WO1998029727A2

WO1998029727A2 - Microsphere-enhanced immunoassay for the detection of veterinary diseases

Info

Publication number: WO1998029727A2
Application number: PCT/US1997/024193
Authority: WO
Inventors: Robert L Stephon
Original assignee: Stephon, Robert, L.
Priority date: 1996-12-31
Filing date: 1997-12-31
Publication date: 1998-07-09
Also published as: AU5811498A

Abstract

Method for diagnosing the presence of veterinary pathogens characterized by pathogen-specific Ag in physiological media of suspected infected mammals and humans comprising contacting a conjugate of a pathogen-specific Ab and a solid support with said physiological media suspected of containing antigen specific to conjugated pathogen-specific Ab to produce a bound agglutinated conjugate-antigen reaction complex, and then determining the presence of antigen in said fluid by measurement of the turbidity associated with the conjugate-antigen reaction complex.

Description

MICROSPHERE-ENHANCED IMMUNOASSAY FOR THE DETECTION OF VETERINARY DISEASES

Field of the Invention

The present invention relates to an immunoassay for the detection of veterinary pathogens in sera, plasma or otherwise physiological media or fluids, and more specifically to a microsphere-enhanced turbidity immunoassay for the diagnosis of various veterinary pathogens which can be diagnosed by complexation with pathogen-specific antibodies, antigens and/or analytes .

Background of the Invention

A variety of diagnostic tests are known for determining the existence of various veterinary diseases, including, for example, canine heartworm infection or Dirofilaria immitis, Brucella abortus, parvovirus, canine distemper, canine hepatitis, feline leukemia, feline infectious peritonitis and foot and mouth disease, all of which can be diagnosed by the demonstration of pathogen- specific antibodies (Ab) or antigens (Ag) or analytes. For example, several methods are known for detection of D . immi tis which are based on the detection of serum antibodies specific for D . immi tis parasite antigens. In one example, U.S. Patent No. 4,842,999 describes a method for diagnosing D . immi tis by, inter alia, immobilizing D . immi tis antigen on a solid surface such as a bead, and incubating the bound antigen with sera suspected of containing anti -D. immi tis antibody. A washing step is required after reaction of antibody with antigen, followed by a second incubation step with a labeled second antibody, which is then followed by an additional washing step.

Another assay for D . immi tis circulating parasite antigens is disclosed in U.S. Patent No. 4,839,275, which can employ either polyclonal or monoclonal anti -D . immi tis antibodies, and is based on imrtvunoelectrophoretic techniques.

Other methods are set forth, for example, in U.S. Patent No. 4,322,493, which describes a sera immunoassay for detection of anti -D. immi tis antibodies by immobilization of D. immi tis microfilaria on a solid support, followed by incubation, washing and labelling steps; and U.S. Patent No. 4,657,850 which describes an immunological serodiagnostic assay for D. immi tis which requires a first step of reacting a biological fluid suspected of containing D. immi tis antibodies with a specific Toxocara caπis-derived antigen, followed by steps of incubation, washing and labelling. The use of the specific T. canis- derived antigens is said to significantly decrease falsely positive results of immunological assays for adult D . iiruπi is-associated antibodies without generating false negative results, or in other words to clear the test sample of non-D. immi tis immune constituents which can cross-react with D . immi tis antigen without depleting the sample of specific D. immitis antibodies .

Thus, as shown, conventional assays of which the above described serum D . immi tis assays are representative exapmles are generally laborious time consuming matters which require multiple incubation, washing and labelling steps, or cumbersome electrophoretic techniques which are labor and equipment intensive. There therefore exists a substantial need for a rapid and accurate diagnostic assay based upon antibody-antigen and/or antigen-antibody interactions for carrying out veterinary diagnostic tests in sera, plasma or other physiological media or fluids to simplify and facilitate the early detection of various veterinary infections . There is also a need for such assays which avoid the requirement for enzyme or radio-labelled antigens, antibodies or complexes which require incubation with specific substrates and measurements of a color endpoint, or measurement of radioactive decay to detect the presence of an analyte being tested, and all of which further require several washing steps, and which are lengthy with involved procedures.

