WO2006135799A2 - Detection de coproantigenes d'ankylostome - Google Patents

Detection de coproantigenes d'ankylostome Download PDF

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Publication number
WO2006135799A2
WO2006135799A2 PCT/US2006/022591 US2006022591W WO2006135799A2 WO 2006135799 A2 WO2006135799 A2 WO 2006135799A2 US 2006022591 W US2006022591 W US 2006022591W WO 2006135799 A2 WO2006135799 A2 WO 2006135799A2
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Prior art keywords
antibody
antigen
hookworm
animal
kit
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PCT/US2006/022591
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English (en)
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WO2006135799A3 (fr
Inventor
Michael Cappello
Robert Bungiro
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Yale University
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Publication of WO2006135799A3 publication Critical patent/WO2006135799A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
    • G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
    • G01N2333/4353—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from nematodes

Definitions

  • Hookworms are bloodfeeding nematodes that infect over 700 million persons worldwide (de Silva et al., 2003, Trends Parasitol. 19:547-551). These intestinal parasites rank among the foremost causes of iron deficiency anemia and malnutrition in the developing world (WHO, 2002, World Health Report).
  • WHO World Health Report
  • hookworm molecular biology, pathogenesis, and immunology Cappello et al., 2003, J. Parasitol. 89:S158-S164; Hotez et al., 2003, Int. J. Parasitol. 33:1245-1258; Brooker et al., 2004, Adv. Parasitol.
  • the present invention features an isolated antibody that specifically binds to a hookworm antigen present in an isolated animal biological sample.
  • the isolated antibody may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a single chain antibody, and combinations thereof, or biologically active fragments, functional equivalents, derivatives, and allelic or species variants thereof.
  • the antibody binds specifically to a hookworm antigen derived from Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma braziliense, Ancylostoma tubaeforme, Ancylostoma caninum, Necator americanus, or Uncinaria stenocephala.
  • the hookworm antigen is a is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the animal biological sample is isolated from an animal selected from the group consisting of a human, a dog, a cat, a horse, a cow, a sheep, a pig, a camel, a rabbit, a mouse, a rat, or a gerbil.
  • the animal biological sample is feces.
  • the present invention features a method of producing an antibody by immunizing an animal with a hookworm antigen under conditions that elicit an antibody response and isolating the antibodies from the animal.
  • the hookworm antigen is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the present invention features a method of producing a monoclonal antibody that specifically binds to a hookwonn antigen by immunizing an animal with a hookworm antigen under conditions that elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody-producing hybridoma cells, culturing the hybridoma cells, and isolating from the culture monoclonal antibodies which bind specifically to the polypeptide.
  • the hookworm antigen is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the present invention features a method of identifying hookworm infection in an animal by detection of hookworm antigen in an animal biological sample, by incubating the biological sample with a first antibody immobilized on a solid support to bind a hookworm antigen, incubating the hookworm antigen bound to the immobilized first antibody with a second antibody to bind said hookworm antigen, and incubating with a detectable third antibody to bind to said second antibody.
  • the animal biological sample is isolated from an animal selected from the group consisting of a human, a dog, a cat, a horse, a cow, a sheep, a pig, a camel, a rabbit, a mouse, a rat, or a gerbil.
  • the animal biological sample is feces.
  • the first, second, or third antibody may be an isolated antibody or a biological sample comprising an antibody.
  • the first, second, or third antibody is an isolated antibody that specifically binds to a hookworm antigen present in an isolated animal biological sample.
  • the first, second, or third antibody may be a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a single chain antibody, or combinations thereof, or biologically active fragments, functional equivalents, derivatives, or allelic or species variants thereof.
  • the biologically active fragment may be a Fab fragment, a F(ab') 2 fragment, or a Fv fragment.
  • the detectable third antibody is coupled to a substrate-modifying enzyme.
  • the substrate-modifying enzyme is horseradish peroxidase.
  • the detectable third antibody is biotinylated.
  • the detectable third antibody emits a detectable signal selected from the group consisting of fluorescent, luminescent, chemiluminescent, and bioluminescent.
  • the hookworm antigen is derived from a hookworm selected from the group consisting of Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma braziliense, Ancylostoma tubaeforme, Ancylostoma caninum, Necator americanus, and Uncinaria stenocephala.
  • the hookworm antigen is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the solid support is selected from the group consisting of a microtiter plate, a microbead, a magnetic bead, a panning surface, a dense particle for density centrifugation, an adsorption column, a dipstick, and an adsorption membrane.
  • the present invention features a method of identifying hookworm infection in an animal by detection of hookworm antigen in an animal biological sample by incubating the biological sample with a first antibody immobilized on a solid support to bind a hookworm antigen, incubating the hookworm antigen bound to the immobilized first antibody with a detectable second antibody to bind said hookworm antigen.
  • the animal biological sample is isolated from a animal selected from the group consisting of a human, a dog, a cat, a horse, a cow, a sheep, a pig, a camel, a rabbit, a mouse, a rat, or a gerbil.
  • the animal biological sample is feces.
