), cats infected with Giardia (Fig. 19, column 12), and two cats infected with parvovirus (Fig. 19, columns 13 and 14). In FIG. 19, columns 1-12 show an image of one microtiter plate, and columns 13 and 14 show an image of another individual microtiter plate.

[044] In FIG. 20 shows microtiter plates in which a series of ELISA assays were performed to detect two or three species of tapeworms of the genus Taenia , e.g. T. pisiformis, T. taeniaeformis , and the genus Dipylidium , e.g. infected dogs and cats as discussed in Example 5. ELISA analyzes were performed on fecal extracts from the following sources: four dogs positive for T. pisiformis (Fig. 20, columns 1, 4, 7 and 10), four cats infected with T. taeniaeformis (Fig. 20, columns 2, 5, 8, and 11), two D. caninum -infected dogs (Fig. 20, columns 3 and 6), two D. caninum -infected cats (Fig. 20, columns 9 and 12). In FIG. 20, columns 1-12 show an image of one microtiter plate.

Detailed description of the preferred embodiments of the invention

I Introduction

[045] The present invention generally relates to methods, devices and kits for detecting and identifying tapeworm species in a fecal sample taken from a mammal. The present invention relates to tapeworm coproantigens of a variety of species, including Taenia pisiformis , Taenia taeniaeformis , and Dipylidium caninum . In particular, the present invention relates to methods, devices and kits for detecting tapeworm infestation and identifying two or more species of tapeworm.

[046] The present invention provides an excellent alternative to existing microscopic examination methods. Indeed, the present invention relates to devices, kits and methods for detecting the presence or absence of tapeworms in a mammalian sample, and these devices, kits and methods: (1) are simple to use and give reasonably reliable results; (2) allow confirmation of the absence or presence of specific species of tapeworms in a mammal, whether or not the mammal is infected with one or more species of tapeworms and/or other parasitic helminths such as hookworms, roundworms, trichocephali and/or roundworms, ( 3) allow the detection of tapeworms even before the appearance of eggs and proglottids in the faeces of an infected host; and (4) distinguish between different types of tapeworms as well as parasitic helminths such as roundworms, trichocephali and hookworms.

[047] The present invention is based in part on the unexpected discovery of properties of the compositions specific to infections caused by tapeworms. In particular, it has been found that antibodies raised against specific tapeworm polypeptides (or raised against whole tapeworm extract, tapeworm E/S product, tapeworm-derived wash, or tapeworm TCA solute) can be used to capture, detection and identification of antigens of tapeworms of various species in a mammal. The discovery that tapeworms of each species could be specifically detected was unexpected because it was assumed that tapeworms were related cestodes and that an antibody raised against a protein isolated from any tapeworm species would cross-react with one or more tapeworms. worms of other species, with host antigens, or with other host components.

[048] In addition, it was found that this antibody can be used to capture and detect tapeworm antigens in a mammal even before the eggs and proglottids are visible in the feces. This ability to detect tapeworms shortly after infection and before any eggs appear in the feces of an infected mammal is highly unexpected, since eggs and proglottids do not usually appear in the feces of an infectious host until several weeks after infection of the host.

[049] Thus, the present invention includes methods, devices, and kits that use antibodies and/or fragments thereof to specifically capture, detect, and identify differences between antigens of different tapeworm species in a mammal. The ability of the present invention to identify and diagnose specific types of tapeworms, even if one or more other types of tapeworms or other types of worms are present, provides the caregiver of the mammal with an optimal choice of treatment to eradicate tapeworms, as well as other worms, such as roundworms, trichocephali and/or hookworms in a mammal. In addition, in some cases, the present invention allows the detection of tapeworms before the appearance of eggs and proglottids in the faeces, which allows the animal caregiver to begin such treatment before the appearance of a serious disease in the mammal. Treatment given before the appearance of eggs and proglottids in the faeces will also significantly reduce or eliminate the chance of spreading infection to other animals or humans.

II. Definition and use of terms

[050] The term “compositions of the invention” refers to all nucleic acids, polypeptides, glycoproteins, carbohydrates, glycolipids, antibodies, and mixtures that include one or more of these nucleic acids, polypeptides, glycoproteins, carbohydrates, glycolipids, and antibodies, and one or more than other compounds that can be used to detect the presence or absence of tapeworms in a sample taken from a mammal, by carrying out the method according to the invention, which is described in detail, by implication or in any other way in the present description.

[051] A “mammalian sample” in which tapeworms according to the invention can be detected includes all components of a living organism and their extracts, such as any liquid, solid, cell or tissue that can have a tapeworm antigen. Therefore, examples of samples include, but are not limited to, faeces, milk, whole blood and parts thereof, including serum, as well as, for example, tissue extracts, including tissue from the breast, intestines, liver, heart, lung, esophagus, brain, muscles and eyes. The sample may be taken directly from the mammal, or it may be taken from any individual who has been in contact with the mammal. For example, the sample may be fresh or rotting feces from a mammal. As another example, the sample may include soil, mud, sand, plant material, or any other material that may be mixed with body components that may be present in a mammal, such as feces. For example, the sample may be taken from an environmental source, including soil, decaying material, or faeces in forests, farms, or areas where animals are kept, including litter boxes, yards, gardens, sandboxes, playgrounds, parks, and beaches. . Regardless of the origin or content of the sample, the sample is sometimes referred to herein as a "sample", "sample taken from a mammal", "test sample" or "test sample".

[052] As used herein, the term "nucleic acid" is synonymous with the terms "gene", "DNA", "cDNA", "EST", "polynucleotide", "oligonucleotide", "polynucleic acid", "RNA", and "mRNA". The nucleic acid may be present in double stranded form or in single stranded form. In addition, the nucleic acid is either naturally isolated, e.g., isolated from the whole or part of a tapeworm, or it can be artificially synthesized in a recombinant host organism or by any other artificial methods known to those skilled in the art, such as the method on based on PCR, carried out by creating a transgenic organism that synthesizes a nucleic acid, using a device for DNA synthesis, or, for example, by any other molecular method.

[053] As used herein, the terms "polypeptide", "peptide", and "protein" are synonymous as used herein to refer to a polymer of amino acid residues. The polypeptide, peptide and protein of the invention may be naturally isolated, for example isolated from the whole or part of a tapeworm, or they may be artificially synthesized in a recombinant host or by any other artificial means known to those skilled in the art. The polypeptide, peptide and protein of the invention may be glycosylated.

[054] the Term "antibody" or "antibody according to the invention" means any antibody that is able to specifically bind to one or more antigens of a particular worm, but not bind to the antigens of other worms. Thus, for example, antibodies to a tapeworm of one species can specifically bind to antigens of tapeworms of other species, but not to any antigens of tapeworms of different species. Antibodies of the invention may be directed against one or more immunogenic polypeptides, glycoproteins, carbohydrates or glycolipids of the invention. Unless otherwise stated, it should be noted that an antibody of the invention may include a mixture of two or more antibodies of different types. For example, an antibody may be a mixture of two types of antibodies, where one antibody of the two types specifically binds to one particular antigen, and the other antibody of the two types specifically binds to some other antigens.

[055] The term "first antibody" as used herein refers to one or more antibodies capable of specifically binding to a first species tapeworm coproantigen, but not to a second species tapeworm coproantigen or a third species tapeworm coproantigen.

[056] As used herein, the term "second antibody" refers to one or more antibodies capable of specifically binding to a second species tapeworm coproantigen, but not a first species tapeworm coproantigen or a third species tapeworm coproantigen.

[057] The term "third antibody" as used herein refers to one or more antibodies capable of specifically binding to a third species tapeworm coproantigen, but not to a first species tapeworm coproantigen or a second species tapeworm coproantigen.

[058] "Immunogenic polypeptide according to the invention" and, more simply, "polypeptide according to the invention" is an immunogen against which antibodies according to the invention can be produced. All "polypeptides of the invention" are immunogenic and therefore can be used to induce an immune response in a host animal to produce antibodies of the invention. Unless otherwise stated, it should be noted that the polypeptide according to the invention may be a single component of a mixed composition of multiple components.

[059] "Immunogen" is any agent, such as an immunogenic extract, polypeptide, glycoprotein, carbohydrate, or glycolipid of the invention, which, for example, is capable of inducing an immune response in an animal exposed to the agent.

[060] As used herein, the term "tapeworm" means flat parasitic helminths such as intestinal tapeworms, which include worms of the genus Taenia and Dipylidium . Thus, the term "tapeworm" as used here does not refer to the entire class of cestodes, but rather it refers to a subclass of eucestodes, including the order pseudophyllidea . Representative examples of tapeworms include Taenia pisiformis, Taenia taeniaeformis, Taenia crassiceps, Dipylidium caninum, Diphyllorobothrium mansonogide, Diphyllorobothrium latum , and Spirometra erinaceieuropaei .

[061] A "tapeworm coproantigen" or "tapeworm-derived coproantigen" is any tapeworm product that is present in the faeces of a mammal infected with tapeworms and that can specifically bind to one or more antibodies of the invention. For example, a tapeworm coproantigen may be, but is not limited to, one or more of the polypeptides of the invention.

[062] As used herein, the term "roundworms" refers to helminths such as intestinal roundworms of the order Ascaris, which include the genus Toxocara , Toxascaris , Baylisascaris , Ascaridia, Parascaris , Ascaris , Anisakis , and Pseudoterranova . Therefore, examples of such roundworms are Toxocara canis, Toxocara cati , and Toxascaris leonina . Thus, the term "roundworms" used here does not refer to the entire nematode species. Therefore, roundworms do not include any member of the genus Ancylostoma, Uncinaria, Necator, Trichuris, Wuchereria, Brugia, or Dirofilaria.

[063] A "roundworm coproantigen" or "roundworm-derived coproantigen" is any roundworm product that is present in the faeces of a mammal infected with roundworms and that can specifically bind to one or more antibodies of the invention. For example, a roundworm coproantigen can be, but is not limited to, the novel 7 kDa C-terminal isoform of DIV6728, which is a T. canis excretory/secretory protein that is present in the faeces of T. canis- infected dogs as early as 38 days. after dogs were first infected with T. canis . Thus, the "roundworm coproantigen" may be the novel 7 kDa C-terminal isoform of DIV6728 (referred to here as "Copro6728") that has been found in dog feces, as discussed in US Patent No. 7951547.

[064] As used herein, the term "trichocephalus" refers to helminths such as intestinal trichocephali of the genus Trichuris and Trichocephalus . Therefore, examples of trichocephalians are Trichuris vulpis, Trichuris campanula, Trichuris serrata, Trichuris felis, Trichuris suis , Trichuris trichiura, Trichuris discolor , and Trichocephalus trichiuris . In addition, the term "trichocephalus" as used herein does not refer to the entire species of nematodes. Thus, for example, trichocephalus does not include any member of the genus Ancylostoma , Uncinaria, Necator, Toxocara , Toxascaris , Ascaris, Wuchereria, Brugia, or Dirofilaria .

[065] A "Trichocephalic coproantigen" or "Trichocephalic coproantigen" is any Trichocephalic product that is present in the faeces of a mammal infected with Trichocephalus and that can specifically bind to one or more antibodies of the invention. Thus, for example, a trichocephalic coproantigen may be, but is not limited to, "DIV6901" or "DIV6902", and hereinafter, this particular antibody will be referred to as "anti-DIV6901 antibody" or "anti-DIV6902 antibody" as discussed in the patent USA No. 7951547.

[066] The term "hookworm" as used herein refers to helminths such as the intestinal hookworm of the genus Ancylostoma, Necator and Uncinaria . Examples of hookworms are Ancylostoma caninum, Ancylostoma braziliense, Ancylostoma duodenal, Ancylostoma ceylanicum, Ancylostoma tubaeforme and Ancylostoma pluridentatum, Necator americanus and Uncinaria stenocephala . In addition, the term "hookworm" as used herein does not refer to the entire species of nematode. Thus, for example, the term "hookworm" does not include any member of the genus Trichuris, Trichocephalus Toxocara, Toxascaris, Ascaris, Wuchereria, Brugia, or Dirofilaria .

[067] "Hookworm coproantigen" or "hookworm-derived coproantigen" is any hookworm product that is present in the feces of a mammal infected with hookworm and that can specifically bind to one or more antibodies of the invention. For example, the hookworm coproantigen can be, but is not limited to, the novel N-terminal 28 kDa isoform of ASP5, which is an Ancylostoma excretory/secretory protein present in the faeces of hookworm-infected dogs as early as 9 days after dogs were first infected with hookworm. Thus, the "hookworm coproantigen" may be a novel N-terminal 28 kDa isoform of ASP5 (referred to here as "CoproASP5") that has been found in dog feces, as discussed in US Patent No. 7951547.

[068] As used herein, the term " Giardia " refers to the protozoa of the genus Giardia . Therefore, examples of the genus Giardia are Giardia lamblia , also known as Giardia intercinalis .

[069] A " Giardia coproantigen" or " Giardia derived coproantigen" is any product of Giardia that is present in the feces of a Giardia infected mammal and that can specifically bind to one or more antibodies of the invention.

[070] "Lectins" are proteins that recognize and bind to specific monosaccharide or oligosaccharide structures (carbohydrates). A lectin usually contains two or more carbohydrate binding sites. The binding specificity of some lectins to carbohydrates is determined by the amino acid residues that bind to the carbohydrate. The strength of lectin-carbohydrate binding can increase with increasing number of molecular interactions. The dissociation constant for lectin-carbohydrate binding is approximately Kd=10 ^-5 -10 ^-7 . Lectins can be labeled with any type of label known to those skilled in the art, including, for example, a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme label, a colloidal metal label, a radioisotope label, and a bioluminescent label.

[071] In some embodiments, the lectins used are lectins that specifically bind to a tapeworm coproantigen. In some embodiments, lectins that specifically bind to O-glycosylated proteins are suitable for use in the present invention. Such lectins include, for example, ECL ( Erythina cristagalli ) lectin, GSL I ( Griffonia Simplicifolia lectin I), GSL II ( Griffonia Simplicifolia lectin II), jacalin, LCA ( Lens culinaris ) lectin, RCA 123 ( Ricinus Communis ), and PSA lectin. ( Phaseolus vulgaris leukoagglutinin), WGA (wheat germ agglutinin) and sWGA (succinylated wheat germ agglutinin). Lectins are commercially available from, for example, Vector Laboratories, Burlingame, CA, USA.

[072] Lectins that specifically bind to carbohydrates on human, dog, cat, horse, cow, sheep, or monkey tapeworm antigens can be used.

[073] The term "specific to", "specifically binds" and "stably binds" means that a particular composition according to the invention, such as, for example, an antibody, polypeptide or oligonucleotide according to the invention recognizes one or more other agents and binds to these agents with greater affinity than at least one other agent. In one example, an antibody of the invention is considered "specific for tapeworm antigens", "specifically binding" and "stably binding" to these antigens, if such an antibody recognizes roundworm antigens and binds to these antigens with greater affinity than to any other antigens of parasitic worms other than tapeworms. Such binding specificity can be tested using techniques well known to those skilled in the art, such as ELISA or radioimmunoassay (RIA). Based on the information about the binding specificity of a particular composition according to the invention, the method according to the invention can be carried out under conditions that allow this composition to bind (and therefore detect such binding) to a particular agent or agents, but not to significantly bind to other agents when keeping the same conditions. In one example, the method of the invention may be carried out under conditions that allow the antibody of the invention to bind (and therefore allow such binding to be detected) to one or more tapeworm antigens of various species present in a particular sample, but not to significantly bind. grade with any antigen of other tapeworm species and/or other parasitic helminths that may be present in this specimen, thus allowing differentiation between tapeworm and roundworm species, trichocephali and hookworm.

[074] "Detection of tapeworms" means the detection of one or more products specific for tapeworms, including, for example, one or more polypeptides, antibodies and nucleic acids according to the invention or one or more tapeworm antigens. The presence of one or more of these tapeworm products in a mammalian sample indicates that the mammal is infected with the tapeworm, regardless of whether any whole tapeworm organism or egg is also present in the sample. Conversely, the absence of one or more of these tapeworm products in a mammalian sample indicates that the mammal is not infected with the tapeworm.

[075] The term “treatment” means administering a therapeutic agent to a patient by any suitable route of administration to reduce or eliminate parasitic worms (helminths) or other internal parasites from the body by reducing, repelling or killing them, and/or reducing or eliminating infestation by intermediate hosts such as insects, such as fleas, which can carry infection with parasitic worms or other internal parasites without significant harm to the patient.

III. Antibodies according to the invention

[076] The present invention further includes antibodies and antigen-binding fragments thereof that are raised against all or part of one or more of the polypeptides of the invention and that specifically bind to them, and also includes compositions that contain said antibodies and antigen-binding fragments thereof. When contacted with a sample taken from a mammal, these antibodies and antigen-binding fragments are able to specifically bind to a particular helminth antigen. For example, immunoglobulin antibodies and tapeworm antigen-binding fragments are able to specifically bind to tapeworm antigens present in the sample, but are not able to specifically bind to any antigen from other worms, such as roundworms, hookworms, or trichocephali, that may be present in the sample. The antibodies of the invention are suitable for use only to capture one or more tapeworm antigens, only to detect one or more tapeworm antigens, or more preferably to capture and detect one or more tapeworm antigens.

[077] Antibodies of the invention may be of any class of antibodies, including, for example, IgG, IgM, IgA, IgD, and IgE, and may be generated by any of a variety of methods known to those skilled in the art. (See for example Dean , Methods Mol. Biol . 80:23-37 (1998); Dean, Methods Mol. Biol . 32:361-79 (1994); Baileg, Methods Mol. Biol . 32:381-88 (1994); Gullick, Methods Mol. Biol . 32:389-99 (1994); Drenckhahn et al. Methods Cell. Biol . 37:7-56 (1993); Morrison, Ann. Rev. Immunol . 10:239- 65 (1992); Wright et al. Crit. Rev. Immunol. 12:125-68(1992); Harlow and Lane, Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory (1988); and Making and Using Antibodies: A Practical Handbook , Howard and Kaser, eds., CRC Press (2006), each of which is hereby incorporated by reference in its entirety).