There also exists a substantial need for a physiological media-based diagnostic assay, such as, for example, specific for D. immitis-associated antigens or other pathogen-specific Ab or Ag or analyte, of reduced complexity of procedures such that they can be readily performed both inside and outside of routine veterinary and clinical practice to simplify and greatly facilitate the early and accurate detection of veterinary pathogenic infections.

Summary of the Invention

In accordance with those needs and desires set forth above, in one aspect of the present invention there is now provided an improved rapid, simple and accurate immunoassay diagnostic method for diagnosing veterinary diseases characterized by pathogen- specific Ag in the sera, plasma, or otherwise physiological media or fluids of suspected infected mammals. The inventive procedure comprises contacting a conjugate of a pathogen-specific Ab attached to or otherwise associated with a solid support with physiological fluid suspected of containing antigen specific to conjugated pathogen-specific Ab to produce a bound agglutinated support- antibody conjugate-antigen reaction complex (MS-Ab-Ag) , and then determining the presence of antigen in the fluid sample by measuring the turbidity associated with the conjugate-antigen reaction complex.

In another aspect, the inventive method for diagnosing veterinary diseases is characterized by pathogen specific Ab in physiological media or fluid of suspected infected mammals, and comprises contacting a solution of known, limiting quantity of pathogen-specific antigen with physiological media or fluid suspected of containing antibodies specific to pathogen-specific Ag to produce bound complexes of Ab with pathogen-specific Ag in solution. The solution containing remaining quantity of pathogen- specific Ag unbound and inversely proportional to the amount of pathogen-specific Ag in the physiological media or fluid of suspected infected mammals is then contacted with pathogen-specific Ab attached to or otherwise associated with a solid support to produce a bound agglutinated support-antibody conjugate-antigen complex (MS-Ab-Ag) . The presence of antibody in the fluid sample is then determined by turbidity measurement thus inversely associated with MS-Ab-Ag formation.

In yet another aspect, the inventive method for diagnosing veterinary diseases is characterized by pathogen specific Ab in physiological media or fluid of suspected infected mammals, and comprises contacting a conjugate of a pathogen-specific Ag attached to or otherwise associated with a solid support with physiological media or fluid suspected of containing antibodies specific to conjugated pathogen-specific Ag to produce a bound agglutinated support-antigen conjugate-antibody complex (MS-Ag-Ab) and then determining the presence of antibody in the fluid sample by turbidity measurement associated with MS-Ag-Ab formation.

In further aspect, the inventive method for diagnosing veterinary diseases is characterized by pathogen specific Ag in physiological media or fluid of suspected infected mammals, and comprises contacting a solution of known, limiting quantity of pathogen-specific antibody with physiological media or fluid suspected of containing antigens specific to pathogen-specific Ab to produce bound complexes of Ag with pathogen-specific Ab in solution. The solution containing remaining quantity of pathogen-specific Ab unbound and inversely proportional to the amount of pathogen- specific Ag in the physiological media or fluid of suspected infected mammals is then contacted with pathogen-specific Ag attached to or otherwise associated with a solid support to produce a bound agglutinated support-antigen conjugate-antibody complex (MS- Ag-Ab) . The presence of antigen in the fluid sample is then determined by turbidity measurement thus inversely associated with MS-Ag-Ab formation.

The invention will be more fully understood from the following detailed description with reference to preferred embodiments, examples and drawings.

Brief Description of the Drawings

FIG. 1 is a schematic representation of one aspect of the assay of the present invention showing positive and negative agglutination results with antibody complexes in the presence and absence of antigen, respectively.

FIG. 2 is a schematic representation of another aspect of the inventive assay showing positive and negative agglutination results with antigen complexes in the presence and absence of antibody, respectfully.

FIG.3 is a schematic representation of another aspect of the invention of a competitive assay for antibody in a sample.