  • the first or second antibody is selected from the group consisting of an isolated antibody and a biological sample comprising an antibody. In one aspect of the invention, the first or second antibody specifically binds to a hookworm antigen present in an isolated animal biological sample. In one aspect of the invention, the first or second antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a single chain antibody, and combinations thereof, or biologically active fragments, functional equivalents, derivatives, and allelic or species variants thereof. In one aspect of the invention, the biologically active fragment is selected from the group consisting of a Fab fragment, a F(ab') 2 fragment, and a Fv fragment.
  • the detectable second antibody is coupled to a substrate-modifying enzyme.
  • the substrate-modifying enzyme is horseradish peroxidase.
  • the detectable second antibody is biotinylated.
  • the detectable second antibody emits a detectable signal selected from the group consisting of fluorescent, luminescent, chemiluminescent, and bioluminescent.
  • the hookworm antigen is derived from a hookworm selected from the group consisting of Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma braziliense, Ancylostoma tubaeforme, Ancylostoma caninum, Necator americanus, and Uncinaria stenocephala.
  • the hookworm antigen is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the solid support is selected from the group consisting of a microtiter plate, a microbead, a magnetic bead, a panning surface, a dense particle for density centrifugation, an adsorption column, a dipstick, and an adsorption membrane.
  • the present invention features a kit for identifying hookworm infection in an animal by detection of hookworm antigen in an animal biological sample, including a first antibody that binds a hookworm antigen, a second antibody that binds said hookworm antigen, a detectable third antibody that binds to said second antibody.
  • the animal biological sample is isolated from a animal selected from the group consisting of a human, a dog, a cat, a horse, a cow, a sheep, a pig, a camel, a rabbit, a mouse, a rat, or a gerbil.
  • the animal biological sample is feces.
  • the first, second, or third antibody is selected from the group consisting of an isolated antibody and a biological sample comprising an antibody.
  • the first, second, or third antibody specifically binds to a hookworm antigen present in an isolated animal biological sample.
  • the first, second, or third antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a single chain antibody, and combinations thereof, or biologically active fragments, functional equivalents, derivatives, and allelic or species variants thereof.
  • the biologically active fragment is selected from the group consisting of a Fab fragment, a F(ab') 2 fragment, and a Fv fragment.
  • the detectable third antibody is coupled to a substrate-modifying enzyme.
  • the substrate-modifying enzyme is horseradish peroxidase.
  • the detectable third antibody is biotinylated.
  • the detectable third antibody emits a detectable signal selected from the group consisting of fluorescent, luminescent, chemiluminescent, and bioluminescent.
  • the hookworm antigen is derived from a hookworm selected from the group consisting of Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma braziliense, Ancylostoma tubaeforme, Ancylostoma caninum, Necator americanus, and Uncinaria stenocephala.
  • the hookworm antigen is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the solid support is selected from the group consisting of a microtiter plate, a microbead, a magnetic bead, a panning surface, a dense particle for density centrifugation, an adsorption column, a dipstick, and an adsorption membrane.
  • the present invention features a kit for identifying hookworm infection in an animal by detection of hookworm antigen in an animal biological sample, including a first antibody that binds a hookworm antigen and a detectable second antibody that binds said hookworm antigen.
  • the animal biological sample is isolated from a animal selected from the group consisting of a human, a dog, a cat, a horse, a cow, a sheep, a pig, a camel, a rabbit, a mouse, a rat, or a gerbil.
  • the animal biological sample is feces.
  • the first or second antibody is selected from the group consisting of an isolated antibody and a Biological sample comprising an antibody. In one aspect of the invention, the first or second antibody specifically binds to a hookworm antigen present in an isolated animal biological sample. In one aspect of the invention, the first or second antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a single chain antibody, and combinations thereof, or biologically active fragments, functional equivalents, derivatives, and allelic or species variants thereof. In one aspect of the invention, the biologically active fragment is selected from the group consisting of a Fab fragment, a F(ab') 2 fragment, and a Fv fragment.
  • the detectable second antibody is coupled to a substrate-modifying enzyme.
  • the substrate-modifying enzyme is horseradish peroxidase.
  • the detectable second antibody is biotinylated.
  • the detectable second antibody emits a detectable signal selected from the group consisting of fluorescent, luminescent, chemiluminescent, and bioluminescent.
  • the hookworm antigen is derived from a hookworm selected from the group consisting of Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma braziliense, Ancylostoma tubaeforme, Ancylostoma caninum, Necator americanus, and Uncinaria stenocephala.
  • the hookworm antigen is a coproantigen.
  • the hookworm antigen is a hookworm excretory/secretory antigen.
  • the hookworm antigen is recombinant Ancylostoma ceylanicum excretory/secretory protein 2.
  • the present invention features an isolated antibody that specifically binds to a helminth antigen present in an isolated animal biological sample, wherein said helminth antigen is a homolog of AceES-2.
  • the present invention features method of producing a monoclonal antibody that specifically binds to a helminth antigen, by immunizing an animal with a helminth antigen under conditions that elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody-producing hybridoma cells, culturing the hybridoma cells, isolating from the culture monoclonal antibodies which bind specifically to the polypeptide, wherein the helminth antigen is a homolog of AceES-2.
  • the present invention features a method of identifying helminth infection in an animal by detection of helminth antigen in an animal biological sample by incubating the biological sample with a first antibody immobilized on a solid support to bind a helminth antigen, incubating the helminth antigen bound to the immobilized first antibody with a second antibody to bind the helminth antigen, and incubating with a detectable third antibody to bind to the second antibody, wherein the helminth antigen is a homolog of AceES-2.