[078] In one method, the polypeptide of the invention is administered to an animal host such as, for example, rabbits, mice, rats, guinea pigs, goats, pigs, cows, sheep, donkeys, dogs, cats, chickens, or horses. An enhanced immune response can be induced in the host animal by binding the polypeptide to a carrier and/or by exposing the host to an adjuvant, however, it should be noted that the present invention does not require that the polypeptide be associated with a carrier or that the host be exposed to adjuvant. An example of a carrier that can be used for this purpose is bovine serum albumin, bovine thyroglobulin and soybean trypsin inhibitor. Examples of adjuvants include complete or incomplete Freund's adjuvant and MDL-TDM adjuvant. Regardless of whether the polypeptide is bound to such a carrier, or whether the host is exposed to an adjuvant, booster immunization may be performed, but not necessarily, followed by blood sampling from the host animal one or more times. Polyclonal antibodies (pAbs) that specifically bind to the polypeptide can then be purified from antisera obtained from blood or bleeding sites. Such purification can be achieved, for example, using affinity chromatography techniques that involve coupling the polypeptide to a solid support. Such affinity chromatography techniques are well known to those skilled in the art.

[079] In several embodiments, an anti-tapeworm antibody of the invention is an antibody that is produced in rabbits by immunizing a host animal with a whole tapeworm extract, a tapeworm E/S product, a tapeworm wash, or a TCA soluble substance, as described below.

[080] It should also be noted that the antibodies of the invention may be, but not necessarily, polyclonal or monoclonal antibodies (mAb), single chain antibodies (scFv), chimeric antibodies, and fragments thereof. Monoclonal antibodies that are specific for the polypeptide of interest can be prepared and purified, for example, by producing cell lines that generate antibodies having the desired specificity for the polypeptide of interest. Cell lines of this type can be derived from cells of a particular type (eg, spleen cells) that are isolated from a host animal already immunized with the polypeptide as previously described. In this case, such cells may then be immortalized, for example, by fusing them with myeloma cells using any of a variety of fusion techniques known to those skilled in the art. In one representative method, cells from an immunized host animal are co-incubated with their fusion partner, such as myeloma cells, in the presence of a detergent for a short period of time before being plated on a medium that supports growth of the hybrid cells (but not the fusion partner with myeloma). Such a choice can be made, for example, using hypoxanthine, aminopterin and thymidine (HAT). If hybrid cells are formed at the time of selection, and possibly one or two weeks after the start of the selection procedure, then hybrid monocolonies (and their supernatants) are tested for their ability to bind to the polypeptide or polypeptides against which the host animal has been immunized. Hybrid colonies having the most optimal binding specificity are the best candidates from which monoclonal antibodies can be isolated. These monoclonal antibodies can, for example, be isolated directly from the supernatant (ie, medium) in which these colonies are grown using any of a variety of methods known to those skilled in the art.

[081] The antibodies of the invention can also be a single chain antibody (scFv) or an antigen-binding fragment of an antibody. Antigen-binding fragments of antibodies are part of an intact antibody containing an antigen-binding site or variable region of an intact antibody, where the specified part does not contain the heavy chain constant domains of the Fc region of the intact antibody. Examples of antibody fragments include Fab-, Fab'-, Fab'-SH-, F(ab') ₂ - and Fv fragments. In addition to production and purification from animal or mammalian cells, antibodies, antibody fragments, or non-antibody backbones can be selected based on various in vitro techniques, including phage presentation, ribosomal presentation, or bacterial presentation.

[082] Antibodies, including second antibodies, can be labeled with any type of label known to those skilled in the art, including, for example, a fluorescent label, a chemiluminescent label, a radioactive label, an enzyme label, a colloidal metal label, a radioisotope label, and a bioluminescent label. In various embodiments of the invention, one or more antibodies according to the invention are labeled with an enzyme, colloidal particle, radionuclide, or fluorophore. The specific label may be, for example, a colored latex particle, a dye ash, or gold ash conjugated to an antibody.

IV. Methods, devices and kits according to the invention

A. Devices and kits according to the invention

[083] In one aspect, the present invention relates to the detection of the presence or absence of one or more tapeworm antigens in a sample, where the specified device contains a solid carrier, where the solid carrier has one or more antibodies immobilized thereon, selected from the group consisting of : (a) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen, and optionally (d) one or more types of Giardia coproantigen, roundworm coproantigen, trichocephalic coproantigen, and/ or a hookworm coproantigen, wherein one or more types of roundworm coproantigens, trichocephalic coproantigens, and hookworm coproantigens specifically bind to antibodies. See US Patent No. 7951547, which is fully incorporated into the present description by reference. The device is designed to specifically bind and separate tapeworm coproantigens from any Giardia , roundworm, trichocephalus, and hookworm antigen in a mammalian specimen.

[084] In one aspect of the invention, the device includes a solid carrier, where one or more antibodies according to the invention are immobilized on a solid carrier. The solid carrier may be, but is not limited to, the inner bottom surface of a microtiter plate well, a microparticle, microfluidization device channels, a cassette, a membrane, or a support that is included, for example, as part of a side flow device. A representative microtiter plate is a 96-well Immulon 1B plate (which is commercially available from Thermo Scientific, Milford, MA), however, it should be noted that one of skill in the art will appreciate that a wide variety of other methods can be used to immobilize antibodies. microtiter plates that are not Immulon 1B 96-well plates, and these plates may be suitable for immobilizing the solid support of the invention.

[085] A representative side flow device is the side flow device described in U.S. Attention No. 5726010, which is fully incorporated into the present description by reference. The sidestream assay device may be a SNAP® device, which is commercially available from IDEXX Laboratories, Inc. Westbrook, M.E. However, one of skill in the art will appreciate that a wide variety of other sidestream devices can be used to immobilize the antibody, other than the SNAP® devices or the devices described in US Patent No. 5,726,010 and which allow antibodies to be immobilized thereon, and therefore these devices may be suitable for use as the device of the invention. These devices may include, for example, side flow devices using colloidal gold technology.

[086] Antibodies used in the device according to the invention can be immobilized on a solid carrier by any known method, including, for example, covalent or non-covalent, direct or indirect binding of antibodies to a solid carrier. Therefore, although these antibodies can be attached to a solid support through physical adsorption (i.e., without the use of chemical linkers), however, it is obvious that these antibodies can be immobilized on a solid support by any method of chemical coupling (i.e., using chemical linkers) well known to the person skilled in the art.

[087] In some embodiments, (a) the first antibody may be raised against a first species whole tapeworm extract, a first species tapeworm E/S product, a first species tapeworm wash, or a first species tapeworm TCA soluble; (b) the second antibody can be raised against a second species whole tapeworm extract, a second species tapeworm E/S product, a second species tapeworm wash, or a second species tapeworm TCA soluble; or (c) the third antibody may be raised against a third species whole tapeworm extract, a third species tapeworm E/S product, a first species tapeworm wash, or a third species tapeworm TCA soluble.

[088] In some embodiments, the solid carrier also has one or more antibodies selected from: an antibody capable of specifically binding to a roundworm coproantigen but not a trichocephalic or hookworm coproantigen; an antibody capable of specifically binding to the trichocephalic coproantigen, but not to the roundworm or hookworm coproantigen; an antibody capable of specifically binding to the hookworm coproantigen, but not to the trichocephalic or roundworm coproantigen; an antibody capable of specifically binding to a Giardia copro antigen, but not to a coproantigen selected from the group consisting of: roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and parvovirus coproantigen; and an antibody capable of specifically binding to a parvovirus coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and Giardia coproantigen. In some embodiments, the hookworm is Ancylostoma, the roundworm is Toxocara , and the trichocephalus is Trichuris . In other embodiments, the hookworms are Ancylostoma caninum or Ancylosloma tubaeforme ; roundworms are Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia ; and parvovirus is feline parvovirus or canine parvovirus.

[089] It should also be noted that the solid support may be any material suitable for immobilizing the antibodies of the invention. So, for example, a solid carrier can be spheres, particles, tubes, wells, probes, indicator strips, pipette tips, glass slides, fibers, membranes, paper, natural and modified celluloses, polyacrylamides, agaroses, glass, polypropylene, polyethylene, polystyrene , dextran, nylon, amylases, plastics, magnetite, or any other suitable material well known to the person skilled in the art. In some embodiments of the invention, the solid carrier may contain many particles, microparticles, chips or spheres. A plurality of particles, microparticles, chips or spheres may be attached to the device or may be loosely associated with the device. A plurality of particles, microparticles, chips or spheres may be present on the surface of the device or within the device. The plurality of particles, microparticles, or spheres may be stationary on or within the device, or they may move within or pass through the device.

[090] The device may optionally include one or more labeled antigen capture reagents that may be mixed with the mammalian sample before it is introduced into the device of the invention. When a labeled antigen capture reagent is included, the labeled antigen capture reagent may or may not be deposited or dried on the solid surface of the device. The term "antigen capture reagent" means any compound that is specific for an antigen of interest or antigens of interest. The labeled antigen capture reagent, whether added to a mammalian sample or pre-deposited on the device, can be, for example, a labeled antibody specific for a roundworm antigen, including but not limited to the antibodies of the present invention.

[091] The device may also optionally include a liquid reagent that provides transport (for example, if the device is, for example, a SNAP® device), or otherwise facilitates removal (for example, if the device includes a microtiter plate ) unbound material (eg, unreacted portions of a mammalian sample, such as, for example, unreacted portions of fecal extract and unbound antigen capture reagent) from the reaction zone (solid phase). The liquid reagent may be a wash reagent and may only serve to remove unbound material from the reaction zone, or it may include a detection reagent and may serve to remove unbound material and facilitate antigen detection. Thus, for example, in the case of an enzyme-conjugated antigen capture reagent, the detection reagent comprises a substrate that produces a detectable signal upon interaction with the enzyme-antibody conjugate in the reaction zone (solid phase). Alternatively, in the case of a labeled antigen capture reagent conjugated to a radioactive, fluorescent, or light-absorbing molecule, the liquid reagent simply acts as a wash solution to facilitate detection of complex formation in the reaction zone by washing away unbound labeled reagent.

[092] The liquid reagent may also include a limited amount of "inhibitor", that is, a substance that blocks the production of a detectable end product. A limited amount is defined as the amount of inhibitor sufficient to block production of the end product until most or all of the excess unbound material has been removed from the second region, at which point a detectable end product is formed.

[093] The device of the invention may also include various binding reagents immobilized at the sites and other than the antigen capture reagent or reagents. For example, an immunoreagent (antibody, antigen, or polypeptide) that recognizes a species-specific (e.g., specific for roundworms) portion of a labeled antibody or antigen capture reagent or enzyme portion of an enzyme-labeled reagent may be included as a positive control to assess viability. reagents in the device. For example, the positive control may be an antibody against horseradish peroxidase, which is produced, for example, in goats or mice. In addition, a reagent, such as an antibody isolated from a non-immune member of the species from which the antibody portion of the antigen-antibody complex is derived, can be included as a negative control to assess the specificity of immunocomplex formation (i.e., antigen-antibody complex).

[094] This device of the invention, in addition to being designed to specifically bind and isolate tapeworm coproantigens in a mammalian sample, may optionally be designed to allow one or more other diagnostic tests to be performed. Thus, for example, the solid carrier may also include reagents for detecting one or more helminths such as roundworms, trichocephali, roundworms and hookworms, one or more non-worm parasites, one or more viruses (such as parvovirus), one or more fungi, one or more protozoa (such as Giardia ), or one or more bacteria. Reagents for detecting one or more non-worm parasites, one or more viruses, one or more fungi, one or more protozoa, or one or more bacteria may be, for example, one or more antibodies or one or more antigens recognized by antibodies, specific for one or more non-worm parasites, one or more viruses, one or more fungi, one or more protozoa, or one or more bacteria.

[095] In one embodiment, the device of the invention is a microtiter plate that includes a plurality of wells, where each well contains a solid carrier having one or more antibodies of the invention immobilized on the carrier.

[096] A microtiter plate can be used in combination with the method of the invention to detect the presence or absence of one or more helminth coproantigens in a sample. For example, a tapeworm infection can be diagnosed in a mammal by detecting one or more tapeworm antigens using an antibody immobilized on a solid support. In one embodiment of the invention, the antigens that are detectable are coproantigens. "Coproantigens" are any product or products of gastrointestinal parasites, such as tapeworms, that are present in a fecal sample of a host species (eg, dogs or cats) and that can specifically bind to antibodies. Thus, the coproantigens may be whole worms, worm eggs, worm fragments, or products secreted, excreted or excreted by worms, or combinations thereof.

[097] In addition to the microtiter plate, there are a large number of other alternative approaches for parallel implementation of multiple immunoassays (ie, multiplex immunoassays) known to those skilled in the art. Typically, multiplex immunoassays can be performed in a single vessel, composition, or device where the components of the multiplex immunoassays are in a dispersed state. These technologies may be based on arrays, microarrays and/or microspheres or microparticles. In arrays or microarrays, the capture reagents are typically spotted or otherwise deposited in discrete regions of a single device, such as a membrane. In sphere-based methods, the capture reagents for each assay are attached to spheres, where spheres for each assay are different from spheres for other assays. Identification may be by light color, physical position, bar code, or the like. See, for example, Tighe, P. J., Ryder, R. R., Todd, I. & Fairclough, L. C. (2015), ELISA in the multiplex era: Potentials and pitfalls. Prot. Clin. Appl., 9: 406-422. doi:10.1002/prca.201400130).

[098] Many such multiplex platforms are commercially available. Examples include Luminex® (see, for example, US Pat. No. 7,523,637); πCode™ MicroDiscs (Plexbio, see e.g. US Patent Application No. US2014/0274778); bar coded magnetic spheres and multiplex assay methods and apparatus (eg Applied BioCode, Inc., Santa Fe Springs, CA, USA); bar coded magnetic chips (eg, Applied BioCode, Inc.; eg, US Pat. No. 8,232,092); test strips in the microfluidization method (for example, LumiraDx®, US patent No. 9919313); and light scattering technologies such as LightDeck® (mBio®, US Patent No. 9739714). Additional multiplex immunoassay technologies include MULTI-ARRAY (Meso Scale Diagnostics, Rockville, MD, USA), Bio-Plex® Multiplex System (Bio-Rad, Hercules, CA, USA), Access 2 technologies (Beckman Coulter, Atlanta, GA); and ProcartaPlex® and ProQuantum® (ThermoFisher Scientific, Waltham, MA, USA) (see Fu Q, Zhu J, Van Eyk JE. Comparison of multiplex immunoassay platforms. Clin Chem. 2010 Feb;56(2):314-8. doi:10.1373/clinchem.2009.135087).

[099] In another aspect of the invention, a device for determining the presence or absence of one or more tapeworm coproantigens in a fecal sample comprises a solid carrier, one or more lectins, and one or more antibodies selected from the group consisting of: (a) a first antibody, capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen. In one embodiment, the solid support has one or more antibodies, two or more antibodies, three or more antibodies, four or more antibodies, five or more antibodies, six or more antibodies, seven or more antibodies, or eight or more antibodies immobilized thereon. antibodies. The labeled lectin can bind to carbohydrates on the tapeworm coproantigen captured by the immobilized antibodies for detection. In another embodiment of the invention, the lectin may be immobilized on a solid support. The immobilized lectin can capture the tapeworm coproantigen, if present, and the resulting complex can be detected using one or more labeled antibodies. In another embodiment, the device further comprises one or more species of tapeworm antigen, wherein the one or more species of tapeworm antigen binds specifically to antibodies.

[0100] In addition, the present invention also includes assay kits (eg, industrial products) for detecting and identifying different types of tapeworms in a mammalian sample. In some embodiments, assay kits can further detect and discriminate coinfection with helminths such as roundworms, trichocephali, and/or hookworms in a mammalian sample. Therefore, the kit may include one or more devices and/or compositions according to the invention. For example, the kit may include antibodies against tapeworms and means for determining the binding of antibodies to tapeworm antigens and means for determining the binding of antibodies to tapeworm antigens. In one particular example, such a kit includes a device having an immobilized antibody against one or more different species of tapeworm, one or more antigen capture reagents (e.g., a non-immobilized labeled antigen capture reagent and an immobilized antigen capture reagent), and a wash reagent, and also the detection reagent and reagents used as positive and negative controls, if desired or necessary. Other components such as buffers, controls, and the like, known to those skilled in the art, may be included in such test kits. The relative amounts of the various reagents can be varied to provide reagent solution concentrations that substantially optimize the sensitivity of the assay. In particular, the reagents may be supplied as dry powders, usually lyophilized, which, when dissolved, provide a reagent solution having appropriate concentrations to be combined with the sample. The kit according to the invention may further include instructions for carrying out one or more methods according to the invention, including instructions for using any device and/or composition according to the invention, which are included in the kit.

B. Methods according to the invention

[0101] The present invention further includes methods for using one or more of the devices, kits and/or compositions of the invention to detect the presence or absence of one or more tapeworm antigens in a sample. Thus, these methods can be carried out to detect the presence or absence of one or more species of tapeworm in a sample, such as, for example, a fecal sample that is obtained from a mammal, including, but not limited to, dogs, cats, pigs, cows, or person. In addition, these methods can be carried out to detect one or more helminths such as roundworms, trichocephali, hookworms and roundworms, non-worm parasites such as Giardia , and viruses such as parvovirus in a sample.