FIG.4 is a schematic representation of another aspect of the invention of a competitive assay for antigen in a sample. FIG. 5 is a representative example of the present inventive assay showing the results of a canine heartworm antigen curve, of heartworm antigen v. absorbance.

Detailed Description of the Invention Many pathogen-specific Ab and Ag diseases including, but not limited to, for example, that of canine heartworm infection, or Dirofilaria immi tis, Brucella abortus, parvovirus, canine distemper, canine hepatitis, feline leukemia, feline infectious peritonitis, foot and mouth disease and various encephalopathies, such as scrapie and "mad cow disease", are present worldwide. Various veterinary diseases have been known to infect humans. See, for example, Lantos, P.L. "From slow virus to prion: a review of transmissible spongiform encephalopathies", Histopath. 20: 1-11 (1992). The prognosis of many of these diseases is mortal and an urgent need therefore exists to assay the physiological media or fluid(s) of suspect mammals and humans for the presence of pathogen to effect timely treatment or to determine prior infection with available medication and the subsequent prevention of the spread of infection, both to other mammals and humans. It has now been found in accordance with the present invention that microsphere enhanced agglutination technology is surprisingly effective for use in the rapid and sensitive diagnosis of various veterinary pathogen-specific Ab and Ag diseases, and which eliminates the need for several steps associated with conventional assays, such as incubation periods and multiple reagent addition and washing steps, thereby significantly reducing overall laboratory and technician time, while concomitantly providing rapid accurate test results.

Microsphere agglutination assays, or latex agglutination, are known. A representative example of such assays is described in „_.-,„„,- _η PCT/US97/24193

U.S. Patent No. 5,086,002 which is directed to an erythrocyte agglutination assay. In this method the presence or amount of an analyte in a whole blood sample is determined by forming a mixture of a sample, erthyrocytes and an agglutination reagent which comprises an erythrocyte binding molecule coupled to at least one specific analyte binding molecule, and in which the erythrocyte binding molecule does not cause agglutination when incubated with erythrocytes in the absence of an analyte or an analyte binding reagent. The observance of agglutination thus determines the presence or absence of the analyte in question.

Other agglutination assays are known in which antibodies are attached to latex beads. A general discussion of latex agglutination assays is provided in Castelan et al . , J.Clin. ; Pathol 21:638 (1968) and Singer & Plotz, Am.J.Med. 888 (1956) . See also, for example, U.S. Patent No. 4,308,026.

In the present inventive method, microsphere agglutination technology is employed in a rapid and accurate homogeneous diagnostic assay for a variety of veterinary pathogen- specific Ab and Ag diseases. In a simplistic illustrative embodiment of one aspect of the present invention, the inventive method comprises contacting a conjugate of a pathogen-specific antibody (Ab) attached to a solid support, or hereinafter "microsphere" (MS-Ab) with physiological media or fluid suspected of containing antigen (Ag) specific to bound Ab to produce a bound agglutinated conjugate-antigen reaction complex (MS-Ab-Ag) . The presence of antigen in the fluid sample is then conveniently determined by measuring the turbidity associated with the conjugate- antigen reaction complex.

In another illustrative embodiment, physiological fluid of interest is contacted with a conjugate of pathogen-specific antigen (Ag) attached to a microsphere (MS-Ag) to produce a bound agglutinated conjugate-antibody reaction complex (MS-Ag-Ab) producing turbidity which can be measured to determine antibody present in the suspected sample.

As shown in FIGs . 1 and 2, agglutination will not result in the absence of antigen or antibody. However, in the presence of antibody or antigen, Ag-Ab or Ab-Ag interactions of MS-Ab or MS-Ag of conjugates via antibody-antigen-antibody or antigen-antibody- antigen interactions is accomplished thereby producing agglutination and concomitant turbidity with the resulting detection of the presence of the antigen or antibody or analyte of interest.