  • the present invention features a method of identifying helminth infection in an animal by detection of helminth antigen in an animal biological sample by incubating the biological sample with a first antibody immobilized on a solid support to bind a helminth antigen, incubating the helminth antigen bound to the immobilized first antibody with a detectable second antibody to bind the helminth antigen, wherein the helminth antigen is a homolog of AceES-2.
  • the present invention features a kit for identifying helminth infection in an animal by detection of helminth antigen in an animal biological sample including, a first antibody that binds a helminth antigen, a second antibody that binds the helminth antigen, and a detectable third antibody that binds to the second antibody, wherein the helminth antigen is a homolog of AceES-2.
  • the present invention features a kit for identifying helminth infection in an animal by detection of helminth antigen in an animal biological sample, including a first antibody that binds a helminth antigen, a detectable second antibody that binds the helminth antigen, wherein the helminth antigen is a homolog of AceES-2.
  • Figure 1 is a graph depicting the detection of known quantities of Ancylostoma ceylanicaum excretory/secretory (ES) products added to PBS-Tween buffer (PBS-T) or 1 :2 uninfected hamster fecal extract (FEX). All points are mean background-subtracted OD values of duplicate samples +/- standard deviation.
  • ES Ancylostoma ceylanicaum excretory/secretory
  • Figure 2 is a graph depicting the detection of Ancylostoma ceylanicum excretory/secretory (ES) coproantigens in hamster fecal extracts at various dilutions. Pooled feces was collected from hamsters infected with Ancylostoma ceylanicum 28 days previously and from age matched uninfected controls. All points are mean background-subtracted OD values of duplicate samples +/-standard deviation.
  • ES Ancylostoma ceylanicum excretory/secretory
  • Figure 3 is a graph depicting the kinetics of excretory/secretory (ES) coproantigen production in Ancylostoma ceylanicum infected hamsters as compared with parasite egg excretion. All points are means of duplicate pooled samples. Error bars did not extend beyond the boundaries of the symbols.
  • Figure 4 is a graph depicting the relationship of excretory/secretory (ES) coproantigen excretion and intestinal hookworm burden in Ancylostoma ceylanicum infected hamsters.
  • Fecal ES values represent means of duplicate samples +/- standard deviation. A best-fit line is indicated.
  • Figure 5 is a graph depicting the effect of sample storage conditions on excretory/secretory (ES) coproantigens. All points are means of duplicate samples +/- standard deviation. Brackets indicate relevant statistically significant pairwise comparisons as described in the Detailed Description. Asterisks below the brackets indicate level of significance: PO.01 (*) or PO.001 (**).
  • Figure 6 is a graph depicting the kinetics of AceES-2 production in
  • FIG. 7 is a graph depicting the detection of Ancylostoma ceylanicum excretory/secretory (ES) coproantigens in hamster fecal extracts using different detectable antibodies.
  • ES Ancylostoma ceylanicum excretory/secretory
  • the present invention features methods and compositions for the detection of hookworm antigen in an animal biological sample, hi one aspect, the present invention features methods and compositions for the detection of hookwo ⁇ n excretory/secretory (ES) antigens in an animal biological sample.
  • ES hookwo ⁇ n excretory/secretory
  • the data presented herein indicate that the present invention may be used for the detection of hookworm antigen in a biological sample isolated from a hookworm infected hamster, and thus can also be used to detect hookwo ⁇ n antigen in a biological sample isolated from other animals that may become infected with hookworm, such as, but not limited to, humans, dogs, cats, horses, cattle, sheep, pigs, camels, rabbits, and mice.
  • an enzyme-linked immunosorbent assay is employed to analyze fecal samples obtained from hamsters infected with Ancylostoma ceylanicum, a model system used for studies of hookworm pathogenesis (Bungiro et al, 2001, J. Infect. Dis. 183:1380-1387; Bungiro et al., 2002, MoI. Biochem. Parasitol. 119:147-151; Bungiro et al., 2003, Infect. Irnmun. 71:1880-1886; Bungiro et al., 2004, Infect. Irnmun. 72:2203-2213; Chu et al., 2004, Infect. Immun.
  • ELISA enzyme-linked immunosorbent assay
  • the methods and compostions described herein also provide a sensitive assay system for identifying hookworm infection in any animal that may become infected with hookworm.
  • the kinetics of fecal ES output is evaluated in an Ancylostoma ceylanicum infected hamster model system and compared to the kinetics of hookworm egg excretion.
  • the relationship of fecal ES with intestinal worm burden is examined.
  • the effects of various sample storage conditions on the ability to detect hookworm antigen in an animal biological sample are evaluated.
  • an element means one element or more than one element.
  • Homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions, e.g., if half ⁇ e.g., five positions in a polymer ten subunits in length) of the positions in two compound sequences are homologous then the two sequences are 50% homologous, if 90% of the positions, e.g., 9 of 10, are matched or homologous, the two sequences share 90% homology.
  • the DNA sequences 3 ⁇ TTGCC5' and 3 1 TATGGC share 50% homology.
  • homology is used synonymously with “identity.”