[0102] In the methods of the invention, detection of one or more species of tapeworm can be accomplished by determining the presence or absence of one or more tapeworm antigens. If the sample being tested for tapeworm coproantigens is feces, then the soluble portion of the feces can be collected according to any protocol known to those skilled in the art. Thus, for example, in addition to the specific protocol described in the Examples section herein, soluble portions of a sample can typically be collected by filtration, extraction, centrifugation, or simple mixing followed by gravimetric precipitation. One skilled in the art will recognize that there are many methods for extracting and preparing non-fecal samples taken from a mammal. For example, the sample may be a bodily fluid that is naturally secreted or otherwise released by a mammal, or that is artificially obtained from a mammal. Such artificial extraction can be done by milking the mammal, or by syringe injection into the mammal's body and drawing the liquid into a syringe. Once the fluid is obtained, it may optionally be fractionated (eg, serum may be fractionated from whole blood and then used as a sample). As another example, the sample can be obtained by taking a swab from a mammal, for example, from the oral cavity of a mammal. In yet another example, tissue sections may be obtained by biopsy.

[0103] The methods include contacting a mammalian sample with one or more antibodies specific for tapeworm coproantigens under conditions conducive to the formation of an antigen/antibody complex, ie, an immunocomplex. That is, the antibody specifically binds to a coproantigen present in the sample. One skilled in the art is familiar with the assays and conditions that can be used to detect such binding of an antigen/antibody complex. For example, an antigen/antibody complex can be detected using a second antibody that binds to the antigen/antibody complex. The formation of a complex between antigen and antibodies in a sample can be detected using any suitable method known to those skilled in the art.

[0104] In addition, to achieve this goal, the relative amount of antibody-antigen complexes that form in one particular reaction can be measured relative to the complexes obtained in any other reaction by any method known to those skilled in the art. If a test specimen has been determined to have specific anti-tapeworm antigen-antibody complexes, then based on the specific complexes formed, it can be deduced that a particular tapeworm is present in the mammalian host and its species can be identified (e.g., Taenia pisiformis and Dipylidium caninum ). If this is confirmed, then it can be concluded that the mammal from which the test sample was taken has an infection caused by intestinal tapeworms. Conclusions that a mammal being tested has an intestinal tapeworm infection can be made by a clinician or medical staff at a diagnostic center or by caregivers of the mammal, such as, for example, a veterinarian. If the caregiver determines (or is otherwise informed) that the mammal has a tapeworm infection and that a particular species of tapeworm is present, the caretaker of the mammal may then administer to the mammal a course of treatment that is optimally designed to rid the mammal of a particular tapeworm infection rather than parasitic worm infection in general. In addition, the present invention can be used to confirm that any animal that has received treatment for a particular tapeworm infection has been free from that infection. A caregiver of a mammal who is aware that the sample includes tapeworms and roundworms but not hookworms can, for example, use this information to treat a mammal that has been sampled specifically for tapeworm detection by administering such to a mammal, a drug that is optimally effective against tapeworms and a second drug that is optimally effective against roundworms. In the absence of such information, the caregiver of the mammal may, for example, otherwise treat the mammal with a drug that is optimally effective only against tapeworms and roundworms only, or against other worms other than tapeworms and roundworms. worms (in such cases, the mammal may be at risk of receiving suboptimal treatment). In addition, people who may come into contact with an infected animal or its feces may be advised to take precautions against the parasite or parasites. In this regard, it is important to accurately and with high specificity identify worm species that can cause serious disease (eg, disease caused by migratory larvae) in humans.

[0105] A patient suffering from infections caused by intestinal parasites may be treated with certain drugs that are known to destroy parasites in the patient. Thus, a patient whose feces have been found to contain an intestinal parasite coproantigen may be treated with a suitable therapeutic agent to reduce or eliminate intestinal parasites.

[0106] Patients suffering from infections caused by intestinal parasitic worms, such as tapeworms, hookworms, trichocephali and/or roundworms, may be treated with anthelmintic drugs, also known as anthelmintic agents (or anthelmintics). Such anthelmintic agents are widely known to those skilled in the art. Tapeworm anthelmintics include, but are not limited to, praziquantel, nitazoxanide, albendazole, epsiprantel, fenbendazole, or combinations thereof. Anthelmintic agents can be administered by various suitable routes, including oral administration, parenteral administration such as subcutaneous, intravenous, intramuscular or intraperitoneal administration, or topical (skin) administration, for example, application directly to the exposed skin surface of a patient in the treatment and/or prevention of intestinal infections. parasites.

[0107] Giardia is a microscopic parasite that causes diarrhea known as giardiasis. Giardia , including Giardia intestinalis , Giardia lamblia , and Giardia duodenalis , are present on surfaces or in soil, food, or water contaminated with the faeces of infected humans or animals. Giardia coproantigen can be detected using a number of commercially available tests, including the rapid canine Giardia VetScan® test (Abaxis, Union City, USA), the Giardia Anigen® rapid CPV-CCV antigen test (BioNote, Seoul, Korea), the Giardia SNAP® (IDEXX, Westbrook, ME, USA) and Giardia antigen ELISA (IDEXX), Giardia /cryptosporidium ProSpecT® microplate assay (Thermo Fisher Scientific, Waltham, MA, USA) and Giardia Witness® test ( Zoetis, Parsippany, NJ, USA). (Barbecho JM, Bowman DD, Liotta JL. Comparative performance of reference laboratory tests and in-clinic tests for Giardia in canine feces. Parasit Vectors . 2018 Aug 1;11(1):444. doi: 10.1186/s13071-018-2990 -6.PMID: 30068364;PMCID: PMC6090814).

[0108] Therapeutic agents for the treatment of Giardia infections are widely known to those skilled in the art. Giardia infections can be treated with one or more of several drugs, including fenbendazole, albendazole, metronidazole, tinidazole, nitazoxanide, paromomycin, quinacrine and furazolidone, febentel, pyrantel pamoate, praziquantel, or combinations thereof. These drugs may be administered by a variety of suitable routes, including oral administration, parenteral administration such as subcutaneous, intravenous, intramuscular or intraperitoneal administration, or topical (skin) administration such as application directly to the exposed skin surface of a patient in the treatment and/or prevention of Giardia . .

[0109] Intermediate hosts may be involved in the transmission of one or more non-worm worms or parasites, fungi, viruses, and bacteria to a patient, and thus successful therapeutic treatment includes a strategy to control or prevent re-infection. For example, since tapeworm infections can be transmitted by intermediate hosts such as fleas and blood-sucking dog lice, successful therapeutic treatment for such an infection involves a strategy to control any intermediate host (eg, fleas) that may be present in the patient. . Thus, the control of intermediate hosts such as fleas helps to prevent re-infection of the patient with tapeworms. Therapeutic agents for the treatment or control of flea infections are well known to those skilled in the art and include selamectin, fipronil, imidacloprid, indoxacarb, pyrethrin, permethrin, flumethrin, spinosad, nitenpyram, afoxolaner, fluralaner, saralan, nitenpyram, methoprene, pyriproxyfen, and lufenuron or combinations thereof. Flea control agents can be administered by a variety of suitable routes, including oral administration, parenteral administration such as subcutaneous, intravenous, intramuscular or intraperitoneal administration, or topical (skin) administration such as application directly to the patient's exposed skin. Representative suitable forms for flea control include sprays, powders, collars, oral care compositions, or topical preparations.

[0110] The steps of the method of the invention may include applying a mammalian sample to a device of the invention that includes: (a) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to the coproantigen of the third tapeworm, but not to the coproantigen of the first tapeworm or to the coproantigen of the second tapeworm, to obtain antibody-coproantigen complexes in the presence of coproantigens, if they are formed in the sample; and detecting the presence or absence of antibody-coproantigen complexes, if any.

[0111] In some embodiments, the device may further comprise one or more antibodies selected from: an antibody capable of specifically binding to a roundworm coproantigen, but not a trichocephalic or hookworm coproantigen; an antibody capable of specifically binding to the trichocephalic coproantigen, but not to the roundworm or hookworm coproantigen; an antibody capable of specifically binding to the hookworm coproantigen, but not to the trichocephalic or roundworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and parvovirus coproantigen; and an antibody capable of specifically binding to a parvovirus coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and Giardia coproantigen. In some embodiments, the hookworm is Ancylostoma, the roundworm is Toxocara , and the trichocephalus is Trichuris . In other embodiments, the hookworms are Ancylostoma caninum or Ancylosloma tubaeforme ; roundworms are Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia ; and parvovirus is feline parvovirus or canine parvovirus.

[0112] In one embodiment of the invention, the step of detecting the presence or absence of antibody-coproantigen complexes, if any, further comprises the step of obtaining one or more lectins that bind to at least one of the complexes. The lectin may be detectably labeled or immobilized on a solid support. Alternatively, the first, second, and third antibodies may be detectably labeled or immobilized on a solid support. In one embodiment of the invention, the first, second and third antibodies can be immobilized on a solid support and the lectin can be detectably labeled. In another embodiment of the invention, the first, second and third antibodies can be detectably labeled and the lectin can be immobilized on a solid support.

[0113] In another embodiment, the step of contacting the mammalian fecal sample with one or more antibodies further comprises the step of contacting the fecal sample with one or more lectins. The lectin may be detectably labeled or immobilized on a solid support. Alternatively, the first, second and third antibodies may be labeled or immobilized on a solid support. In one embodiment of the invention, the first, second and third antibodies can be immobilized on a solid support and the lectin can be detectably labeled. In another embodiment of the invention, the first, second and third antibodies can be detectably labeled and the lectin can be immobilized on a solid support.

[0114] Antibodies and lectins can be directly or indirectly attached to a solid carrier or support, such as a well of a microtiter plate, an antibody-immobilizing part of a SNAP® device, a magnetic sphere, a non-magnetic sphere, a column, a matrix, a membrane, a fibrous material composed of synthetic or natural fiber (e.g. glass or materials based on cellulose or thermoplastic polymers such as polyethylene, polypropylene or polyester), a sintered glass structure consisting of particulate materials (e.g. glass or various thermoplastic polymers), or a film from a molded membrane, consisting of nitrocellulose, nylon, polysulfone or the like. (usually synthetic in nature). All of these substrate materials can be used in suitable forms such as films, sheets or plates, or they can be applied to or attached to appropriate inert carriers or laminated to such carriers such as paper, glass, plastic films or fabrics. Suitable methods for immobilizing antibodies, peptides, and lectins on solid phases include ionic, hydrophobic, covalent interactions, and the like.

[0115] However, the methods of the invention do not require the use of solid phases or substrates. Those skilled in the art will recognize that there are a number of methods by which the method of the invention can be carried out to detect the presence or absence of roundworms without the use of solid phases or supports. Thus, for example, immunoprecipitation methods can be carried out that do not require the use of solid phases or supports.

[0116] In some embodiments, an antigen/antibody complex is detected if an indicator reagent, such as an enzyme conjugate that is bound to the antibody, catalyzes the detectable reaction. An indicator reagent, including but not necessarily a signal-generating compound, may be applied to the antigen/antibody complex under conditions that allow a detectable antigen/antibody/indicator complex to be obtained. The antibody may optionally be labeled with an indicator reagent prior to formation of the antigen/antibody complex.

[0117] The formation of an antigen/antibody complex or an antigen/antibody/indicator complex in some of the methods of the invention can be specifically detected, e.g. resonance or deposition methods. Detection of the antigen/antibody complex can also be accomplished by adding a second antibody that is coupled to an indicator reagent comprising a signal-generating compound. Indicator reagents comprising signal-generating compounds (labels) associated with the polypeptide/antibody complex can be detected using the methods described above and may include chromogenic agents, catalysts such as enzyme conjugates, fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridines, phenanthridines, ruthenium and luminol, radioactive elements, direct visual markers, as well as cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The choice of a particular label is not critical, but it can form a signal either by itself or in combination with one or more additional substances.

[0118] The methods of the invention include, but are not limited to, competitive binding methods, direct reactions, or sandwich assays, including, but not limited to, ELISA, RIA, immunofluorescent assays (IFA), haemagglutination (HA), immunoassay with fluorescence polarization (FPIA); and microtiter plate assays (ie, any assay performed in one or more wells of a microtiter plate). One assay according to the invention includes a reverse flow chromatographic column binding assay, which can be carried out, for example, using a SNAP® device. See US Patent No. 5726010.

[0119] In some embodiments of the invention, the method according to the invention facilitates the conduct of sandwich assays or assays for competitive specific binding. In a sandwich assay, antigen capture reagents are immobilized in the reaction zone. These antigen capture reagents can specifically bind to antigens in the sample being tested for tapeworm. After the antigen of the sample has been bound, the antigen capture reagent/antigen complex is detected by any suitable method. Thus, for example, the complex can be reacted with labeled specific binding reagents (eg, with an enzyme-antibody conjugate) and with a detectable antigen (eg, after interaction with a substrate).

[0120] In other embodiments of the method according to the invention, a competitive binding assay is performed. In a competitive binding assay, antigen capture reagents are immobilized in the reaction zone and simultaneously contacted with the sample antigen and a labeled antigen (eg, an antigen-enzyme conjugate). The amount of label detected in the reaction zone is inversely proportional to the amount of antigen in the sample.

[0121] In some embodiments of the method, antibodies specific for tapeworm coproantigens are attached to a solid phase or to a support. A sample is added to the substrate, which may include tapeworm antigen. Then antibodies are added that specifically bind to tapeworm antigens. The antibodies may be the same antibodies used in the solid phase, or they may be from different sources or species. In addition, these antibodies may be linked to an indicator reagent such as an enzyme conjugate. Washing steps may be carried out prior to each addition. A chromophore or enzyme substrate may then be added to develop the color. The color reaction may be terminated and the color may be quantified, for example with a spectrophotometer, and/or visually assessed by eye.

[0122] In other embodiments of this method, antibodies specific for tapeworm coproantigens are attached to a solid phase or to a support. A sample is added to the substrate, which may include tapeworm antigen. Second antibodies against other species are then added that specifically bind to the coproantigens. These second antibodies, unlike the solid phase antibodies, come from other species. Thereafter, third antibodies against other species are added that specifically bind to the second antibodies and do not specifically bind to the solid phase antibodies. Third antibodies may include an indicator reagent such as an enzyme conjugate. Washing steps may be carried out prior to each addition. A chromophore or enzyme substrate may then be added to develop the color. The color reaction may be terminated and the color may be quantified, for example with a spectrophotometer, and/or visually assessed by eye.

[0123] In a specific example, the method of the invention is carried out using a device that is a side flow assay device by adding the obtained mammalian sample to the flow matrix of the device in the first region (sample application zone). The resulting sample is transferred into a channel with a liquid flow under the action of capillaries into the second region of the flow matrix, where there is a label in the form of large particles capable of binding and forming the first complex with the antigen in the sample. The large particle label can be, for example, a colored latex particle, a dye ash, or gold ash conjugated to an antibody specific for a roundworm antigen. The first complex is transferred to a third region of the flow matrix where an antibody that specifically binds to the roundworm antigen is immobilized at a specific site. The second complex is formed between the immobilized antibody and the first complex. The large particle label, which is part of the second complex, can be directly assessed by eye.

[0124] Each worm specific antibody can be an immobilized antigen capture reagent in the reaction zone (solid phase). A second antigen capture reagent, i.e., a second specific anti-tapeworm antibody that has been conjugated to the label, can either be added to the sample before it is added to the device, or a second antigen capture reagent can be injected into the device. Thus, for example, the labeled antigen capture reagent can be deposited and dried in a liquid path that permits such liquid passage between the sample application area and the solid phase. Contacting the labeled antigen capture reagent with the test sample may cause the labeled antigen capture reagent to dissolve.

[0125] In one embodiment of the method according to the invention, a specific worm coproantigen is detected using ELISA. Specific examples of the ELISA method according to the invention are described here in the "Examples" section. Although the present invention has been described with reference to specific ELISA methods, it should be noted that the person skilled in the art is aware of alternative, additional or other ELISA steps that can be carried out without deviating from the main goal achieved using this method according to the invention.

[0126] In another embodiment, the tapeworm coproantigen is detected using a sidestream device such as, for example, a SNAP® device.

[0127] In addition, the methods of the invention for detecting tapeworm infestation can be combined with other diagnostic assays to detect the presence of other organisms or conditions. Thus, for example, the assays of the invention may be performed in combination with reagents that detect one or more helminths, non-worm fecal parasites, one or more viruses, one or more fungi, one or more protozoa, one or more bacteria, one or more parasites in the blood or occult blood, or a combination thereof. When there are two or more unique binding sites on a single assay device (such as, for example, two unique spots on a SNAP® assay device), the present invention allows the detection of two or more organisms in a single sample. In one embodiment of the invention, there are three unique spots to detect past or present infections or infestations with three organisms (spots represent either antigen or antibody binding reagents) in a single sample (i.e., where the same single sample has been exposed to three capture reagents on a single device). In yet another embodiment of the invention, there are four unique spots to detect former or present infections or infestations with four organisms (the spots represent antigen- or antibody-binding reagents) in a single sample (i.e., where the same single sample has been exposed to four capture reagents on one device). However, it should be noted that the same device may include more than four unique spots and/or detect more than four organisms.

[0128] Reagents for detecting one or more helminths, non-worm parasites, one or more viruses, one or more fungi, one or more protozoa, or one or more bacteria can be, for example, one or more antibodies or one or more antigens recognized by antibodies specific for one or more helminths, non-worm parasites, one or more viruses, one or more fungi, or one or more bacteria. In some embodiments, the reagents may include one or more antibodies or one or more antigens recognized by antibodies specific to one or more helminthic parasites (e.g., roundworms, trichocephalus, hookworms, and roundworms), to parasites that are not worms, one or more viruses (eg, parvoviruses, such as canine parvovirus or feline parvovirus), one or more fungi, one or more protozoa (eg, Giardia , such as Giardia lamblia ), or one or more bacteria.