The present invention also provides for "competitive" assays for diagnosing veterinary diseases characterized by pathogen specific Ab or Ag in physiological media or fluid of suspected mammals. In an illustrative embodiment of a competitive assay, a solution of known, limiting quantity of pathogen-specific antigen is contacted with physiological media or fluid suspected of containing antibodies specific to pathogen-specific Ag to produce bound complexes of Ab with pathogen- specific Ag in solution. The solution containing the remaining quantity of pathogen-specific Ag unbound and inversely proportional to the amount of pathogen-specific Ag in the physiological media or fluid of suspected infected mammals is next contacted with pathogen-specific Ab attached to or otherwise associated with a solid support to produce a bound agglutinated support -an ibody conjugate complex (MS-Ab-Ag) . Turbidity measurement can then be carried out to determine the presence of antibody in the fluid sample which is inversely proportional to MS- Ab-Ag formation. A schematic diagram of this illustrative competitive assay is shown in FIG. 3.

In another illustrative embodiment of a competitive assay of this invention a solution of known, limiting quantity of pathogen-specific antibody is contacted with physiological media or fluid suspected of containing antigens specific to pathogen-specific Ab to produce bound complexes of Ag with pathogen-specific Ab in solution. The solution containing remaining unbound pathogen- specific Ab, and which quantity is inversely proportional to the amount of pathogen-specific Ag in the physiological media or fluid of the suspected infected animal, is then contacted with pathogen- specific Ag attached to or otherwise associated with a solid support to produce a bound agglutinated support-antigen conjugate-antibody complex (MS-Ag-Ab) . The presence of antigen in the fluid sample which is inversely proportional with MS-Ag-Ab formation is then determined by turbidity measurement. A schematic diagram of this competitive assay embodiment is provided in FIG.4.

Turbidity may be measured by any conventional means, such as, for example, spectrophotometric techniques including automated clinical analyzers capable of dispensing, mixing and incubating a specimen, reaction buffer and conjugate reagent. Calibrations and/or controls, for example, for use in this example, contain known concentrations of Ag and Ab, which can be obtained from commercial sources . As set forth hereinabove, this inventive assay is not limited to the detection of any particular analyte or antigen, and may be used in the diagnostic determination of any veterinary pathogen-specific Ab and/or Ag or analyte. The analyte or pathogen may be any substance normally found in sera, plasma or other physiological media or fluid of mammals and humans such as, for instance, urine, and which has a preferential affinity for the analyte binding molecule, including monoclonal antibodies, or polyclonal antibodies, IgM, IgG and IgE, and Fab/Fab^' fragments thereof, which may have repeating epitopes recognizable by one analyte binding molecule. As representative examples, the present inventive assay is effective, but not limited to, determination of the presence of pathogen specific to the following veterinary disease states: heartworm antigens (D. immi tis excretory/ secretory products); heartworm-specific IgE Ab; heartworm-specific antibodies; simian immunodeficiency virus; feline immunodeficiency virus; feline leukemia; feline lymphosarcoma; feline panleukopenia virus; feline infectious anemia; feline thrombocytopenia; feline infectious peritonitis; feline enteric coronavirus; feline respiratory disease complex; equine infectious anemia; equine ehrlichial colitis (equine monocytic ehrlichiosis, potomac horse fever); equine uveitis equine encephalomyelitis ; contagious equine metritis; equine herpesvirus 1 (EHV-1); bovine leukemia; bovine immunodeficiency- like virus; bovine spongiform encephalopathy (mad cow disease) ; bovine thrombocytopenia; virus-induced bovine pneumonia; bovine respiratory syncytial virus (BRSV) ; sporadic bovine encephalomyelitis; infectious bovine rhinotracheitis (IBR) ; contagious bovine pleuropneumonia; bovine herpevirus 1 (BHV-l) ; progesterone in cow's milk; brucella abortus; brucella suis ; brucella melitensis; brucella canis; bovine genital campylobacteriosis ,- chlamydial abortion; enzootic abortion; canine rheumatoid arthritis; canine systemic lupus erythematosus ; canine malignant lymphoma; canine thrombocytopenia; canine hepatitis; canine coronavirus; canine parvovirus; canine distemper; canine ehrlichiosis; porcine thrombocytopenia; porcine streptococcal lymphadenitis; porcineepidemic diarrhea; african swine fever; hog cholera; swine vesicular disease; swine influenza; trichinosis; glasser' s disease; rabies; pseudorabies ; anaplasmosis ; babesiosis; trypanosomiases ; campylobacteriosis; giardiasis; enteric colibacillosis ; rotaviral enteritis; transmissible gastroenteritis; thryroxine (T4), 3,5,3'- triiodothyronine (T3) , and thyroid stimulating hormone (TSH) in animals; chlamydial conjunctivitis; chlamydial pneumonia; bacillary hemoglobinuria; blackleg (clostridium chauvoei) ; akabane disease; erysipelas; foot-and-mouth disease; aspergillosis ; Lyme disease (borreliosis) (in animals); melioidosis; neosporosis; Q-fever; toxoplasmosis; vesicular stomatitis; bluetongue; hemorrhagic fevers such as crimean-congo hemorrhagic fever and Restin hemorrhagic fever; heartwater (cowdriosis) ; malignant catarrhal fever; paratuberculosis ; rift valley fever; Rocky Mountain spotted fever; scrapie; pneumonic pasteurellosis ; hemorrhagic septicemia; infectious pustular vulvovaginitis (IPV) ,- mycoplasmal pneumonia; pasteurellosis; tracheobronchitis ; allergy testing in general (allergen-specific reaginic antibody (IgE)); chlamydiosis ; corynebacterium kutscheri infection; murine respiratory mycoplasmosis ; Tyzzer's disease; sendai virus infection; tuberculosis; viral hemorrhagic disease; chich anemia virus; hemorrhagic enteritis of turkeys; avian paramyxovirus type 3 (PMV- 3); avian spirochetosis; fowl cholera; goose viral hepatitis; avian leukosis; mycoplasma gallisepticum (MG) infection; mycoplasma synoviae (MS) infection; Marek' s disease; reticuloendotheliosis ; Newcastle disease; viral arthritis; egg drop syndrome (EDS) ; infectious bronchitis; infectious coryza; fowl plague; turkey bordetellosis ; and turkey rhinotracheitis .