  • identity refers to the nucleic acids and proteins, it should be construed to be applied to homology at both the nucleic acid and the amino acid levels.
  • the determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm.
  • a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. MoI. Biol.
  • NCBI National Center for Biotechnology Information
  • BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).
  • PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (id.) and relationships between molecules which share a common pattern.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
  • “Mutants,” “derivatives,” and “variants” of the peptides of the invention are peptides which may be altered in one or more amino acids (or in one or more base pairs) such that the peptide (or nucleic acid) is not identical to the sequences recited herein, but has a property similar to that of the protein to which it is compared.
  • a “functional derivative” of a sequence, either protein or nucleic acid is a molecule that possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of protein or a nucleic acid sequence encoding the protein, or a portion thereof.
  • a functional derivative of a protein may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function.
  • the term “functional derivative” is intended to include the “fragments,” “variants,” or “analogs,” of a molecule.
  • a “variant” or “allelic or species variant” of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid.
  • two molecules possess a common activity and may substitute for each other they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary structure of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.
  • antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
  • isolated antibody refers to an antibody that has been separated from that with which it is naturally associated in an organism.
  • synthetic antibody refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amnio acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • the term “heavy chain antibody” or “heavy chain antibodies” comprises immunoglobulin molecules derived from camelid species, either by immunization with an antigen and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies.
  • the term “heavy chain antibody” or “heavy chain antibodies” further encompasses immunoglobulin molecules isolated from an animal with heavy chain disease, or prepared by the cloning and expression of V H (variable heavy chain immunoglobulin) genes from an animal.
  • hookworm a parasitic nematode such as, but not limited to, Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma braziliense, Ancylostoma tubaeforme, Ancylostoma caninum, Necator americanus, and Uncinaria stenocephala.
  • hookworm antigen any hookworm biological material, including natural, recombinant or synthetic materials, that can stimulate an immune response in an animal.
  • coproantigen an antigen that is present in feces.
  • excretory/secretory antigen any hookworm biological material that can be excreted, secreted, ejected, released, or otherwise discharged from a live or a dead hookworm into its environment and that is capable of stimulating an immune response in an animal.
  • Antibodies As will be understood by one skilled in the art, any antibody that can recognize and specifically bind to a hookworm antigen is useful in the present invention.
  • the invention should not be construed to be limited to any one type of antibody, either known or heretofor unknown, provided that the antibody can specifically bind to a hookworm antigen.
  • Methods of making and using such antibodies are well known in the art.
  • the generation of polyclonal antibodies can be accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom.
  • Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al.
  • the present invention encompasses the use of antibodies derived from camelid species. That is, the present invention includes, but is not limited to, the use of antibodies derived from species of the camelid family.
  • camelid antibodies differ from those of most other mammals in that they lack a light chain, and thus comprise only heavy chains with complete and diverse antigen binding capabilities (Hamers-Casterman et al., 1993, Nature 363:446-448).
  • heavy-chain antibodies are useful in that they are smaller than conventional mammalian antibodies, they are more soluble than conventional antibodies, and further demonstrate an increased stability compared to some other antibodies.
  • Camelid species include, but are not limited to Old World camelids, such as two-humped camels (C. bactrianus) and one humped camels (C. dromedarius).
  • the camelid family further comprises New World camelids including, but not limited to llamas, alpacas, vicuna and guanaco.
  • the use of Old World and New World camelids for the production of antibodies is contemplated in the present invention, as are other methods for the production of camelid antibodies set forth herein.
  • the production of polyclonal sera from camelid species is substantively similar to the production of polyclonal sera from other animals such as sheep, donkeys, goats, horses, rabbits, mice, chickens, rats, and the like.
  • Camelid species for the production of antibodies and sundry other uses are available from various sources, including but not limited to, Camello Fataga S.L. (Gran Canaria, Canary Islands) for Old World camelids, and High Acres Llamas (Fredricksburg, TX) for New World camelids.
  • camelid antibodies from the serum of a camelid species like the isolation of antibodies from the serum of other animals such as sheep, donkeys, goats, horses, rabbits, mice, chickens, rats, and the like, can be performed by many methods well known in the art, including but not limited to ammonium sulfate precipitation, antigen affinity purification, Protein A and Protein G purification, and the like.
  • a camelid species may be immunized to a desired antigen, for example, a hookworm antigen, or fragment thereof, using techniques well known in the art. The whole blood can them be drawn from the camelid and sera can be separated using standard techniques.
  • the sera can then be absorbed onto a Protein G-Sepharose column (Pharmacia, Piscataway, NJ) and washed with appropriate buffers, for example 20 mM phosphate buffer (pH 7.0).
  • the camelid antibody can then be eluted using a variety of techniques well known in the art, for example 0.15M NaCl, 0.58% acetic acid (pH 3.5).
  • the efficiency of the elution and purification of the camelid antibody can be determined by various methods, including SDS- PAGE, Bradford Assays, and the like.
  • the fraction that is not absorbed can be bound to a Protein A-Sepharose column (Pharmacia, Piscataway, NJ) and eluted using, for example 0.15M NaCl, 0.58% acetic acid (pH 4.5).
  • a Protein A-Sepharose column Puracia, Piscataway, NJ
  • acetic acid pH 4.5
  • the present invention further contemplates the production of camelid antibodies expressed from nucleic acid.