[0129] The method may also include, but is not limited to, using one or more tapeworm nucleic acids, including but not limited to nucleic acids of the invention, to determine the presence or absence of one or more tapeworm species in a sample taken from a mammal. Such use of these nucleic acids to detect the presence of helminths may be done before, during, or after any other aspects of the method, including detection of one or more species of tapeworm by antibody. Thus, in one aspect of the invention, after one or more species of tapeworms are found or not found in a particular sample, and after the mammal from which the sample was taken is diagnosed as infected or not infected with tapeworms, that sample (or later obtained sample from a diagnosed mammal) can be tested for the presence or absence of any one or more nucleic acids, including any one or more nucleic acids according to the invention. Any absence of a specific helminth in a specific mammal using one or more nucleic acids (after the helminth has been detected using one or more antibodies) should be considered as the probability that the antibodies detected the helminth corpo antigen before the presence of the detectable helminth nucleic acid in the sample. . In such a case, the mammal's caregiver may ignore the fact that the nucleic acid failed to detect the helminth and treat the mammal, in particular the helminth infection, on the basis that the antibodies do detect the helminth. In another aspect of the invention, nucleic acids are used to determine the presence or absence of helminths in a particular mammal, and then the presence or absence of helminths is further assessed using antibodies of the invention. Detection of one or more helminthic nucleic acids can be carried out using any nucleic acid detection methods known to the person skilled in the art. Thus, for example, such detection can be carried out by performing a PCR-based method, such as, but not limited to, for example, a real-time PCR-based method. Examples of PCR-based methods are described, for example, in PCR Protocols (Methods in Molecular Biology) , 2nd ^ed ., Bartlett and Stirling, eds., Humana Press (2003); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), each of which is incorporated herein by reference in its entirety.

[0130] The present invention is specifically described with reference to the following Examples; however, they should not be construed as limiting the scope of the invention.

Example A

[0131] Unless otherwise stated, the materials and methods described below were used to obtain the data described in one or more of Examples 1-5 below.

[0132] Preparation of Taenia pisiformis and Dipylidium caninum worm extracts. Taenia pisiformis and Dipylidium caninum worm extracts were purchased from Antibody systems, Inc. Hurst, Texas, USA. The worm extracts were centrifuged at 10,000×g for 30 minutes at 4°C. The supernatant was harvested, dialyzed in PBS, pH 7.0 (membrane molecular weight cutoff 12-14 kDa, part 132678, Spectrum, Repligen, Waltham MA, USA) and protein concentration determined by Bradford assay.

[0133] Preparation of Taenia taeniaeformis worm extracts . Taenia taeniaeformis was obtained from the laboratory of the Department of Veterinary Medicine at Ross University in St. Kitts. All whole worms were washed several times with cold PBS, pH 7.0 to remove any faecal material and mucus from the host at room temperature, and homogenized at 4°C with a tissue grinder until appreciable pieces of tissue disintegrated. into pieces not visible to the naked eye. The homogenized materials were transferred to a 50 ml Falcon tube and the chopper was washed 2-3 times with cold PBS, pH 7.0. The homogenized tapeworms, together with the chopper washes, were centrifuged at 10,000×g for 30 minutes at 4°C, and the supernatants were collected before dialysis in PBS, pH 7.0 (membrane cutoff 12-14 kDa, part 132678, Spectrum, Repligen, Waltham MA, USA). Protein concentration was determined using the Bradford assay.

[0134] Preparation of the soluble fraction of Dipylidium caninum TCA. This antigen was obtained by destroying the worms in an aqueous buffer solution (phosphate buffer, pH 7.2), followed by centrifugation to remove insoluble substances and large particles, adding dropwise 30% trichloroacetic acid (TCA) to a final concentration of 15%, stirring for another 15 minutes at 18-27°C after adding all of the TCA, storage on a laboratory table for another 45 minutes at 18-27°C at rest, centrifugation to remove insoluble components, dialysis against an aqueous buffer solution (0 .01 M phosphate buffer, pH 7.2) using a Spectrapor® dialysis tube, MWCO: 6-8 K, and lyophilization on a lyophilizer.

[0135] Preparation of the worm wash . Frozen stock worm samples were washed 4 times in PBS buffer at pH 7.2. Buffer solutions from the first 3 washing steps were discarded. The buffer solution in the fourth washing step was centrifuged at 10,000×g for 20 minutes and the supernatant was collected. The supernatant was concentrated on an iCON concentrator (MWCO: 20 ml/9K; Thermo Scientific). The resulting concentrate was termed "worm wash" (WW).

[0136] E/S product preparation . Excreted/secreted products (E/S) were collected by keeping live worms in a T-150 flask with tissue culture medium (EMEM with D-glucose, gentamicin and fungizone, pH 7.2-7.3) in an incubator at 37°C with 5% CO ₂ for two weeks. Briefly, live tapeworms were washed several times with warm medium to remove any fecal debris and then placed in a T-150 tissue culture flask containing 100 ml warm medium (EMEM). The viability of the tapeworms was checked daily and the medium changed three times a day. Used media was pooled and concentrated on an Icon concentrator (MWCO: 9K; Thermo Scientific) and then the resulting E/S concentrated product was used to generate antibodies.

[0137] Obtaining polyclonal antibodies (pAb) . Polyclonal antibodies were produced in specific pathogen free (SPF) rabbits at SDIX, LLC (Windham, Maine, USA). The immunogens are whole worm extracts ( Dipylidium caninum and Taenia pisiformis from Antibody Systems Inc. Hurst, Tex.; T. taeniaeformis from IDEXX Laboratories, Inc) or an E/S product (IDEXX Laboratories, Westbrook, Maine). Briefly, rabbits were challenged by subcutaneous injection of the same immunogen in different adjuvants four times over 50 days. Serum was collected at the end of the immunization procedure.

[0138] Obtaining a monoclonal antibody (mAb) . Mouse monoclonal antibodies were obtained according to standard procedures, unless otherwise noted. Briefly, 3-5 Balb/c mice were immunized with the immunogen, and after completion of the immunization schedule, spleen cells were harvested. Spleen cells were fused with myeloma cells. After several rounds of screening and selection with HAT medium, isotyping and subcloning, specific hybridoma cell lines were obtained that secreted the desired mAb.

[0139] Purification and isolation of antibodies . Rabbit polyclonal antibody and mouse monoclonal antibody were purified by affinity chromatography on a G-protein Sepharose 4 Fast Flow column (Thermo Fisher Scientific) using an AKTA purification system. Briefly, rabbit serum or TCF (final culture fluid) was diluted with wash buffer and loaded onto a G-protein column. The column was thoroughly washed with wash buffer before the antibody was eluted from the column. The eluted antibody was neutralized with 1M Tris buffer, pH 8.0, then dialyzed in 10 mM PBS, pH 7.2, and stored at -20°C for later use.

[0140] Preparation of fecal extract . Fresh, unpreserved faecal samples of dogs or cats (1 gram) were suspended in 4 ml diluent solution (diluent solution is: 0.05M Tris base; 1 mM EDTA; 0.45% Kathon; 16 mg/ml gentamicin sulfate; 0.05 % Tween-20; 40% fetal bovine serum; 10% rabbit serum; and 5% mouse serum). The suspension was centrifuged at 4000 rpm for 20 minutes to obtain the first supernatant. The first supernatant was centrifuged at 10,000×g for 10 minutes to obtain a second supernatant, which is referred to here as "fecal extract".

[0141] ELISA assays . Purified polyclonal Abs or monoclonal Abs (100 μl/well and 3 μg/ml) were immobilized by physical adsorption onto Immulon 1B 96-well plates overnight at 4°C. The plates were then blocked with 1% BSA in 0.1 M Tris pH 7.0 at 4° C. overnight followed by drying at room temperature. Approximately 100 ml of fecal extract was added to each well and incubated at room temperature for one hour. The wells were then washed five times with PBS-Tween-20 according to standard methods known to those skilled in the art. In a separate reaction vessel, free rabbit pAb (another antibody, in the case of mAb against the same target used) was labeled with horseradish peroxidase (HRP) using a crosslinker, namely succinimidyl-4-[N-maleimidomethyl]cyclohexane-1 -carboxylate (SMCC) to obtain a conjugate, and 3 μg/ml of this conjugate was added to each well having an immobilized pAb or mAb. After a 30 minute incubation period at room temperature, unbound conjugate was removed from the wells by washing with a solution of PBS-Tween-20 in accordance with standard methods known to those skilled in the art. Then 50 μl of TMBLUE™ peroxidase substrate (IDEXX Laboratories, Westbrook, ME) was added to each well and the plates were incubated for 10 minutes at room temperature. After stopping each enzyme reaction by adding 0.1% sodium dodecyl sulfate (SDS), after a 10-minute incubation period, the optical density (OD) was measured in each well of a 96-well plate at A650 using standard spectrophotometric methods in an ELISA plate reader to calculate "OD650 values" (or simply OD values) for each well. In this format, the OD value obtained for any particular well of a 96-well plate is directly proportional to the amount of specifically bound antigen present in the well. OD values of 0.1 or below were considered as negative results, and other OD values were considered as positive results, unless otherwise indicated in the Examples.

[0142] Carbohydrate/Glycosylation Characterization by Carbohydrate ELISA Using Jacalin-Biotin . An immulon 1B plate coated with mouse IgM mAb ADX226 was used for carbohydrate ELISA. Approximately 100 μl of fecal extract was added to each well and incubated at room temperature for one hour. The wells were then washed five times with PBS-Tween-20 according to standard methods known to those skilled in the art. Then, yacalin-biotin (0.25 μg/ml) was added to each well, followed by incubation for one hour at room temperature. Unbound yacalin-biotin (Vector Laboratories, 30 Ingold Road, Burlingame, CA) was removed from the wells by washing with PBS-Tween-20 solution according to standard methods known to those skilled in the art. Streptavidin-HRP conjugate (Vector Laboratories, 30 Ingold Road, Burlingame, CA) was added to each well and incubated at room temperature for 30 minutes. The unbound streptavidin-HRP conjugate was washed again with PBS-Tween-20 solution. Then 50 μl of TMBLUE™ peroxidase substrate (IDEXX Laboratories, Westbrook, ME) was added to each well after incubation for 30 minutes with streptavidin-HRP conjugate and the plates were incubated for 1 minute at room temperature. After stopping each enzyme reaction by adding 0.1% sodium dodecyl sulfate (SDS) after a 1-minute incubation period, the optical density (OD) was measured in each well of a 96-well plate at A650 as illustrated above in the section where faecal ELISA is described.

Example 1A

[0143] Generation and screening of mouse monoclonal antibodies against Taenia pisiformis . Mouse mAbs (monoclonal antibodies) were induced by immunizing mice with T. pisiformis worm extract (WE) (hereinafter referred to as T. pisiformis extract or T. pisiformis extract ) and hybridomas were obtained. Candidate hybridomas were screened for the ability of secreted mAbs to bind to the immunogen (ie, T. pisiformis extract) coated on microtiter plates in a capture ELISA assay. Based on this screen, one hundred (100) hybridomas (ie, 100 mAb) that were positive in this screen (ie, able to bind to the T. pisiformis extract) were selected for further analysis. Then, 100 mAb candidates were screened again for their ability to function in a coproantigen ELISA assay using a capture screening assay. Microtiter plates were coated with a rabbit polyclonal antibody that was produced by standard immunization of rabbits with T. pisiformis extract. Then FEX (fecal extract) obtained from dog fecal samples was added. Fecal samples were obtained from dogs known to be infected with T. pisiformis and these samples were obtained from IDEXX Reference Laboratories. One of 100 candidate mAb supernatants was added to each well and then goat anti-mouse IgG was labeled conjugated. In this assay, five mAbs (02D09, 03H02, 07C02, ADX131, and ADX13) were particularly carefully evaluated and then selected for further analysis.

[0144] Five mAbs were further screened for their ability to function when paired with each other in a sandwich assay. For this purpose, each of the five mAbs were loaded onto wells of microtiter plates and incubated with FEX ( Taenia Pisiformis positive samples: n=10; T. pisiformis negative samples: n=10) followed by the addition of one of the five labeled mAbs. , or mouse pAb against WE Taenia pisiformis . Five mAbs functioned well in combination with at least one of the other mAbs, and therefore they were selected for further study. Of the five mAbs that functioned well, two antibodies (ADX131 and ADX132) were selected for further studies because they produced low background and strong signal when paired with each other in a coproantigen ELISA.

[0145] ADX131 and ADX132 each paired with HRP conjugates 02D09, 03H02, 07C02, ADX131 and ADX132 in a different ELISA assay. An additional mating with a rabbit anti-WE T. pisiformis pAb was used as a positive control. ADX131 and ADX132 were separately applied to the wells of microtiter plates and then incubated with FEX in dogs infected with T. pisiformis ; dogs not infected with T. pisiformis or with T. pisiformis worm extract; and with conjugates and HRP substrate. In each of the five antibody pairs, ADX131 gave a strong signal when incubated with FEX infected dogs and worm extract. In four of the five antibody pairs, ADX131 was negative (i.e., signal below cut-off) when incubated with FEX in uninfected dogs, except for mating with 03H02, which gave a signal slightly above the selected cut-off of 0.1 OD (Fig. 1 ). In all five antibody pairs, ADX132 gave a strong signal when incubated with FEX infected dogs and worm extract. In all five antibody pairs, ADX132 produced a low background signal when uninfected dogs were incubated with FEX (FIG. 2).

Example 1B

[0146] ELISA assay for specificity to Taenia pisiformis coproantigen and evaluation of the sensitivity of such an assay . To evaluate the specificity of the assay, an ELISA assay for T. pisiformis coproantigen (using plate-coated ADX131 and ADX132-HRP conjugate) was performed in which FEX testing was performed on dogs infected with hookworm Ancylostoma caninum (n=44), roundworms Taxocara canis (n=9), Trichuris vulpis (n=36), D. caninum (n=44) or Taenia pisiformis (n=5). The result (see FIG. 3) indicated that all 5 T. pisiformis samples were positive in this assay and all others were negative. ELISA analysis for T. pisiformis coproantigen showed no cross interaction with hookworm Ancylostoma caninum , roundworms Taxocara canis , trichocephalus Trichuris vulpis , or D. caninum . Thus, the T. pisiformis coproantigen ELISA assay had 100% sensitivity and specificity in this experiment. To assess the specificity of the assay, a coproantigen sandwich ELISA was performed using ADX131 coated on a microtiter plate and ADX132-HRP conjugate applied after addition of a patient sample. Analysis was performed on WE D. caninum , WE T. pisiformis , WE T. crassiceps , WE T. taeniaeformis , E/S T. taeniaeformis , and FEX dogs positive for D. caninum ; dogs negative for D. caninum ; cats positive for D. caninum ; cats negative for D. caninum ; dogs positive for T. pisiformis ; dogs negative for T. pisiformis ; a group of three cats positive for T. taeniaeformis; and a group of three cats negative for T. taeniaeformis . The above WE were used at a concentration of 1 μg/ml of protein. The analysis was additionally performed on several WE samples at 2 μg/ml protein and/or 10 μg/ml protein. WE specimens having these higher concentrations are D. caninum WE, T. pisiformis WE, T. crassiceps WE, Ancylostoma caninum hookworm WE, Toxocara canis WE, and Trichuris vulpis trichocephalus WE. Among these samples, the assay was only positive for T. pisiformis WE and in dogs positive for T. pisiformis (FIG. 4). Thus, the ADPX131/ADX132-HRP ELISA was highly specific in this experiment.

[0147] To evaluate the specificity of the T. pisiformis coproantigen ELISA assay (using plated ADX131 and ADX132-HRP conjugate), FEX 822 was analyzed on dog samples. Of these samples, 1 sample was confirmed as T. pisiformis positive by microscopy (i.e., the proglottids were observed under a microscope), and 821 samples were confirmed as T. pisiformis negative by microscopy. The results showed that out of 821 samples that were negative on microscopy, 818 samples were negative in the Taenia pisiformis coproantigen ELISA and 3 samples were positive. A sample that was positive on microscopy was ELISA positive for T. pisiformis coproantigen. Thus, the specificity of the ELISA assay for the T. pisiformis coproantigen was 99.7% in this experiment.

Example 1C

[0148]Antigen characterization: glycosylation of antigens associated with mAb ADX131 against T. pisiformis . To determine if the coproantigen associated with mAb ADX131 is glycosylated, 21 different lectins were tested for their ability to bind to the coproantigen using a commercially available kit (biotinylated lectin kits I, II and III from Vector Laboratories, Burlingame, CA ). Analyzes were performed according to the manufacturer's instructions. Briefly, FEX dogs positive forT. pisiformiswere added to ADX131 coated plates. After washing, lectin:biotin conjugates were added, followed by streptavidin-HRP and color substrate. In this test, the following lectins, such as WGA, succinylated WGA, PSA, GSLII, and LCA, produced strong signal and low background, indicating that they bound to the coproantigen.T. pisiformis.

[0149]Antigen characterization: glycosylation of antigens associated with mAb ADX132 against T. pisiformis . To determine if the coproantigen associated with mAb ADX132 is glycosylated, the ability of ADX132 to bind to FEX products associated with 21 different lectins was tested using a commercially available kit (biotinylated lectin kits I, II and III from Vector Laboratories, Burlingame, CA). Analyzes were performed according to the manufacturer's instructions. Briefly, wells of microtiter plates were coated with one of the lectins and FEX positive for dogs was added.T. pisiformisand then the ADX132-HRP conjugate and color substrate were added. The lectins WGA, UEA, and GSLII associated with a coproantigen were used in this test.

[0150] The lectin binding data showed that the T. pisiformis coproantigen associated with ADX131/ADX132 is glycosylated. The lectin binding data also showed that the ADX131/132 coproantigen contains the following molecules: unreduced GlcNac; restored GlcNac; fucose; GSLII; and mannose.

Example 2A

[0151]Obtaining rabbit polyclonal antibodies against T. taeniaeformis . Worms extracts (WE), product E/S and worm washings (WW)T. taeniaeformis received as described above. Rabbits were immunized with WET. taeniaeformis, E/ST. taeniaeformis or W.W.T. taeniaeformis as described above for the production of polyclonal antibodies, namely rabbit pAb against W.E.T. taeniaeformis, rabbit pAb against E/ST. taeniaeformis and rabbit pAb vs. WWT. taeniaeformis.