Antigens and antibodies useful in this invention can be obtained from virtually any source, many of which are readily obtainable from commercial sources or from known materials.

For example, the preparation and characterization of D . immi tis specific monoclonal antibodies is discussed in detail in U.S. Patent No. 4,839,275, the entire disclosure of which is incorporated herein by reference. Other sources of commercially available antibodies and antigens include, for example, OEM Concepts, Inc. Toms River, N.J. (HW D . immi tis) , and National Veterinary Services Laboratory, Ames, Iowa (a source of antigen for brucella abortus test, employing antigen coated onto microspheres).

The size of the microspheres used in this invention is not critical, with any size effective for forming turbidity causing MS-Ab-Ag or MS-Ag-Ab reactions suitable for use herein. However, from a more practical standpoint directed to ease of handling and result detection considerations, a range of sizes between about 0.010 urn to about 2.000 urn in diameter is preferred, and especially preferred is about 0.010 urn to about 0.30 um. Microspheres useful herein are those known and used in the field of latex agglutination immunoassays and are effective to serve as a support for antibody/antigen reagent for the assay and to undergo agglutination in the presence of an agglutinator reagent sufficient for analytical purposes. A general discussion of such particles or microspheres can be found in U.S. Patent No. 4,847,209, the entire disclosure of which is incorporated herein by reference.

The microspheres may be of any conventional material, for example, including but not limited to spherical polystyrene microspheres (latex), charcoal particles, or particles manufactured from glass, polyacrylamide, cellulose, nylon and silica microspheres, microspheres formulated with a polynapthalene core- chloromethyl shell, kaolin, or various ion exchange resins. Further, the microspheres may be of any color and/or may be phosphorescent or fluorescent in nature, all of which are commercially available. Sources include, for example, Bangs

Laboratories, Inc., Fishers, IN, Polysciences, Inc., Warrington, PA and Seradyne, Inc., Indianapolis, IN.