  • camelid antibodies expressed from nucleic acid Such methods are well known in the art, and are detailed in, for example U.S. Patents 5,800,988; 5,759,808; 5,840,526, and 6,015,695, which are incorporated herein by reference in their entirety.
  • cDNA can be synthesized from camelid spleen mRNA. Isolation of RNA can be performed using multiple methods and compositions, including TRIZOL (Gibco/BRL, La Jolla, CA) further, total RNA can be isolated from tissues using the guanidium isothiocyanate method detailed in, for example, Sambrook et al.
  • RNAse hr RNAse hr and E. coli DNA polymerase I according to techniques well known in the art.
  • VHH variable heavy immunoglobulin chains
  • the clones can be expressed in any type of expression vector known to the skilled artisan. Further, various expression systems can be used to express the V HH peptides of the present invention, and include, but are not limited to eukaryotic and prokaryotic systems, including bacterial cells, mammalian cells, insect cells, yeast cells, and the like.
  • V HH immunoglobulin proteins isolated from a camelid species or expressed from nucleic acids encoding such proteins can be used directly in the methods of the present invention, or can be further isolated and/or purified using methods disclosed elsewhere herein.
  • the present invention is not limited to V HH proteins isolated from camelid species, but also includes V HH proteins isolated from other sources such as animals with heavy chain disease (Seligmann et al., 1979, Immunological Rev. 48: 145-167, incorporated herein by reference in its entirety).
  • the present invention further comprises variable heavy chain immunoglobulins produced from mice and other mammals, as detailed in Ward et al.
  • V H genes were isolated from mouse splenic preparations and expressed in E. coli.
  • the present invention encompasses the use of such heavy chain immunoglobulins in the treatment of various autoimmune disorders detailed herein. expression of V H (variable heavy chain immunoglobulin) genes from an animal.
  • Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12:125-168) and the references cited therein. Further, the antibody of the invention may be "humanized” using the technology described in Wright et al. (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77:755-759).
  • a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting DNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
  • the procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York).
  • Bacteriophage which encode the desired antibody may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed.
  • the bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell.
  • Bacteriophage which do not express the antibody will not bind to the cell.
  • panning techniques are well known in the art and are described for example, in Wright et al. (supra).
  • a cDNA library is generated from mRNA obtained from a population of antibody-producing cells.
  • the mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same.
  • Amplified cDNA is cloned into Ml 3 expression vectors creating a library of phage which express human Fab fragments on their surface.
  • Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin.
  • this procedure immortalizes DNA encoding human immunoglobulin rather than cells which express human immunoglobulin.
  • Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHl) of the heavy chain.
  • Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment.
  • An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein.
  • Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al. (1991, J. MoI. Biol. 222:581-597). Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.
  • the invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al., 1995, J. MoI. Biol. 248:97-105).
  • the invention encompasses polyclonal, monoclonal, synthetic antibodies, and the like.
  • the crucial feature of the antibody of the invention is that the antibody specifically bind with a hookworm antigen.
  • immunoassay formats including competitive and noncompetitive immunoassay formats, antigen capture assays, two-antibody sandwich assays, and three-antibody sandwich assays are useful methods of the invention (Self et al., 1996, Curr. Opin. Biotechnol. 7:60-65).
  • the invention should not be construed to be limited to any one type of known or heretofor unknown immunoassay, provided that the immunoassay is able to detect hookworm antigen in an animal biological sample.
  • the method of the invention relies on one or more antigen capture assays.
  • antigen capture assay antibody is bound to a solid support, and sample is added such that hookworm antigen is bound by the antibody. After unbound proteins are removed by washing, the amount of bound antigen can be quantified, if desired, using, for example, but not limited to, a radioassay (Harlow et al., 1989, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, New York).
  • Enzyme-linked immunosorbent assays are useful in the methods of the invention.
  • An enzyme such as, but not limited to, horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase or urease can be linked, for example, to an anti- hookworm antigen antibody or to a secondary antibody for use in a method of the invention.
  • a horseradish-peroxidase detection system may be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB chromogenic substrate tetramethylbenzidine
  • enzyme-linked systems include, for example, the alkaline phosphatase detection system, which may be used with the chromogenic substrate p-nitrophenyl phosphate to yield a soluble product readily detectable at 405 nm.
  • a beta-galactosidase detection system may be used with the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside (ONPG) to yield a soluble product detectable at 410 nm.
  • a urease detection system may be used with a substrate such as urea-bromocresol purple (Sigma Immunochemicals, St. Louis, MO).
  • Useful enzyme-linked primary and secondary antibodies can be obtained from any number of commercial sources. Chemiluminescent detection is also useful for detecting hookworm antigen or for determining a quantity of hookworm antigen according to a method of the invention. Chemiluminescent secondary antibodies may be obtained from any number of commercial sources.
  • Fluorescent detection is also useful for detecting hookworm antigen or for determining a level of hookworm antigen in a method of the invention.
  • Useful fluorochiOmes include, but are not limited to, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B- phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine- Fluorescein- or rhodamine-labeled hookworm antigen-specific antibodies.