A. Initial evaluation by ELISA assay using T. taeniaeformis rabbit anti- WE pAb and T. taeniaeformis E/S rabbit pAb conjugates

[0152] For the initial assessment, conducted using coproantigen ELISA assays conducted using T. taeniaeformis rabbit anti-WE pAb and T. taeniaeformis E/S rabbit pAb , cat fecal extracts from seven cats positive for T. taeniaeformis were tested, and six cats negative for T. taeniaeformis .

[0153]ELISA assay using rabbit pAb conjugates against W.E. T. taeniaeformis : In this ELISA, the rabbit anti-WE pAbT. taeniaeformis plated, contacted with FEX and then with rabbit pAb-HRP conjugate against WET. taeniaeformis and with color substrate. In this assay, all seven samples positive forT. taeniaeformis,were considered as positive, and all six samples negative forT. taeniaeformis,viewed as negative. Therefore, in this experiment, the sensitivity and specificity of an ELISA assay performed using rabbit pAb conjugates W.E.T. taeniaeformis, was 100%.

[0154]ELISA assay using rabbit pAb conjugates against E/S T. taeniaeformis : In this ELISA, rabbit anti-E/S pAbT. taeniaeformis plated, contacted with FEX and then with rabbit anti-E/S pAb conjugateT. taeniaeformis and with color substrate. In this assay, all seven samples positive forT. taeniaeformis,were considered as positive, and all six samples negative forT. taeniaeformis,viewed as negative. Therefore, in this experiment, the sensitivity and specificity of an ELISA assay performed using rabbit pAb conjugates against E/ST. taeniaeformis, was 100%.

B. Performance of an ELISA assay using rabbit anti - WE T. taeniaeformis pAb conjugates in dog and cat faecal samples

[0155] Efficacy of coproantigen ELISA assay using rabbit pAb against W.E.T. taeniaeformis (as described in section A) was further evaluated on dog and cat faecal extracts. The set of canine samples included faecal extracts from 31 dogs positive forT. taeniaeformis, and 74 dogs negative forT. taeniaeformis. The feline sample set included faecal extracts from 13 cats positive forT. taeniaeformis, and 39 cats negative forT. taeniaeformis.

[0156] For dogs, from 31 positive samples, ELISA assay using rabbit pAb conjugates against W.E.T. taeniaeformis, gave a positive signal only in 8 samples. Of 74 negative canine samples, ELISA assay using rabbit pAb conjugates against W.E.T. taeniaeformis, gave a positive signal only in 6 samples. Therefore, in this experiment, the sensitivity was 25.8% and the specificity was 91.9%.

[0157] For cats, out of 13 positive samples, ELISA assay using rabbit pAb conjugates against W.E.T. taeniaeformis, gave a positive signal in 12 samples. Of 39 negative samples, ELISA assay using rabbit pAb conjugates against W.E.T. taeniaeformis, gave a positive signal only in 4 samples. Therefore, in this experiment, the sensitivity was 92.3% and the specificity was 89.7%.

C. Performance of an ELISA assay using rabbit anti - E/S T. taeniaeformis pAb conjugates in dog and cat faecal samples

[0158] Efficacy of coproantigen ELISA assay using rabbit pAb against E/ST. taeniaeformis (as described in section A) were further evaluated on dog and cat faecal extracts. The set of canine samples included faecal extracts from 31 dogs positive forT. taeniaeformis, and 74 dogs negative forT. taeniaeformis. The feline sample set included faecal extracts from 13 cats positive forT. taeniaeformis, and 39 cats negative forT. taeniaeformis.

[0159] For dogs, from 31 positive samples, ELISA assay using rabbit pAb conjugates against E/ST. taeniaeformis, gave a positive signal only in 4 samples. Of 74 negative canine samples, ELISA assay using rabbit pAb conjugates against E/ST. taeniaeformis, gave a positive signal in only 1 sample. Therefore, in this experiment, the sensitivity was 12.9% and the specificity was 98.6%.

[0160] For cats, out of 13 positive samples, ELISA assay using rabbit pAb conjugates against E/ST. taeniaeformis, gave a positive signal in 12 samples. Of 39 negative samples, ELISA assay using rabbit pAb conjugates against E/ST. taeniaeformis, gave a positive signal only in 3 samples. Therefore, in this experiment, the sensitivity was 92.3% and the specificity was 92.3%.

D. Performance of an ELISA assay using rabbit anti - WW T. taeniaeformis pAb conjugates in dog and cat faecal samples

[0161] Efficiency of a coproantigen ELISA assay using a rabbit pAb against WWT. taeniaeformiswere evaluated on extracts of faeces from dogs and cats. The set of canine samples included faecal extracts from 31 dogs positive forT. taeniaeformis, and 74 dogs negative forT. taeniaeformis. The feline sample set included faecal extracts from 13 cats positive forT. taeniaeformis, and 39 cats negative forT. taeniaeformis.

[0162] For dogs, from 31 positive samples, ELISA assay using rabbit pAb conjugates against WWT. taeniaeformis, gave a positive signal in only 16 samples. Of 74 negative canine samples, ELISA assay using rabbit pAb conjugates against WWT. taeniaeformis, gave a positive signal only in 3 samples. Therefore, in this experiment, the sensitivity was 51.6% and the specificity was 98.6%.

[0163] For cats, out of 13 positive samples, ELISA assay using rabbit pAb conjugates against WWT. taeniaeformis, gave a positive signal in 12 samples. Of 39 negative samples, ELISA assay using rabbit pAb conjugates against WWT. taeniaeformis, gave a positive signal only in 3 samples. Therefore, in this experiment, the sensitivity was 92.3% and the specificity was 95.9%.

Example 2B

[0164]Obtaining mouse polyclonal and monoclonal antibodies against WE T. taeniaeformis and E/S T. taeniaeformis . Worms extract (WE)T. taeniaeformis and E/S productT. taeniaeformis received as described above. Mice were immunized with WET. taeniaeformis or E/ST. taeniaeformis, as described above for the production of mouse polyclonal antibodies, namely mouse pAb against W.E.T. taeniaeformis and mouse pAb against E/ST. taeniaeformis. These polyclonal antibodies specifically bind to their respective immunogens when these immunogens have been applied to microtiter plates.

A. Preparation of anti-WE mAb T. taeniaeformis

[0165] Hybridomas were obtained from mice immunized with T. taeniaeformis WE as described above. From the obtained mouse monoclonal antibody (mouse mAb) secreting hybridomas, two mouse anti- T. taeniaeformis WE mAbs, such as ADX184 (IgG) and ADX185 (IgM), were selected because they gave a strong signal and low background in ELISA assays.

B. Efficiency of np coproantigen ELISA assay using mouse mAbs against T. taeniaeformis WE

[0166] To assess sensitivity, a coproantigen sandwich ELISA was performed using ADX185 coated on a microtiter plate, followed by the addition of a patient sample, and then with the addition of a rabbit pAb-HRP anti-WE conjugateT. taeniaeformis. The analysis was performed on FEX obtained from 7 cats positive forT. taeniaeformis, and 3 cats negative forT. taeniaeformis. As shown in FIG. 5, analysis detected coproantigen in 7 of 7 positive samples and none of 3 negative samples. Thus, in this experiment, the sensitivity of the ELISA assay performed using ADX185/rabbit pAb-HRP against WET. taeniaeformis was 100%.

[0167] To assess specificity, a coproantigen sandwich ELISA was performed using ADX184 coated on a microtiter plate and ADX193-HRP conjugate applied after addition of a patient sample.

ADX193 is a mouse mAb against E/ST. taeniaeformisdescribed below. The analysis was carried out on WED. caninum, WET. pisiformis, WET. crassips, WET. taeniaeformis,E/S T. taeniaeformis, as well as on extracts of faeces of dogs positive forD. caninum; dogs negative forD. caninum; cats positive forD. caninum; cats negative forD. caninum; dogs positive forT. pisiformis; dogs negative forT. pisiformis; group of three cats positive forT. taeniaeformis, and a group of three cats negative forT. taeniaeformis. The above WE were used at a concentration of 1 μg/ml of protein. The assay was further run on several WE samples at 10 μg/ml protein and 2 μg/ml protein. WE samples having these higher concentrations are WED. caninum, WET. pisiformis, WET. taeniaeformis,W.E.T. crassips, WE hookwormsAncylostoma caninum, WE roundwormsToxocara canis and WE trichocephalusTrichuris vulpis. Among these samples, as shown in FIG. 6, the analysis was positive only for WET. taeniaeformis (at 10 µg/mL) and for the group of cats positive forT. taeniaeformis. Thus, the ADX184/ADX193-HRP ELISA was highly specific in this experiment.

C. Preparation and screening of mouse anti- E/S T. taeniaeformis mAb

[0168] Hybridomas were obtained from mice immunized with E/S T. taeniaeformis as described above. From the obtained hybridomas secreting mouse monoclonal antibody (mouse mAb), five mouse mAbs against E/S T. taeniaeformis were selected: ADX190, ADX191, ADX192, ADX193, ADX194. These five mouse mAbs were further evaluated for their potency in the ELISA assay when each was paired with a mouse pAb-HRP anti-E/S conjugate. Five tests for E/S T. taeniaeformis were performed, that is, analyzes of faecal extracts from seven T. taeniaeformis positive cats and three T. taeniaeformis negative cats. In each of the five assays, all 7 positive feline samples were detected. Each was also negative for the three negative feline samples, although ADX193 and ADX194 had higher background in one of the three negative feline samples.

Example 2C

Performance of ELISA performed using ADX184 with ADX193-HRP

[0169] ELISA was performed using ADX184 coated on microtiter plates and ADX193 conjugated to HRP. Both mouse mAbs were used at a concentration of 5 μg/ml.

[0170] The ADX 184/ADX193-HRP ELISA was performed on faecal extracts of six T. taeniaeformis positive cats, faecal extracts of four T. taeniaeformis negative cats, and one E/S T protein sample. taeniaeformis . As shown in FIG. 7, this analysis detected a coproantigen in all six positive fecal extracts and in the E/S sample, but did not detect a coproantigen in any of the four negative fecal extracts.

[0171] The ADX 184/ADX193-HRP ELISA was performed on fecal extracts from 68 T. taeniaeformis positive cats and 108 T. taeniaeformis negative cats that were positive for D. caninum. The assay was positive in 55 of 68 T. taeniaeformis positive specimens (sensitivity 80.9%) and positive in 1 of 108 T. taeniaeformis negative specimens (99.1% specificity; cross-reactivity with D. caninum , 0.9%).

2D example

Performance of ELISA performed using ADX191 with ADX194-HRP

[0172] An ELISA assay for coproantigen was performed using ADX191 coated on microtiter plates and ADX194 conjugated to HRP. Both mouse mAbs were used at a concentration of 3 μg/ml. ELISA analysis was performed on faecal extracts of six T. taeniaeformis positive cats, on fecal extracts of four T. taeniaeformis negative cats, and on one T. taeniaeformis E/S protein sample. As shown in FIG. 8, this analysis detected coproantigen in all six positive fecal extracts and in the E/S sample, but did not detect coproantigen in any of the four negative fecal extracts.

Example 3A

A. Preparation of rabbit pAb against TCA soluble fraction Dipylidium caninum (rabbit anti-TCA pAb D. caninum )

[0173] The TCA fraction of D. caninum worms was obtained as described above and used to immunize two rabbits. Antisera from one of these rabbits were collected for further analysis.

B. Evaluation by ELISA assay using rabbit anti-TCA D. caninum pAb on dog and cat samples.

[0174] In this coproantigen ELISA, rabbit pAb against TCAD. caninum plated, contacted with FEX and then with rabbit pAb-HRP anti-TCA conjugateD. caninum and with color substrate as described above.

[0175] For dogs: ELISA assay using rabbit pAb against TCAD. caninumwere performed on fecal extracts from 58 dogs positive forD. caninum,and this ELISA gave a positive signal in 57 out of 58 samples. This assay was also performed on faecal extracts from 27 dogs negative forD. caninum; and this ELISA gave a positive signal in 8 out of 27 samples. Therefore, in this experiment, the sensitivity of the assay was 98.3% and the specificity was 70.4%.

[0176] For cats: ELISA assay using rabbit pAb against TCAD. caninumwere performed on fecal extracts from 31 cats positive forD. caninum,and this ELISA gave a positive signal in 28 out of 31 samples. This assay was also performed on faecal extracts from 13 cats negative forD. caninum; and this ELISA gave a positive signal in 5 out of 13 samples. Therefore, in this experiment, the sensitivity of the assay was 90.3% and the specificity was 61.5%.

Example 3B

A. Preparation of rabbit pAb against the WE fraction D. caninum (rabbit pAb against W.E. D. caninum )

[0177] WE was prepared from D. caninum worms (Antibody Systems) as described above and used to immunize two rabbits. Antisera from one of these rabbits were collected for further analysis.

B. ELISA Assay Using Rabbit Anti -WE D. caninum pAb

[0178] In this ELISA, the rabbit pAb against W.E.D. caninum applied to plates, subjected to contact with FEX, at 5 μg/ml, and then with rabbit pAb-HRP anti-WE conjugateD. caninum at 3 µg/ml and with color substrate as described above. This ELISA assay using a rabbit pAb against W.E.D. caninum, carried out on extracts of worms isolated from helminths of several species: WED. caninum, WET. pisiformis, WET. crassips, WE hookwormAncylostoma caninum, WE roundwormsToxocara canis and WE trichocephalusTrichuris vulpis. As shown in FIG. 9, WE onlyD. caninum gave a positive signal in this ELISA assay, indicating the specificity of such an assay in this experiment.

[0179] Performance of ELISA assay using rabbit pAb against W.E.D. caninum, was evaluated in an additional experiment as follows. The analysis was performed on fecal extracts of 44 dogs positive forD. caninum, and 48 dogs negative forD. caninum. This assay gave a positive signal in 16 of the 44 positive samples, but not in one of the negative samples. Therefore, in this experiment, the sensitivity was 36.4% and the specificity was 100%.

Example 3C

A. Preparation of rabbit anti-E/S D. caninum pAb (rabbit anti -E/S D. caninum pAb )

[0180] E/S was prepared from D. caninum worms in dogs as described above and used to immunize two rabbits. Antisera from one of these rabbits were collected for further analysis.

B. ELISA Assay Using Rabbit Anti -E/S D. caninum pAb

[0181] In this coproantigen ELISA, rabbit pAb against E/SD. caninum plated, contacted with FEX and then with rabbit pAb-HRP anti-E/S conjugateD. caninum and with color substrate as described above.

[0182] The assay was performed on fecal extracts from 7 D. caninum positive dogs. This assay gave a positive signal in 4 out of 7 positive samples. This test gave a positive signal in 1 out of four D. caninum negative dogs. Therefore, in this experiment, the sensitivity was 57.1% and the specificity was 75%.

3D example

A. Preparation of rabbit anti-WE D. caninum mAb (rabbit anti-WE D. caninum mAb )

[0183] WE was prepared from whole canine D. caninum worms as described above. Rabbits were immunized with WE Dipylidium caninum according to the standard immunization procedure (SDIX, Windham, Maine, USA). Following a booster dose at least 3 weeks after the first immunization, a rabbit blood sample was used to produce monoclonal antibodies (ImmunoPrecise Antibodies, Ltd. (IPA), Victoria, British Columbia, Canada). Rabbit B cells were harvested and B cell culture supernatants were evaluated in 96-well plates coated with D. caninum WE and probed with a second anti-rabbit IgG antibody. Approximately 50 ml of B cell supernatants from positive wells were tested for specific binding in a second screen (IDEXX) with various fecal extracts. B cells of the best 32 candidates from this second screen were stored in lysis buffer for cloning purposes. From these 32 candidates, RNA was isolated and the variable regions of the heavy and light (kappa) rabbit antibody chains were cloned into separate mammalian expression vectors (IPA). Tissue culture supernatants harvested from mammalian cells transfected with antibody heavy and light chain vectors were evaluated again for various faecal extracts. The top five clones of the recombinant DNA constructs encoding the rabbit mAb were sequenced (IPA). Two of these five rabbit mAb clones, namely RDX13 and RDX12, were selected for further analysis.

B. Evaluation by ELISA assay using rabbit anti-WE D. caninum mAb on dog and cat samples.

[0184] In this series of coproantigen ELISAs, mouse ADX226 mAb was plated, contacted with FEX, and then with RDX13-HRP and/or RDX12-HRP conjugates and color substrate as described above.

[0185] In the first experiment, RDX13-HRP and RDX12-HRP conjugates were mixed and analyzed on fecal extracts of 38 D. caninum positive dogs, 28 D. caninum negative dogs, 20 D. caninum positive cats, and 7 cats negative for D. caninum . This test gave a positive signal in 28 of 38 dogs that tested positive, 2 of 28 dogs that tested negative, 18 of 20 cats that tested positive, and none of the 7 cats that tested negative. Thus, in this experiment, the assay has a sensitivity of 73.7% and specificity of 92.9% for canine samples and a sensitivity of 90.0% and specificity of 100% for feline samples.

[0186] In the second, third, and fourth experiments, RDX13-HRP and RDX12-HRP conjugates were used both alone and in admixture, and analysis was performed on faecal extracts of 37 D. caninum positive dogs, 28 D. caninum negative dogs. caninum , 21 D. caninum positive cats and 7 D. caninum negative cats.

[0187] In the second experiment, where RDX13-HRP was used alone, the assay gave a positive signal in 20 of 37 dogs that tested positive, 1 of 28 dogs that tested negative, 15 of 21 cats that tested positive, and none of the cats from 7 cats tested negative. Thus, in this experiment, the assay has a sensitivity of 54.1% and specificity of 96.4% for canine samples and a sensitivity of 71.4% and specificity of 100% for feline samples.

[0188] In the third experiment, where RDX12-HRP was used alone, the assay gave a positive signal in 13 of 37 dogs that tested positive, 1 of 28 dogs that tested negative, 16 of 21 cats that tested positive, and none of the cats from 7 cats tested negative. Thus, in this experiment, the assay has a sensitivity of 35.1% and a specificity of 96.4% for canine samples and a sensitivity of 76.2% and a specificity of 100% for feline samples.