  • Radioimmunoassays are also useful in the methods of the invention. Such assays are well known in the art, and are described for example in Brophy et al. (1990, Biochem. Biophys. Res. Comm. 167:898-903) and Guechot et al. (1996, Clin. Chem. 42:558- 563). Radioimmunoassays are performed, for example, using Iodine-125-labeled primary or secondary antibody (Harlow et al., supra, 1999).
  • a signal emitted from a detectable antibody is analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation, such as a gamma counter for detection of Iodine- 125; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a spectrophotometer to detect color from a chromogenic substrate
  • a radiation counter to detect radiation, such as a gamma counter for detection of Iodine- 125
  • a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • quantitative analysis of the amount of hookworm antigen is perfo ⁇ ned using a spectrophotometer. It is understood that the assays of the invention can be performed manually or, if desired, can be automated and that the signal emitted from multiple samples can be detected simultaneously in many systems available commercially.
  • the methods of the invention also encompass the use of capillary electrophoresis based immunoassays (CEIA), which can be automated, if desired. Immunoassays also may be used in conjunction with laser-induced fluorescence as described, for example, in Schmalzing et al. (1997, Electrophoresis 18:2184-2193) and Bao (1997, J. Chromatogr. B. Biomed. Sci. 699:463-480).
  • Liposome immunoassays such as flow-injection liposome immunoassays and liposome immunosensors, may also be used to detect hookworm antigen according to the methods of the invention (Rongen et al., 1997, J. Immunol. Methods 204:105-133).
  • Sandwich enzyme immunoassays may also be useful in the methods of the invention, hi a two-antibody sandwich assay, a first antibody is bound to a solid support, and the antigen is allowed to bind to the first antibody. The amount of hookworm antigen is quantified by detecting and measuring the amount of a detectable second antibody that binds to the complex of the hookworm antigen and the first antibody.
  • a three-antibody sandwich assay a first antibody is bound to a solid support, and the antigen is allowed to bind to the first antibody. Then a second antibody is added and is allowed to bind to the antigen, which is bound to the first antibody.
  • the amount of hookworm antigen is quantified by detecting and measuring the amount of a detectable third antibody that binds to the second antibody. Quantitative western blotting may also be used to detect hookworm antigen or to determine a level of hookworm antigen in a method of the invention. Western blots are quantified using well known methods such as scanning densitometry (Parra et al., 1998, J. Vase. Surg. 28:669-675).
  • a hookworm antigen, or the presence of hookworm infection may be conducted by detecting a hookworm antigen.
  • a hookworm antigen, or the presence of hookworm infection may be conducted by detecting at least two hookworm antigens.
  • multiple unique hookwork antigens, or the presence of a hookworm infection may be detected by using multiple unique detecting molecules, hi an embodiment, multiple unique antigens are detected in a single sample. In another embodiment, multiple unique antigens are detected in multiple samples.
  • a hookworm antigen is Ancylostoma ceylanicum excretory/secretory protein 2 ("AceES-2")-
  • the nucleic acid sequence of AceES- 2 is set forth in SEQ ID NO:1
  • the polypeptide sequence of AceES-2 is set forth in SEQ ID NO:2.
  • a hookworm antigen is recombinant AceES-2. It will be understood by the skilled artisan, when armed with the disclosure set forth herein, that the invention also applies to other hookworm antigens that are found to be substantially antigenic in the presence or pathogenesis of a hookworm-related disease.
  • any antigen that is associated with the presence of a parasitic nematode or a parasitic helminth can be used according to the methods and compositions of the present invention, to provide a diagnosis of parasitic nematode or parasitic helminth infection in the animal from which it is detected,
  • a hookworm antigen is a mutant, variant, or fragment of AceES-2.
  • a hookworm antigen is a variant or an allelic species variant of AceES-2. The skilled artisan will know how to identify such an antigen, how to make an antibody to such an antigen, and how to detect such an antigen, based on the disclosure set forth herein.
  • AceES-2 homologs include AceES-2-like proteins from other nematode or helminthic organisms (e.g., Ascaris, Trichuris, or Haemonchus), recombinant forms of AceES-2 (e.g., hexahis-tagged polypeptides, polypeptides with conservative amino acid mutations, truncated polypeptides representing antigenic regions of polypeptides), and fusion proteins comprising AceES-2 or an AcesES-2 homolog.
  • AceES-2-like proteins from other nematode or helminthic organisms e.g., Ascaris, Trichuris, or Haemonchus
  • recombinant forms of AceES-2 e.g., hexahis-tagged polypeptides, polypeptides with conservative amino acid mutations, truncated polypeptides representing antigenic regions of polypeptides
  • fusion proteins comprising AceES-2 or an AcesES-2 homolog.
  • an AceES-2 homolog shares 80% homology with AceES- 2. In another embodiment, an AceES-2 homolog shares 85% homology with AceES-2. In yet another embodiment, an AceES-2 homolog shares 90% homology with AceES-2. In another embodiment, an AceES-2 homolog shares 95% homology with AceES-2. In yet another embodiment, an AceES-2 homolog shares 96% homology with AceES-2, more preferably, 97% homology with AceES-2, more preferably, 98% homology with AceES-2, more preferably, 98% homology with AceES-2, more preferably, 99% homology with AceES-2, and even more preferably, 99.9% homology with AceES-2.
  • an antigen useful in the present invention is an antigen from any helminth.
  • any antigen that is associated with the presence of a parasitic helminth can be used according to the methods and compositions of the present invention, to provide a diagnosis of parasitic helminth infection in the animal from which it is detected.
  • a helminthic antigen is a mutant, variant, or fragment of AceES-2.
  • a helminthic antigen is a variant or an allelic species variant of AceES-2. The skilled artisan will know how to identify such an antigen, how to make an antibody to such an antigen, and how to detect such an antigen, based on the disclosure set forth herein.
  • the invention includes various kits for identifying hookworm infection by detection of hookworm antigen in a biological sample. Although exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in light of the present disclosure. Each of these kits is included within the invention.
  • the invention features a kit for identifying hookworm infection by detection of hookworm antigen in a biological sample.
  • the kit comprises a first antibody that binds a hookworm antigen, a second antibody that binds hookworm antigen, and a detectable third antibody that binds to the second antibody.
  • the invention features a kit for identifying hookworm infection by detection of hookworm antigen in a biological sample.
  • the kit comprises a first antibody that binds a hookworm antigen, and a detectable second antibody that binds hookworm antigen.
  • Live hookworms were rinsed with phosphate buffered saline (PBS) and used to prepare excretory/secretory (ES) products by incubation in sterile PBS (10 worms per ml) for 6 hr at 37 0 C. The worms were removed and the raw ES products centrifuged at 3,300 x g for 15 min to remove particulates. The ES was then concentrated approximately 100-fold using a centrifugal concentrator with a 5 kDa molecular weight cutoff (Millipore Corp., Bedford MA) and protein content determined using the BCA system (Pierce Chemical Co., Rockford IL) with a bovine serum albumin standard curve.
  • PBS phosphate buffered saline
  • ES excretory/secretory
  • rAceES-2 The recombinant Ancylostoma ceylanicum ES protein 2 (rAceES-2) was expressed in E. coli using the PET-32 vector (Novagen, Inc., Madison WI) and purified as previously described (Bungiro et al., 2004, Infect. Immun. 72:2203-2213).
  • Antibodies Rabbit antiserum was prepared by subcutaneously immunizing New Zealand rabbits with 400 ug ES or 500 ug rAceES-2 in complete Freund's adjuvant, followed by 2 booster immunizations at 3 week intervals of 100 ug antigen in incomplete Freund's adjuvant.
  • Rabbit immunization, bleeding, and serum preparation were conducted by Veterinary Clinical Services, Section of Comparative Medicine, Yale University School of Medicine. Rabbit IgG was purified from hyperimmune serum using HiTrap Protein G HP columns (Amersham Biosciences, Piscataway, NJ) according to manufacturer's instructions. Immune hamster serum was prepared from animals infected 102 days previously with Ancylostoma ceylanicum (Bungiro et al., 2001, J. Infect. Dis. 183:1380-1387). Fecal Extracts Hamsters were group housed in wire-bottomed cages and fecal pellets collected onto damp cardboard, then frozen at -2O 0 C until use (unless noted otherwise).
  • Soluble fecal extracts were prepared by homogenizing thawed feces in PBS/0.05% Tween (PBS-T; 2 ml per gram wet weight). The homogenate was centrifuged for 15 minutes at 3,300 x g to remove particulates and the supernatant stored at -2O 0 C. Immediately prior to use thawed extracts were centrifuged for 5 minutes at 12,000 x g to remove any precipitated material.
  • PBS-T PBS/0.05% Tween
  • Immulon-2 microtiter plates (Dynex, Chantilly, VA) were incubated overnight at 4 0 C with 100 ul/well rabbit anti-ES or anti-rAceES-2 capture IgG diluted to 10 ug/ml in PBS. The plates were rinsed 4X with PBS-T and blocked for one hour at room temperature (RT) with 1% nonfat dry milk in PBS. Plates were rinsed 4X with PBS-T following blocking and each subsequent step. Fecal extracts or purified ES were diluted in PBS-T to a final volume of 100 ul per well and incubated for 2-3 hours at RT.
  • captured antigens were then detected by incubation with 1 : 1000 infected hamster serum for 1-2 hours at RT, followed by 1 : 1000 horseradish peroxidase (HRP) conjugated goat anti-hamster IgG (ICN Biochemicals, Irvine, CA) for 30-60 min at RT.
  • HRP conjugated rabbit anti-goat IgG ICN
  • captured antigens were then detected by incubation with biotin-labeled rabbit anti-ES IgG for 1 hr at RT followed by streptavidin-conjugated horseradish peroxidase for 30 min at RT.
  • Bound HRP was visualized by the addition of 100 ul/well ABTS substrate solution (1 mg/ml ABTS [2,2'-Azino-bis-3- ethylbenzthiazoline-6-sulfonic acid; Sigma] in 0.1 M citrate buffer, pH 5.0, 0.03% H 2 O 2 ). After 30-60 min at RT the A 405 was recorded using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale CA).
  • Hookworm eggs were evaluated in hamster feces by a flotation method in which wet fecal pellets were mixed to assure uniform distribution of eggs and an aliquot suspended in saturated NaCl solution (1 ml per 100 mg feces).
  • the sample was vortexed, strained through gauze into a 15 ml conical tube, and filled to the top with saturated NaCl.