[0189] In the fourth experiment, where the RDX13-HRP and RDX12-HRP conjugates were mixed, the assay gave a positive signal in 22 of 37 dogs with a positive result, in 1 of 28 dogs with a negative result, in 19 of 21 cats with a positive result, and none in one cat out of 7 cats with a negative result. Thus, in this experiment, the assay has a sensitivity of 59.5% and specificity of 96.4% for canine samples and a sensitivity of 90.5% and specificity of 100% for feline samples.

Example 3E

A. Preparation of mouse anti-WE D. caninum pAb (mouse anti-WE D. caninum pAb )

[0190] WE was prepared from wormsD. caninum dogs as described above and used to immunize mice. The resulting antiserum, i.e., mouse anti-WE pAbD. caninum, used in the ELISA experiment described below.

B. ELISA assay using mouse anti -WE D. caninum pAb on dog and cat samples

[0191] In this coproantigen ELISA, the mouse pAb against W.E.D. caninum plated, contacted with FEX and then with mouse pAb-HRP conjugate against WED. caninum and with color substrate as described above.

[0192] This assay was performed on faecal extracts from four D. caninum positive dogs and 3 D. caninum positive cats; one cat negative for D. caninum and infected with Giardia and T. taeniaeformis ; one cat negative for D. caninum and infected with T. taeniaeformis ; and one dog infected with Toxocara and Taenia pisiformis . As shown in FIG. 10, the assay was positive in 4 out of 4 dogs that tested positive, none of the dogs tested negative, 3 of 3 cats tested positive, and none of the 2 cats tested negative. Hence, in this experiment, the assay showed a high degree of specificity and sensitivity.

Example 3F

A. Obtaining mouse mAb against the soluble fraction of TCA D. caninum (mouse anti-TCA mAb D. caninum )

[0193] A soluble fraction of TCA was obtained from D. caninum worms from dogs as described above and used for mouse immunization (MBS). The spleen of an immunized mouse was used to generate a mouse mAb, ADX251, which was used in the following ELISA experiments.

B. Evaluation by ELISA assays using mouse anti-TCA mAb D. caninum on samples taken from dogs and cats

[0194] In the first coproantigen ELISA configuration, mouse ADX226 mAb was plated, contacted with FEX, and then with mouse ADX251-HRP mAb conjugate and color substrate.

[0195] The assay was performed on fecal extracts from 3 D. caninum positive dogs, 5 D. caninum negative dogs, 3 D. caninum positive cats, and 1 D. caninum negative cat. As shown in FIG. 11, the assay was positive in 3 of 3 dogs that tested positive, none of the 5 dogs that tested negative, 3 of 3 cats that tested positive, and no cats that tested negative. Hence, in this experiment, the assay showed a high degree of specificity and sensitivity.

[0196] In the second configuration of the coproantigen ELISA, mouse mAb ADX251 was plated, contacted with FEX and then mouse mAb ADX227-HRP conjugate and color substrate. The assay was performed on fecal extracts from 3 D. caninum positive dogs; 5 dogs negative for D. caninum ; 3 D. caninum positive cats and 1 D. caninum negative cat. As shown in FIG. 12, the assay was positive in 3 of 3 dogs that tested positive, none of the 5 dogs that tested negative, 3 of 3 cats that tested positive, and no cats that tested negative. Hence, in this experiment, the assay showed a high degree of specificity and sensitivity.

[0197] In the third configuration of the coproantigen ELISA, mouse ADX251 mAb was plated, contacted with FEX and then with rabbit anti-WE D. caninum pAb-HRP conjugate and color substrate. The assay was performed on fecal extracts from 3 D. caninum positive dogs; 5 dogs negative for D. caninum; 3 cats positive for D. caninum; and 1 cat negative for D. caninum . As shown in FIG. 13, the assay was positive in 3 of 3 dogs that tested positive, none of the 5 dogs that tested negative, 3 of 3 cats that tested positive, and no cats that tested negative. Hence, in this experiment, the assay showed a high degree of specificity and sensitivity.

3G example

A. Preparation of mouse anti-WE D. caninum mAb (mouse anti-WE D. caninum mAb )

[0198] D. caninum WE was prepared from D. caninum worms from dogs as described above and used to immunize mice. Four monoclonal antibodies (ADX224, ADX225, ADX226 and ADX227) were obtained from these mice as described above and these antibodies were selected for further analysis.

B. Evaluation by ELISA assays using mouse anti-WE D. caninum mAb on dog and cat samples

[0199] ELISA assays in the following configurations were performed on fecal extracts of 3 D. caninum positive dogs, 4 D. caninum negative dogs , 3 D. caninum positive cats , and 1 D. caninum negative cat.

[0200] In the first series of coproantigen ELISA configurations, mouse anti-WE D. caninum mAbs were plated separately, contacted with FEX and then rabbit anti-WE D. caninum pAb-HRP conjugate and color substrate. The results are shown in FIG. 14. Thus, the assay performed using ADX224 on the plate detected coproantigen in 5 out of six positive samples and its absence in all negative samples. Analysis using ADX225 on a plate detected coproantigen in 6 out of six positive samples and its absence in all negative samples. Analysis using ADX226 on a plate detected coproantigen in 6 out of six positive samples and its absence in all negative samples. Analysis using ADX227 on a plate detected coproantigen in six out of six positive samples and its absence in all negative samples.

[0201] In a second series of coproantigen ELISA configurations, each of the four mouse anti-WE D. caninum mAbs was plated separately, contacted with FEX, and then with mouse anti-WE D. caninum mAb-HRP conjugates and a color substrate. The results are shown in FIG. 15. From these test combinations, two pairs were selected. Analysis using ADX226 on plate and ADX227-HRP detected coproantigen in six of six positive samples and absent from all negative samples, and analysis using ADX227 on plate and ADX227-HRP detected coproantigen in six of six positive samples. samples and its absence in all negative samples. This successful pairing of ADX227 with itself suggests that the coproantigen detected by ADX227 contains a repeating epitope.

Example 3H

A. Antigen characterization: Glycosylation of antigens associated with the D. caninum antibody ADX226 .

[0202] In order to determine whether the coproantigen associated with mouse mAb ADX226 against WE D. caninum is glycosylated, the ability of 21 different lectins to bind to coproantigen was tested using a commercially available kit (kits with biotinylated lectins I, II and III from Vector Laboratories, Burlingame, CA). Analyzes were performed according to the manufacturer's instructions. Briefly, ADX226 was applied to Immulon lb. plates. FEX dogs positive or negative for D. caninum were added to the plates. After washing, lectin:biotin conjugates were added, followed by streptavidin-HRP and color substrate. The results are shown in FIG. 16. In this assay, the following lectins PSA, LCA, yacalin, ECL, GSL1, RCA123 and WGA specifically and strongly bind to ADX226 against the coproantigen. These lectin binding data indicated that the D. caninum coproantigen associated with ADX226 is glycosylated.

B. Antigen characterization: glycosylation of antigens associated with rabbit anti-WE D. caninum pAb .

[0203] In order to determine whether the coproantigen associated with the rabbit anti-WE D. caninum pAb is glycosylated, the ability of 21 different lectins to bind to the coproantigen was tested using a commercially available kit (kits with biotinylated lectins I, II and III from Vector Laboratories, Burlingame, CA). Analyzes were performed according to the manufacturer's instructions. Briefly, rabbit anti-WE D. caninum pAb was applied to Immulon lb. plates. FEX dogs positive or negative for D. caninum were added to the plates. After washing, lectin:biotin conjugates were added, followed by streptavidin-HRP and color substrate. In this assay, the following lectins yacalin, WGA, WGA succinylated, GSL II were associated with a rabbit pAb against D. caninum WE coproantigen. The results are shown in FIG. 17. These lectin binding data indicated that the D. caninum coproantigen associated with the rabbit anti-WE D. caninum pAb is glycosylated. These data also showed that the lectin antibody sandwich assay could be used to detect the D. caninum coproantigen.

C. Antigen characterization: glycosylation of antigens associated with anti- D. caninum antibodies , namely rabbit anti-WE D. caninum pAb and ADX187

[0204] An analysis was performed in a similar configuration in order to determine whether the coproantigen(s) associated with the rabbit anti-WE D. caninum pAb and ADX187 (mouse anti-WE D. caninum mAb), glycosylated(s), and the ability of 21 different lectins to bind to coproantigen was tested in this assay using a commercially available kit (biotinylated lectin kits I, II and III from Vector Laboratories, Burlingame, CA). In this configuration, 21 different biotinylated lectins were loaded onto Immulon lb. plates. FEX dogs positive or negative for D. caninum were added to the plates. After washing, rabbit pAb-HRP or ADX187-HRP anti-WE D. caninum conjugates were added, followed by streptavidin-HRP and color substrate. The results are shown in FIG. 18. In this assay, the following lectins: jacalin, WGA, and succinylated WGA were bound to a rabbit pAb against D. caninum WE coproantigen. The same series of lectins: yacalin, WGA, and succinylated WGA bind to ADX187 against a coproantigen. These lectin binding data confirm that the D. caninum coproantigen(s) bound to the rabbit anti-WE D. caninum pAb and ADX187 are glycosylated(s). These data also showed that the lectin antibody sandwich assay could be used to detect the D. caninum coproantigen.

[0205] Further testing for O-glycosylation of coproantigen recognized by ADX226

In this experiment, ADX226 was loaded onto microtiter plates followed by FEX, yacalin-biotin, streptavidin-HRP, and finally HRP substrate. FEX was prepared from fecal extracts of 20 D. caninum positive dogs, 24 D. caninum negative dogs, 12 D. caninum positive cats, and 3 D. caninum negative cats. The assay was positive in 18 out of 20 dogs positive (95% sensitivity), none out of 24 dogs negative (100% specificity), 11 out of 12 cats positive (91.7% sensitivity) and none. in one of 3 cats with a negative result (100% specificity). These data indicated that the coproantigen associated with ADX226 is O-glycosylated and that a sandwich immunoassay using an antibody and a lectin can be performed to detect the tapeworm coproantigen.

Example 4

[0206] A series of faecal sandwich ELISAs were performed in microtiter plates essentially as described in Example A above to confirm the specificity of the ELISA assays for the detection of T. pisiformis tapeworms, T. taeniaeformis tapeworms, D tapeworms caninum , hookworms Ancylostoma caninum ( A. caninum ), hookworms Ancylostoma tubaeforme ( A. tubaeforme ), roundworms Toxocare canis ( T. сanis ), roundworms Toxocara cati ( T. cati ), trichocephalus Trichuris vulpis ( T. vulgaris ), the trichocephalus Trichuris felis ( T. felis ) and the protozoan Giardia Lamblia ( Giardia ).

[0207] Fecal extracts from the following sources were tested in ELISAs: T. pisiformis positive dogs (Figure 19, column 1), T. taeniaeformis infected cats (Figure 19, column 2), D. caninum (Fig. 19, column 3), cats infected with D. caninum (Fig. 19, column 4), dogs infected with A. caninum hookworm (Fig. 19, column 5), cats infected with A. tubaeforme hookworm ( Fig. 19, column 6), dogs infected with T. canis roundworms (Fig. 19, column 7), cats infected with T. cati roundworms (Fig. 19, column 8), dogs infected with T. vulpis trichocephalus (Fig. 19, column 9), cats infected with trichocephalus T. felis (Fig. 19, column 10), dogs infected with Giardia (Fig. 19, column 11), cats infected with Giardia (Fig. 19, column 12) , and two cats infected with parvovirus (Fig. 19, columns 13 and 14). In FIG. 19, columns 1-12 are an image of one microtiter plate, and columns 13 and 14 are an image of another individual microtiter plate.

[0208] T. pisiformis tapeworm coproantigen ELISA assay (Figure 19, row A) . A sandwich ELISA was performed to detect the T. pisiformis coproantigen. Briefly, mAb ADX131 was applied to microtiter plates, incubated with fecal extract, and then incubated with mAb ADX132-HRP conjugate followed by detection using a peroxidase substrate. ADX131 was applied at a concentration of 3 μg/ml. ADX132-HRP was used at a concentration of 3 μg/ml. The results showed that this T. pisiformis coproantigen ELISA signaled positive for FEX from T. pisiformis infected dogs. However, this T. pisiformis ELISA did not detect a coproantigen in the FEX of any of the following animals: cats infected with T. taeniaeformis tapeworms; dogs infected with D. caninum; cats infected with D. caninum; dogs infected with A. caninum hookworm; cats infected with A. tubaeforme hookworm; dogs infected with T. canis roundworms; cats infected with T. cati roundworms; dogs infected with trichocephalus T. vulpis; cats infected with trichocephalus T. felis; dogs infected with Giardia; to puss infected with Giardia; and cats infected with parvovirus (Fig. 19, row A). Thus, this T. pisiformis ELISA specifically detected a T. pisiformis coproantigen that did not cross-react with any of the following coproantigens: T. taeniaeformis tapeworms, D. caninum tapeworms, A. caninum hookworm, T roundworms canis , roundworms T. cati , trichocephalus T. vulpis , trichocephalus T. felis, protozoa Giardia Lamblia and feline parvovirus. Thus, this T. pisiformis ELISA was highly species specific. This T. pisiformis ELISA is suitable for detecting or diagnosing T. pisiformis infection or infection. This T. pisiformis ELISA can be used to distinguish T. pisiformis infection from infection with T. taeniaeformis tapeworms, D. caninum tapeworms, A. caninum hookworm, A. tubaeforme hookworm, T. canis roundworms , T. cati roundworms, T. vulpis trichocephalus, T. felis trichocephalus, Giardia lamblia protozoa, canine parvovirus, and feline parvovirus.

[0209] T. taeniaeformis tapeworm coproantigen ELISA assay (Figure 19, row B) . A sandwich ELISA was performed to detect the Taenia taeniaeformis coproantigen . Briefly, mAb ADX184 was applied to microtiter plates, incubated with fecal extract, and then incubated with mouse mAb ADX193-HRP conjugate, followed by detection using a peroxidase substrate. ADX184 was applied at a concentration of 5 μg/ml. ADX193-HRP was used at a concentration of 3 μg/ml. The results showed that this T. taeniaeformis coproantigen ELISA signaled positive for FEX from T. taeniaeformis infected dogs. However, this T. taeniaeformis ELISA did not detect a coproantigen in the FEX of any of the following animals: cats infected with Taenia pisiformis tapeworms; dogs infected with D. caninum; cats infected with D. caninum; dogs infected with A. caninum hookworm; cats infected with A. tubaeforme hookworm; dogs infected with T. canis roundworms; cats infected with T. cati roundworms; dogs infected with trichocephalus T. vulpis; cats infected with trichocephalus T. felis; dogs infected with Giardia; to puss infected with Giardia; and cats infected with parvovirus (Fig. 19, row B). Thus, this T. taeniaeformis ELISA specifically detected a T. taeniaeformis coproantigen that did not cross-react with any of the following coproantigens: T. pisiformi tapeworms, D. caninum tapeworms, A. caninum hookworm, A. tubaeforme, T. canis roundworms, T. cati roundworms, T. vulpis trichocephalus, T. felis trichocephalus, Giardia Lamblia protozoa , and feline parvovirus. Thus, this T. taeniaeformis ELISA was highly species specific. This T. taeniaeformis ELISA is suitable for detecting or diagnosing T. taeniaeformis infection or infection. This T. taeniaeformis ELISA can be used to distinguish T. taeniaeformis infection from infection with T. pisiformis tapeworms, D. caninum tapeworms, A. caninum hookworm, A. tubaeforme hookworm, T. canis roundworms , T. cati roundworms, T. vulpis trichocephalus, T. felis trichocephalus, Giardia lamblia protozoa, canine parvovirus, and feline parvovirus.

[0210] D. caninum tapeworm coproantigen ELISA assay (Figure 19, row C) . A sandwich ELISA was performed to detect D. caninum coproantigen. Briefly, mAb ADX226 was applied to microtiter plates, incubated with fecal extract, and then incubated with mouse mAb RDX5-HRP conjugate, followed by detection using a peroxidase substrate. ADX226 was applied at a concentration of 3 μg/ml. RDX5-HRP was used at a concentration of 3 μg/ml. The results showed that this D. caninum coproantigen ELISA signaled positive for FEX from D. caninum infected dogs and D. caninum infected cats . However, this D. caninum ELISA did not detect coproantigen in the FEX of any of the following animals: dogs infected with T. pisiformis tapeworms; cats infected with T. taeniaeformis, dogs infected with A. caninum hookworm; cats infected with A. tubaeforme hookworm; dogs infected with T. canis roundworms; cats infected with T. cati roundworms; dogs infected with trichocephalus T. vulpis; cats infected with trichocephalus T. felis; dogs infected with Giardia; to puss infected with Giardia; and cats infected with parvovirus (Fig. 19, row C). Thus, this D. caninum ELISA specifically detected a D. caninum coproantigen that did not cross-react with any of the following coproantigens: T. taeniaeformis tapeworms; tapeworms T. pisiformi , hookworm A. caninum , hookworm A. tubaeforme; roundworms T. canis ; roundworms T. cati ; trichocephala T. vulpis ; trichuria T. felis; protozoan Giardia lamblia and feline parvovirus. Thus, this D. caninum ELISA was highly species-specific and is suitable for detecting or diagnosing D. caninum infection or infection. This D. caninum ELISA can be used to distinguish D. caninum infection from infection with T. pisiformis tapeworms, T. taeniaeformis tapeworms , A. caninum hookworm, A. tubaeforme hookworm, T. canis roundworms , roundworms T. cati , trichocephalus T. vulpis , trichocephalus T. felis, protozoa Giardia Lamblia; canine parvovirus and feline parvovirus.