  • a glass coverslip was placed on the meniscus for 30 minutes, then gently removed and placed on a slide for microscopic counting of eggs. A fresh coverslip was placed on the meniscus, incubated, and counted as before; this was repeated until no additional eggs were recovered.
  • Statistical Analysis of Data are presented in the text and figures as means +/- standard deviation.
  • fecal ES concentration was evaluated by a Spearman rank correlation test.
  • the effect of various sample storage conditions on fecal ES concentration was evaluated by an analysis of variance (ANOVA) followed by a Tukey- Kramer multiple comparisons post test, hi each case P values of ⁇ 0.05 were considered significant.
  • ES immunogenic Ancylostoma ceylanicum excretory/secretory
  • a sandwich ELISA protocol was developed employing a polyclonal rabbit anti-ES capture IgG coupled with detectable antibodies obtained from hookworm-infected hamsters.
  • this method allowed ES proteins to be detected over a range of 10 ng/ml to 10 ug/ml when concentrated ES was serially diluted in PBS-T buffer or buffer containing 1:2 diluted uninfected hamster fecal extract.
  • Comparison of OD values obtained under the two reaction conditions indicated that fecal material had a modest inhibitory effect on the assay; this was found to be more pronounced at higher ES concentrations.
  • ES remained below 1 ug/ml in fecal extracts until day 10, following which concentrations steadily increased, reaching a maximal value of 4.5 ug/ml by day 17.
  • Fecal ES output remained above 4 ug/ml until day 19, then declined somewhat over the next two days, coincident with the death of one of the animals on day 20.
  • AceES-2 a highly immunogenic 11.7 kDa ES protein previously cloned from Ancylostoma ceylanicum cDNA (Bungiro et al., 2004, Infect Irnmun. 72:2203-2213), was selected for use in the study.
  • the fecal ELISA was modified by substituting capture antibodies raised against recombinant AceES-2, increasing fecal extract dilutions fiom 1:2 to 1:5 (to minimize fecal interference observed when rAceES-2 was used to generate standard curves), and adding an additional detectable antibody step to the protocol (to enhance sensitivity).
  • the modified fecal ELISA allowed rAceES-2 to be detected at concentrations as low as 2 ng/ml (approximately 66 nM) and native AceES-2 to be detected in hookworm-infected fecal extracts at up to 1:128 dilution (not shown).
  • the captured ES antigen was then detected with Ancylostoma ceylanicum infected hamster serum followed by horseradish peroxidase conjugated goat anti-hamster IgG detectable antibody, as well as with biotin- labeled rabbit anti-ES IgG detectable antibody followed by streptavidin-conjugated horseradish peroxidase.
  • Absorbance values at 405 nm were measured 30 min following the addition of ABTS substrate, and the difference in signal between fecal extract only and fecal extract plus ES is indicated by brackets.

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Abstract

L'invention concerne des procédés et des compositions permettant de détecter une infection par l'ankylostome chez un animal. L'invention concerne également des procédés et des compositions permettant de détecter une infection par l'helminthe chez un animal.
PCT/US2006/022591 2005-06-10 2006-06-09 Detection de coproantigenes d'ankylostome WO2006135799A2 (fr)

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US9746469B2 (en) 2007-06-15 2017-08-29 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm, whipworm, and hookworm
US9239326B2 (en) 2007-06-15 2016-01-19 Idexx Laboratories, Inc. Compositions, devices, kits and methods for detecting hookworm
US9063129B2 (en) 2007-06-15 2015-06-23 Idexx Laboratories, Inc. Device, kit and method for hookworm antigen capture and detection
US11267879B2 (en) 2007-06-15 2022-03-08 Idexx Laboratories, Inc. Compositions, devices, kits and methods for detecting hookworm
US10942180B2 (en) 2007-06-15 2021-03-09 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm, whipworm and hookworm
US10640551B2 (en) 2007-06-15 2020-05-05 Idexx Laboratories, Inc. Compositions, devices, kits and methods for detecting hookworm
US8895294B2 (en) 2007-06-15 2014-11-25 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm, whipworm, and hookworm
US9040245B2 (en) 2007-06-15 2015-05-26 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm, whipworm, and hookworm
US10429388B2 (en) 2007-06-15 2019-10-01 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm, whipworm and hookworm
US8580518B2 (en) 2008-05-19 2013-11-12 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm
US9212220B2 (en) 2008-05-19 2015-12-15 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm
US7993861B2 (en) 2008-05-19 2011-08-09 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting whipworm
US8105795B2 (en) 2008-05-19 2012-01-31 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm
US7993862B2 (en) 2008-05-19 2011-08-09 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm
US8367808B2 (en) 2008-05-19 2013-02-05 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting whipworm
US8268574B2 (en) 2008-05-19 2012-09-18 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm
US9103823B2 (en) 2008-05-19 2015-08-11 Idexx Laboratories, Inc. Methods, devices, kits and compositions for detecting roundworm
WO2022120423A1 (fr) * 2020-12-08 2022-06-16 James Cook University Peptides et leurs utilisations
CN114807389A (zh) * 2022-06-16 2022-07-29 云南省寄生虫病防治所 一种检测美洲钩虫的引物混合物、试剂盒及检测方法

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