[0211]ELISA assay for hookworm coproantigen Ancylostoma (Fig. 19, row D). A sandwich ELISA was performed to detect coproantigenA. caninum. Briefly, a rabbit anti-ASP5-l polyclonal antibody was plated on microtiter plates, incubated with fecal extract, and then incubated with a mouse anti-ASP5-l mAb-HRP conjugate followed by detection using a peroxidase substrate as discussed in US Pat. No. 7,951,547, which is fully volume is included in the present description by reference. Rabbit polyclonal antibody was applied at a concentration of 5 μg/ml. The mouse mAb was used at a concentration of 4 μg/ml. The results showed that this coproantigen ELISAAncylostoma gives a positive signal for FEX from infected dogsD. caninum, and cats infectedA. tubaeforme. However, this ELISA assayAncylostoma did not detect coproantigen in FEX of any of the following animals: tapeworm-infected dogsT. pisiformis; cats, infected with tapewormsT. taeniaeformis; dogs infected with tapewormsD. caninum; cats infected with tapewormsD. caninum,dogs infected with roundwormsT. canis; cats infected roundwormsT. cati;, dogs infected with trichocephalusT. vulpis; cats infected with trichocephalusT. felis; dogs infectedGiardia; tooats infectedGiardia; and cats infected with parvovirus (Fig. 19, row D). Therefore, this ELISA assay onAncylostoma specifically detected coproantigenA. caninum andA. tubaeforme, which does not cross-react with any of the following coproantigens: tapewormsT. taeniaeformis; tapewormsT. pisiformi, tapewormsD. caninum; roundwormsT. canis; roundwormsT. cati; trichocephalusT. vulpis; trichocephalusT. felis; protozoaGiardia Lambliaand feline parvovirus. Thus, this ELISA assay forAncylostomawas highly specific and is suitable for detecting or diagnosing hookworm infection or infestationA. caninum andA. tubaeforme. In addition, this ELISA assay onAncylostoma can be used to distinguish hookworm infectionA. caninum orA. tubaeforme from tapeworm infectionT. pisiformis, tapewormsT. taeniaeformis, tapewormsD. caninum;roundwormsT. canis, roundwormsT. cati, trichocephalomaT. vulpis, trichocephalomaT. felis, protozoaGiardia Lamblia; canine parvovirus and feline parvovirus.

[0212]ELISA assay for roundworm coproantigen Toxocara (Fig. 19, row E). A sandwich ELISA was performed to detect coproantigenT. canis andT. cati. Briefly, mouse mAb against DIV6728C proteinT. canis were applied to microtiter plates, incubated with faecal extract, and then incubated with a conjugate of HRP and another mouse mAb against DIV6728C proteinT. canis followed by detection using a peroxidase substrate as discussed in US Pat. No. 7,951,547, which is incorporated herein by reference in its entirety. ADX5 was applied at a concentration of 6 μg/ml. ADX10-HRP was used at a concentration of 5 μg/ml. The results showed that this coproantigen ELISAToxocaragives a positive signal for FEX from infected dogsT. canis andcats, infectedT. cati. However, this ELISA assayToxocaradid not detect coproantigen in FEX of any of the following animals: tapeworm-infected dogsT. pisiformis; cats, infectedT. taeniaeformis; dogs infected with tapewormsD. caninum; cats infected with tapewormsD. caninum,dogs infected with hookwormA. caninum; cats infected hookwormA. tubaeforme; dogs infected with trichocephalusT. vulpis; cats infected with trichocephalusT. felis; dogs infectedGiardia; tooats infectedGiardia; and cats infected with parvovirus (Fig. 19, row E). Thus, this ELISA assay forToxocaraspecifically detected coproantigentoxocara,which does not cross-react with any of the following coproantigens: tapewormsT. taeniaeformis; tapewormsT. pisiformis, tapewormsD. caninum; hookwormsA. caninum;hookwormsA. tubaeforme; trichocephalusT. vulpis; trichocephalusT. felis; protozoaGiardia Lambliaand feline parvovirus. Thus, this ELISA assay forToxocarawas highly specific and is suitable for detecting or diagnosing infection or infectionT. canis andT. cati. This roundworm ELISAToxocaracan be used to distinguish roundworm infectionT. canis orT. cati from tapeworm infectionT. pisiformis, tapewormsT. taeniaeformis, tapewormsD. caninum;hookwormA. caninum; hookwormA. tubaeforme; trichocephalusT. vulpis, trichocephalomaT. felis, protozoaGiardia Lamblia; canine parvovirus and feline parvovirus.

[0213]ELISA assay for trichocephalus coproantigen Trichuris (Fig. 19, row F).

[0214] A coproantigen sandwich ELISA was performed to detect coproantigenT. vulpis. Briefly, mouse mAb against DIV6901 protein (ADX6)T. vulpis applied to microtiter plates, incubated with faecal extract, and then incubated with a conjugate of HRP and another mouse anti-DIV6901 (ADX14) mAbT. vulpis followed by detection using a peroxidase substrate as discussed in US Pat. No. 7,951,547, which is incorporated herein by reference in its entirety. ADX6 was applied at a concentration of 3 μg/ml. ADX10-HRP was used at a concentration of 3 μg/ml. The results showed that this coproantigen ELISATrichurisgives a positive signal for FEX from infected dogsT. vulpis, andcats, infectedT. felis. However, this ELISA assayTrichurisdid not detect coproantigen in FEX of any of the following animals: tapeworm-infected dogsT. pisiformis; cats, infectedT. taeniaeformis; dogs infected with tapewormsD. caninum; cats infected with tapewormsD. caninum,dogs infected with hookwormA. caninum; cats infected hookwormA. tubaeforme; dogs infected with roundwormsT. canis;cats infected with roundwormsT. cati; dogs infectedGiardia; tooats infectedGiardia; and cats infected with parvovirus (Fig. 19, row F). Thus, this ELISA assay forTrichurisspecifically detected coproantigenT. vulpisandT. felis,which does not cross-react with any of the following coproantigens: tapewormsT. taeniaeformis; tapewormsT. pisiformis, tapewormsD. caninum; hookwormsA. caninum;hookwormsA. tubaeforme; roundwormsT. canis;roundwormsT. cati; protozoaGiardia Lambliaand feline parvovirus. Thus, this ELISA assay forTrichuriswas highly specific and is suitable for detecting or diagnosing infection or infectionT. vulpisandT. felis. This ELISA test for trichocephalusTrichuriscan be used to distinguish trichocephalus infectionT. vulpisorT. felisfrom tapeworm infectionT. pisiformis, tapewormsT. taeniaeformis, tapewormsD. caninum;hookwormA. caninum; hookwormA. tubaeforme; roundwormsT. canis;roundwormsT. cati; protozoaGiardia Lamblia; canine parvovirus and feline parvovirus.

[0215]ELISA for coproantigen Giardia (Fig. 19, row G). A sandwich ELISA was performed to detect coproantigenGiardia lamblia using an antibody pair that specifically detects a soluble antigen (SNAP® Giardia Test, IDEXX Laboratories, Inc. Westbrook, ME, USA) (Olson ME, Leonard NJ, Strout J. Prevalence and Diagnosis of Giardia Infection In dogs and cats Using a coryal antigen test and fail mass Can Vet J. 2010 Jun;51(6):640-2.PMID:20808578). Briefly, a rabbit polyclonal antibody againstGiardia applied to microtiter plates, incubated with fecal extract, and then incubated with a conjugate of HRP and mouse monoclonal antibody againstGiardia followed by detection using a peroxidase substrate. The polyclonal antibody was applied at a concentration of 3 μg/ml. mAb-HRP was used at a concentration of 3 μg/ml. The results showed that this coproantigen ELISAGiardia gives a positive signal for FEX from infected dogsGiardia,and cats, infectedGiardia. However, this ELISA assayGiardia did not detect coproantigen in FEX of any of the following animals: tapeworm-infected dogsT. pisiformis; cats, infected with tapewormsT. taeniaeformis; dogs infected with tapewormsD. caninum; cats infected with tapewormsD. caninum,dogs infected with hookwormA. caninum; cats infected hookwormA. tubaeforme; dogs infected with roundwormsT. canis; cats, infected roundwormsT. cati; dogs infected with trichocephalusT. vulpis; cats infected with trichocephalusT. felis; and cats infected with parvovirus (Fig. 19, row G). Thus, this ELISA assay forGiardia specifically detected coproantigenGiardia lamblia, which does not cross-react with any of the following coproantigens: tapewormsT. taeniaeformis; tapewormsT. pisiformi, tapewormsD. caninum; hookwormsA. caninum;hookwormsA. tubaeforme; roundwormsT. canis; roundwormsT. cati; trichocephalusT. vulpis; trichocephalusT. felis; and feline parvovirus. Thus, this ELISA assay forGiardia was highly species-specific and suitable for detecting or diagnosing infectionGiardia lamblia. This ELISA assay forGiardia can be used to distinguish between infectionGiardia lamblia from tapeworm infectionT. pisiformis, tapewormsT. taeniaeformis, tapewormsD. caninum;hookwormA. caninum; hookwormA. tubaeforme;roundwormsT. canis, roundwormsT. cati, trichocephalomaT. vulpis, trichocephalomaT. felis; canine parvovirus and feline parvovirus.

[0216] The parvovirus positive infection status of cats (FIG. 19, columns 13 and 14) was confirmed using the SNAP® parvovirus assay (M. Abd-Eldaim, M. Beall and MA Kennedy. “Detection of feline panleukopenia virus using a commercial ELISA for canine parvovirus" Vet Ther. 2009 Winter; 10(4): E1-6) according to the manufacturer's instructions (IDEXX Laboratories Inc., Westbrook, Maine, USA). This multiplex faecal ELISA allows simultaneous detection of T. pisiformis tapeworm, T. taeneaeformis tapeworm, D. caninum tapeworm, A. caninum hookworm; hookworm A. tubaeforme ; roundworms T. canis; roundworms T. cati ; the trichocephalus T. vulpis, the trichocephalus T. felis , and the protozoan Giardia Lamblia .

[0217] These data have shown that the methods of the invention can be used to facilitate the detection and species-specific differentiation of infections caused by at least eight different intestinal parasites.

Example 5

[0218] A number of faecal ELISAs have been developed capable of detecting two or three species of tapeworms of the genus Taenia , eg, T. pisiformis , T. taeniaeformis , and the genus Dipylidium , eg, D. caninum . ELISA was carried out in microtiter plates, essentially as described above in the "Example A" section.

[0219] Fecal extracts from the following sources were analyzed in ELISA assays: four dogs positive for T. pisiformis (Fig. 20, columns 1, 4, 7, and 10), four cats infected with T. taeniaeformis (Fig. 20, columns 2, 5, 8 and 11), two dogs infected with D. caninum (Fig. 20, columns 3 and 6), two cats infected with D. caninum (Fig. 20, columns 9 and 12). In FIG. 20, columns 1-12 represent an image of a single microtiter plate.

[0220] Coproantigen ELISA for the detection of tapeworms of the genus Taenia (Fig. 20, row A) . A sandwich ELISA was performed to detect coproantigen in animals infected with Taenia tapeworms. Briefly, mAb ADX131 and mAb ADX184 were applied to microtiter plates at 3 µg/ml, incubated with fecal extract, and then incubated with mAb ADX132-HRP and ADX193-HRP conjugates at 3 µg/ml each, followed by detection using substrate peroxidases. The results showed that this Taenia coproantigen ELISA signaled positive for FEX from T. pisiformis infected dogs (Fig. 20, row A, columns 1, 4, 7, and 10) and T. taeniaeformis infected cats (Fig. 20, row A, columns 2, 5, 8 and 11). However, this Taenia ELISA did not detect coproantigen in FEX in dogs infected with D. caninum tapeworms (Fig. 20, row A, columns 3 and 6) or cats infected with D. caninum (Fig. 20, row A , columns 9 and 12). Therefore, this Taenia ELISA allows specific detection of T. pisiformis and T. taeniaeformis coproantigen , which does not cross-react with D. caninum coproantigen. This Taenia ELISA is suitable for detecting or diagnosing infection or infection with one or more Taenia tapeworms, including T. pisiformis and T. taeniaeformis . This Taenia ELISA can be used to distinguish infection with one or more Taenia tapeworms, including T. pisiformis and T. taeniaeformis , from infection with Dipylidium tapeworms, including D. caninum .

[0221] Coproantigen ELISA for the detection of T. pisiformis and D. caninum tapeworms (Figure 20, row B) . A sandwich ELISA was performed to detect coproantigen in animals infected with T. pisiformis and D. caninum tapeworms. Briefly, mAb ADX131 and mAb ADX226 were applied to microtiter plates at a concentration of 3 μg/ml, incubated with fecal extract, and then incubated with conjugates of mAb ADX132-HRP and mAb RDX5-HRP at a concentration of 3 μg/ml each, followed by detection using peroxidase substrate. The results showed that this ELISA signaled positive for FEX from dogs infected with T. pisiformis (Fig. 20, row D, columns 1, 4, 7 and 10), dogs infected with D. caninum tapeworms (Fig. 20 , row B, columns 3 and 5) and cats infected with D. caninum (Fig. 20, row B, columns 9 and 12). However, this ELISA did not detect coproantigen in FEX in cats infected with T. taeniaeformis (FIG. 20, row B, columns 2, 5, 8 and 11). Therefore, this ELISA allows the specific detection of T. pisiformis and D. caninum coproantigen that does not cross-react with T. taeniaeformis coproantigen . This ELISA is suitable for detecting or diagnosing infection or infection with T. pisiformis and/or D. caninum tapeworms and can be used to distinguish infection with T. pisiformis and/or D. caninum tapeworms from infection with T. taeniaeformis .

[0222] Coproantigen ELISA for the detection of T. taeniaeformis and D. caninum tapeworms (Figure 20, row C) . A sandwich ELISA was performed to detect coproantigen in animals infected with T. taeniaeformis and D. caninum tapeworms. Briefly, mAb ADX184 and mAb ADX226 were applied to microtiter plates at 3 μg/ml, incubated with fecal extract, and then incubated with mAb ADX193-HRP and RDX5-HRP conjugates at 3 μg/ml each, followed by detection using substrate peroxidases. The results showed that this ELISA signaled positive for FEX from cats infected with T. taeniaeformis (Fig. 20, row C, columns 2, 5, 8 and 11), dogs infected with D. caninum tapeworms (Fig. 20 , row C, columns 3 and 6), and cats infected with D. caninum (Fig. 20, row C, columns 9 and 12). However, this ELISA did not detect coproantigen in FEX in dogs infected with T. pisiformis (FIG. 20, row C, columns 1, 4, 7 and 10). Therefore, this ELISA allows specific detection of T. taeniaeformis and D. caninum coproantigen that does not cross-react with T. pisiformis coproantigen. This ELISA is suitable for detecting or diagnosing infection or infection with T. taeniaeformis and/or D. caninum tapeworms and can be used to distinguish infection with T. taeniaeformis and/or D. caninum tapeworms from infection with T. pisiformis .

[0223]Coproantigen ELISA for the detection of tapeworms T. pisiformis, T. taeniaeformis and D. caninum (Fig. 20, row D). A sandwich ELISA was performed to detect coproantigen in animals infected with tapeworms.T. pisiformis, T. taeniaeformis andD. caninum. Briefly, mAb ADX131, mAb ADX184, and mAb ADX226 were applied to microtiter plates at a concentration of 3 μg/mL, incubated with fecal extract, and then incubated with mAb ADX132-HRP, mAb ADX193-HRP, and RDX5-HRP at 3 μg/mL. each, followed by detection using a peroxidase substrate. The results showed that this ELISA was positive for FEX from dogs infected withT. pisiformis(Fig. 20, row D, columns 1, 4, 7 and 10), cats infected with tapewormsT. taeniaeformis (Fig. 20, row D, columns 2, 5, 8, and 11), tapeworm-infected dogsD. caninum (Fig. 20, row D, columns 3 and 6), and cats infectedD. caninum (Fig. 20, row D, columns 9 and 12). Therefore, this ELISA assay allows the detection of coproantigenT. pisiformis, T. taeniaeformis andD. caninum. This ELISA is suitable for detecting or diagnosing tapeworm infection or infestation.T. pisiformis, T. taeniaeformis and/orD. caninum. The data obtained showed that this ELISA could be used to facilitate the detection of tapeworm infection.Taenia and/ordipylidium in dogs and cats.

Claims (145)

1. A method for detecting the presence or absence of one or more flatworm antigens in a fecal sample, comprising: (a) contacting a mammalian fecal sample with one or more antibodies selected from the group consisting of: (i) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (ii) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (iii) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen; (b) detecting the presence or absence of antibody-coproantigen complexes, if any. 2. A method for diagnosing infection of a mammal with one or more flatworms, comprising the steps: (a) contacting a mammalian fecal sample with one or more antibodies selected from the group consisting of: (i) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (ii) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (iii) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen; and (b) forming antibody-coproantigen complexes in the presence of coproantigens, if any, in the sample; (c) detecting the presence or absence of antibody-coproantigen complexes, if any; and (d) diagnosing the presence in a mammal of: (i) infection with a first tapeworm if an anti-first tapeworm antibody-coproantigen complex is present; (ii) infection with a second tapeworm if an anti-second tapeworm antibody-coproantigen complex is present; and (iii) infection with a third tapeworm if an anti-third tapeworm antibody-coproantigen complex is present. 3. A method for diagnosing and treating a mammal infected with one or more flatworms, comprising the steps of: (a) contacting a mammalian fecal sample with one or more antibodies selected from the group consisting of: (i) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (ii) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (iii) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen; and (b) forming antibody-coproantigen complexes in the presence of coproantigens, if any, in the sample; (c) detecting the presence or absence of antibody-coproantigen complexes, if any; and (d) diagnosing the presence in a mammal of: (i) infection with a first tapeworm if an anti-first tapeworm antibody-coproantigen complex is present; (ii) infection with a second tapeworm if an anti-second tapeworm antibody-coproantigen complex is present; and (iii) infection with a third tapeworm if an anti-third tapeworm antibody-coproantigen complex is present; and (e) administering an effective amount of one or more therapeutic agents for treating a mammal having a first tapeworm infection, a second tapeworm infection, or a third tapeworm infection, or a combination thereof. 4. The method of claim 3, wherein step (e) further comprises administering one or more additional therapeutic agents for: (a) treating the infection with one or more parasitic helminth worms, one or more non-worm parasites, one or more viruses, one or more fungi, one or more protozoa, or one or more bacteria. 5. The method according to any one of paragraphs. 3 or 4, wherein step (e) further comprises administering one or more therapeutic agents to (b) eradicate, repel, or kill the intermediate host of parasitic flatworms, parasitic helminth worms, non-worm parasites, viruses, fungi, protozoa, or bacteria . 6. The method according to any one of paragraphs. 1-5, where the fecal sample is obtained from a mammal that is a dog or cat. 7. The method according to any one of paragraphs. 1-6 where the first tapeworm is Taenia pisiformis. 8. The method of claim 7, wherein the first antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia taeniaeformis tapeworm, Dipylidium tapeworm, hookworm, roundworm, trichocephalus, Giardia , and parvovirus. 9. The method of claim 8, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 10. The method of claim 9 wherein the Dipylidium tapeworm is Dipylidium caninum ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 11. The method according to any one of paragraphs. 1-10 where the second tapeworm is Taenia taeniaeformis . 12. The method of claim 11, wherein the second antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia taeniaeformis tapeworm coproantigen, Dipylidium tapeworm coproantigen, hookworm coproantigen, roundworm coproantigen, trichocephalus coproantigen , Giardia coproantigen and parvovirus coproantigen. 13. The method of claim 12, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 14. The method of claim 13 wherein the Dipylidium tapeworm is Dipylidium caninum ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 15. The method according to any one of paragraphs. 1-14 where the third tapeworm is Dipylidium caninum . 16. The method of claim 15, wherein the third antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia tapeworm coproantigen, hookworm coproantigen, roundworm coproantigen, trichocephalic coproantigen, Giardia coproantigen, and parvovirus coproantigen . 17. The method of claim 16, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 18. The method of claim 17 wherein the Taenia tapeworms are Taenia pisiformis or Taenia taeniaeformis ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 19. The method according to any one of paragraphs. 1-6, where the first tapeworm and the second tapeworm belong to the species Taenia. 20. The method according to any one of paragraphs. 1-6 where the first tapeworm is Taenia pisiformis and the second tapeworm is Dipylidium caninum . 21. The method according to any one of paragraphs. 1-6 where the first tapeworm and the second tapeworm are Taenia and the third tapeworm is Dipylidium caninum . 22. The method according to any one of paragraphs. 1-6 where the first tapeworm is Taenia pisiformis , the second tapeworm is Taenia taeniaeformis , and the third tapeworm is Dipylidium caninum . 23. The method according to any one of paragraphs. 1-6, wherein the first, second, and third antibodies do not specifically bind to any coproantigen derived from at least one worm selected from the group consisting of roundworms, trichocephalus, and hookworm. 24. The method of claim 23 wherein the roundworms are Toxocara canis, Toxocara cati, Toxocara vitulorum, Toxascaris leonina, Baylisascaris procyonis, Ascaridia galli, Parascaris equorum, Ascaris suum , Ascaris lumbricoides , Anisakis simplex , or Pseudoterranova decipiens. 25. The method of claim 23 wherein the trichocephalus is Trichuris vulpis, Trichuris campanula, Trichuris serrata, Trichuris felis, Trichuris suis , Trichuris trichiura, Trichuris discolor, or Trichocephalus trichiuris . 26. The method of claim 23 wherein the hookworm is Ancylostoma caninum, Ancylostoma braziliense, Ancylostoma duodenal, Ancylostoma ceylanicum, Ancylostoma tubaeforme, Ancylostoma pluridentatum, Necator americanus, or Uncinaria stenocephala . 27. The method according to any one of paragraphs. 1-26, where the group of step (a) further consists of: (i) an antibody capable of specifically binding to a roundworm coproantigen, but not to a coproantigen derived from the group consisting of trichocephalus, hookworm, tapeworm, Giardia , and parvovirus; (ii) an antibody capable of specifically binding to a trichocephalic coproantigen, but not to a coproantigen derived from the group consisting of roundworms, hookworm, tapeworms, Giardia , and parvovirus; and (iii) an antibody capable of specifically binding to the hookworm coproantigen but not to the coproantigen derived from the group consisting of trichocephalus, roundworms, tapeworms, Giardia and parvovirus; (iv) an antibody capable of specifically binding to a Giardia ' coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and parvovirus coproantigen; and (v) an antibody capable of specifically binding to a parvovirus coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and Giardia coproantigen. 28. The method of claim 27 wherein the hookworms are Ancylostoma, the roundworms are Toxocara and the trichocephalus is Trichuris . 29. The method of claim 28, wherein the hookworms are Ancylostoma caninum or Ancylosloma tubaeforme ; roundworms are Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia ; and parvovirus is feline parvovirus or canine parvovirus. 30. The method according to any one of paragraphs. 2-29, where step (d) further includes diagnosis: (iv) infections with roundworms, if an anti-roundworm antibody-coproantigen complex is present, (v) infection with trichocephalus if an antibody-coproantigen complex against trichocephalus is present; (vi) infection with hookworm if an anti-hookworm antibody-coproantigen complex is present; (vii) infection with Giardia if an anti- Giardia antibody-coproantigen complex is present; and (viii) parvovirus infection if an anti-parvovirus antibody-coproantigen complex is present. 31. The method according to any one of paragraphs. 1-30 wherein the fecal sample is contacted with two or more antibodies, three or more antibodies, four or more antibodies, five or more antibodies, six or more antibodies, seven or more antibodies, or eight or more antibodies. 32. The method according to any one of paragraphs. 1-31, wherein the step of detecting the presence or absence of complexes further comprises the step of generating at least one second antibody that binds to one or more complexes. 33. The method of claim 32, wherein the second antibody is labeled or attached to a solid support. 34. The method according to any one of paragraphs. 1-32, wherein one or more of the first, second, and third antibodies are labeled. 35. The method according to any one of paragraphs. 1-32, where the first, second and third antibodies are immobilized on a solid support. 36. The method according to p. 30, where one or more antibodies that can specifically bind to coproantigen roundworms; antibodies capable of specifically binding to the trichocephalic coproantigen; antibodies capable of specifically binding to hookworm coproantigen; antibodies capable of specifically binding to Giardia coproantigen; and antibodies capable of specifically binding to parvovirus coproantigen are labeled. 37. The method according to p. 30, where the antibody is able to specifically bind to coproantigen of roundworms; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen; and an antibody capable of specifically binding to the parvovirus coproantigen are immobilized on a solid support. 38. The method according to any one of paragraphs. 35 or 37, where the solid support forms part of the ELISA device. 39. The method of claim 38, wherein the ELISA device is a sidestream immunoassay device. 40. The method according to any one of paragraphs. 1-39, further comprising the step of contacting the sample with one or more reagents to detect one or more groups consisting of: one or more parasitic helminth worms; one or more non-worm parasites; one or more viruses; one or more fungi; one or more protozoa and one or more bacteria. 41. The method according to p. 40, where the reagents contain one or more antibodies or one or more antigens recognized by antibodies specific to one or more parasitic worms; to parasites that are not worms; to one or more viruses; to one or more fungi; to one or more protozoa or to one or more bacteria. 42. The method according to any one of paragraphs. 40 or 41, wherein the one or more parasitic worms include at least one of roundworms, trichocephali, hookworms, and roundworms. 43. The method of claim 41, wherein the one or more reagents is an antibody that specifically binds to a coproantigen of non-worm Giardia parasites. 44. The method of claim 43 wherein Giardia is Giardia lamblia . 45. The method according to claim 44, where the antibody does not bind to a coproantigen derived from T. pisiformis , Taenia taeniaeformis , D. caninum , A. caninum , with which dogs were infected, A. tubaeforme , T. canis , T. cati, T vulpis, T. felis and parvovirus. 46. The method according to any one of paragraphs. 1-45, further comprising the step of determining the presence or absence of a roundworm, trichocephalic, or hookworm nucleic acid. 47. The method of claim 46, wherein the step of determining the presence or absence of a nucleic acid is carried out using a polymerase chain reaction (PCR) assay. 48. The method according to any one of paragraphs. 1-47, where at least one of the coproantigens is glycosylated. 49. The method according to any one of paragraphs. 1-48, wherein the step of detecting the presence or absence of complexes further comprises the step of obtaining one or more lectins that bind to at least one of the complexes. 50. The method of claim 49 wherein the lectin is detectably labeled or immobilized on a solid support. 51. The method according to p. 49, where the first, second, third antibodies; an antibody capable of specifically binding to roundworm coproantigen; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen and an antibody capable of specifically binding to a parvovirus coproantigen are detectably labeled or immobilized on a solid support. 52. The method according to any one of paragraphs. 48 or 49, where the first, second and third antibodies; an antibody capable of specifically binding to roundworm coproantigen; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen and an antibody capable of specifically binding to a parvovirus coproantigen are immobilized on a solid support, and the lectin is subjected to a detectable label. 53. The method according to any one of paragraphs. 48 or 49, where the first, second and third antibodies; an antibody capable of specifically binding to roundworm coproantigen; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen and an antibody capable of specifically binding to a parvovirus coproantigen are detectably labeled, and the lectin is immobilized on a solid support. 54. The method of claim 48, wherein before the step of contacting the mammalian fecal sample with one or more antibodies, the step of contacting the fecal sample with one or more lectins is further carried out. 55. The method of claim 54, wherein one or more lectins are detectably labeled or immobilized on a solid support. 56. The method according to p. 54, where the first antibody, the second antibody, the third antibody; an antibody capable of specifically binding to roundworm coproantigen; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen and an antibody capable of specifically binding to a parvovirus coproantigen are detectably labeled or immobilized on a solid support. 57. The method according to p. 54, where the first antibody, the second antibody and the third antibody; an antibody capable of specifically binding to roundworm coproantigen; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen and an antibody capable of specifically binding to a parvovirus coproantigen are immobilized on a solid support, and one or more lectins are subjected to detectable labeling. 58. The method according to p. 54, where the first antibody, the second antibody and the third antibody; an antibody capable of specifically binding to roundworm coproantigen; an antibody capable of specifically binding to a trichocephalic coproantigen; an antibody capable of specifically binding to hookworm coproantigen; an antibody capable of specifically binding to a Giardia coproantigen and an antibody capable of specifically binding to a parvovirus coproantigen are detectably labeled and one or more lectins are immobilized on a solid support. 59. A device for detecting the presence or absence of one or more tapeworm coproantigens in a fecal sample, wherein said device contains a solid carrier that has one or more antibodies immobilized thereon, selected from the group consisting of: (a) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen. 60. A device for detecting the presence or absence of one or more tapeworm coproantigens in a fecal sample, wherein said device contains a solid support, one or more lectins, and one or more antibodies selected from the group consisting of: (a) a first antibody capable of specifically binding to a first tapeworm coproantigen, but not to a second tapeworm coproantigen or a third tapeworm coproantigen; (b) a second antibody capable of specifically binding to a second tapeworm coproantigen, but not to a first tapeworm coproantigen or a third tapeworm coproantigen; and (c) a third antibody capable of specifically binding to a third tapeworm coproantigen, but not to a first tapeworm coproantigen or a second tapeworm coproantigen, wherein the solid support has one or more lectins or one or more antibodies immobilized thereon. 61. The device of claim 60 wherein the solid support has two or more antibodies immobilized thereon. 62. The device of claim 60 wherein one or more lectins are immobilized on a solid support. 63. The device according to any one of paragraphs. 59 or 60, further comprising (d) one or more types of Giardia coproantigen, roundworm coproantigen, trichocephalic coproantigen, and/or hookworm coproantigen, wherein one or more types of roundworm coproantigen, trichocephalic coproantigen, and/or hookworm coproantigen are specifically associated with the antibodies. 64. The device according to any one of paragraphs. 59 or 60, where the solid carrier has two or more antibodies immobilized thereon. 65. The device according to any one of paragraphs. 59-64 further comprising one or more species of tapeworm antigen, wherein the one or more species of tapeworm antigen is specifically associated with the antibodies. 66. The device according to any one of paragraphs. 59-65, where (a) the first antibody was induced against a first species whole worm extract, a first species tapeworm E/S product, a first species tapeworm wash, or a first species tapeworm TCA soluble product. (b) a second antibody was raised against a second species whole worm extract, a second species tapeworm E/S product, a first species tapeworm wash, or a second species tapeworm TCA soluble product; or (c) a third antibody was raised against a third species whole worm extract, a third species tapeworm E/S product, a first species tapeworm wash, or a third species tapeworm TCA soluble product; 67. The device according to any one of paragraphs. 59-66 where the first species of tapeworm is Taenia pisiformis . 68. The device of claim 67, wherein the first antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia taeniaeformis tapeworm coproantigen, Dipylidium tapeworm coproantigen, hookworm coproantigen, roundworm coproantigen, trichocephalus coproantigen , Giardia coproantigen and parvovirus coproantigen. 69. The apparatus of claim 68, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 70. The device of claim 68, wherein the Dipylidium tapeworms are Dipylidium caninum ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 71. The device according to any one of paragraphs. 59-70 where the second species of tapeworm is Taenia taeniaeformis . 72. The device of claim 71, wherein the second antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia pisiformis tapeworm coproantigen, Dipylidium tapeworm coproantigen, hookworm coproantigen, roundworm coproantigen, trichocephalic coproantigen , Giardia coproantigen and parvovirus coproantigen. 73. The apparatus of claim 72, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 74. The device of claim 72, wherein the Dipylidium tapeworms are Dipylidium caninum ; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 75. The device according to any one of paragraphs. 59-74 where the third species of tapeworm is Dipylidium caninum . 76. The device of claim 75, wherein the third antibody does not specifically cross-react with one or more coproantigens selected from the group consisting of: Taenia tapeworm coproantigen, hookworm coproantigen, roundworm coproantigen, trichocephalic coproantigen, Giardia coproantigen, and parvovirus coproantigen . 77. The apparatus of claim 76, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 78. The device according to claim 76, where the Taenia tapeworms are Taenia pisiformis or Taenia taeniaeformis; the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 79. The device according to any one of paragraphs. 59-66 where the first and second species of tapeworms are Taenia . 80. The apparatus of claim 59-66, wherein the first species of tapeworms are Taenia pisiformis and the second species of tapeworms are Taenia taeniaeformis . 81. The device according to any one of paragraphs. 59-66 where the first and second species of tapeworms are Taenia and the third species of tapeworms are Dipylidium caninum . 82. The device according to any one of paragraphs. 59-66 where the first species of tapeworms are Taenia pisiformis , the second species of tapeworms are Taenia taeniaeformis , and the third species of tapeworms are Dipylidium caninum . 83. The device of claim 82, wherein the first, second, and third antibodies do not specifically bind to any coproantigen derived from at least one member selected from the group consisting of: hookworm coproantigen, roundworm coproantigen, trichocephalic coproantigen, coproantigen Giardia and parvovirus coproantigen. 84. The device according to any one of paragraphs. 59-83, where the device further includes one or more reagents for detecting one or more groups consisting of: one or more parasitic helminth worms; one or more non-worm parasites; one or more viruses; one or more fungi; one or more protozoa and one or more bacteria. 85. The device according to any one of paragraphs. 59-84, wherein the solid support further has one or more antibodies selected from the group consisting of: (i) an antibody capable of specifically binding to a roundworm coproantigen, but not to a coproantigen derived from the group consisting of trichocephalus, hookworm, tapeworm, Giardia , and parvovirus; (ii) an antibody capable of specifically binding to a trichocephalic coproantigen, but not to a coproantigen derived from the group consisting of roundworms, hookworm, tapeworms, Giardia , and parvovirus; and (iii) an antibody capable of specifically binding to the hookworm coproantigen but not to the coproantigen derived from the group consisting of trichocephalus, roundworms, tapeworms, Giardia and parvovirus; (iv) an antibody capable of specifically binding to a Giardia ' coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and parvovirus coproantigen; and (v) an antibody capable of specifically binding to a parvovirus coproantigen, but not to a coproantigen selected from the group consisting of roundworm coproantigen, trichocephalic coproantigen, hookworm coproantigen, Taenia tapeworm coproantigen, Dipylidium tapeworm coproantigen, and Giardia coproantigen. 86. The device of claim 85, wherein the solid carrier contains two or more antibodies, three or more antibodies, four or more antibodies, five or more antibodies, six or more antibodies, seven or more antibodies, or eight or more antibodies. 87. The apparatus of claim 85, wherein the hookworm is Ancylostoma , the roundworms are Toxocara , and the trichocephalus is Trichuris . 88. The device according to claim 87, where the hookworm is Ancylostoma caninum or Ancylostoma tubaeforme ; the roundworm is Toxocara canis or Toxocara cati ; trichocephalus is Trichuris vulpis or Trichuris felis; Giardia is Giardia lamblia and parvovirus is feline parvovirus or canine parvovirus. 89. The device according to any one of paragraphs. 59-88, where the specified device is a device for enzyme-linked immunosorbent assay. 90. The device of claim 89, wherein the ELISA device is a sidestream immunoassay device. 91. The device according to any one of paragraphs. 59-90, where the sample is taken from dogs or cats. 92. The device according to any one of paragraphs. 59-91, where at least one of the coproantigens is glycosylated. 93. A kit for the detection of one or more coproantigens of flat helminths in a sample taken from a mammal, where the specified kit contains the device according to p. 59-92 and one or more reagents sufficient to detect one or more coproantigens. 94. Set according to item 93, where one or more reagents are selected from the group consisting of one or more indicator reagents; one or more antibodies for labeling the compounds; one or more antibodies; one or more antigen capture reagents; one or more inhibitors; and one or more wash reagents.

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