Antibody or antigen can be attached to microspheres to form conjugate reagents of this invention either covalently or non- covalently by any conventional methodology. For example, chemical linking of anti-D. immi tis Ab to spherical polystyrene microspheres with pendant carboxyl moieties can be accomplished by conventional carbodiimide techniques. Alternatively, non-functionalized polystyrene may be employed to form a conjugate reagent by simple adsorption of the Ab's on the latex support. Other methods may employ carboxyl, amino (aliphatic/aromatic), aldehydes, amides, hydrazides, hydroxides, hydroxyl, thiol, epoxide, chloro-methyl , and vinyl benzyl chloride functionality, and still further may include streptavidin/avidin, and protein A, protein B and G technology.

To further exemplify the present invention, the following embodiment of an aspect of the invention for the detection of canine heartworm D . immi tis , is provided. This and other examples herein provided are intended for illustrative purposes only, and are not intended to limit the scope of the invention or claims, or the spirit thereof, in any way.

EXAMPLES

1. Preparation of Antibodies

An extract of the female heartworm (Dirofilaria Immitis, the Antigen, Ag) is injected along with adjuvants and stabilizers into chickens in order to raise antibodies (Ab) used in the assay. Chickens are preferred in the raising of these particular antibodies as the antibodies derived from chickens exclusively, when used in homogeneous microsphere based (MS) immunoassays , are not subject to interferences (in the form of "autoagglutination" or background absorbance) by rheumatoid factor (RF) in serum specimens. Antibodies derived from sources other than chickens may be subject to such interference in this type of assay. The employ of Fab/Fab^' fragments of IgG-derived antibodies is also contemplated to avoid the autoaggulatination phenomenon. The technique of injection into animal hosts produces polyclonal antibodies to heartworm antigen (HW) , as opposed to monoclonal antibodies which are typically used in solid phase, heterogeneous immunoassays for HW. Polyclonal antibodies are preferred for use with this assay because multiple epitopes enable the formation of multiple Ab-Ag-MP agglutinated complexes .

2.1. Conjugation of Purified Ab's to

Microspheres (MS)

Polyclonal Ab's from chicken egg yolks (known as "IgY", as compared to the "IgG" fraction typically obtained from serum and which are also contemplated for use herein as well as other sources of antibodies) are isolated and purified according to standard techniques. The Ab is chemically linked to 0.042 urn polystyrene (latex) microspheres by formation of an amide bond between e-amino groups on the Ab with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer (2- [N-morpholino] ethanesulfonic acid), pH=6.1, is added to purified IgY. The MS, IgY, and buffer solution is mixed for 15 minutes and EDAC ( l-ethyl-3 - (3- dimethylaminopropyl) carbodiimide HC1) is added. The solution is mixed for 1 hour and the reaction is stopped by ultracentrifugation @ 40,000 x g for 1 hour. The conjugate is resuspended by ultrasonication and washed x 2 in 50 mM MES buffer, pH=8.4, and stored in a buffered protein solution @ 2-8 C.

2.2. Conjugation of Purified Ab's to MS

In this example, an IgY Ab is chemically linked to .190 um polystyrene (latex) microspheres (MS) by formation of an amide bond between e-amino groups on the Ab with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH=6.1, is added to purified IgY. The MS, IgY, and buffer solution is mixed for 15 minutes and EDAC is next added. The solution is mixed for 1 hour and the reaction is stopped by ultracentrifugation @ 40,000 x g for 1 hour. The conjugate is resuspended by ultrasonication and washed x 2 in 50 mM MES buffer, pH=8.4, and stored in a buffered protein solution @ 2-8 C.

2.3. Conjugation of Purified Ab's to MS Polyclonal Ab's from chicken egg yolks, "IgY", are isolated and purified according to standard techniques. The Ab is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e-amino groups on the Ab with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1, is mixed with N-hydroxysuccinimide (NHS) and EDAC is then added to the purified IgY. The solution is mixed for 1 hour and the reaction is stopped by ultracentrifugation @ 40,000 x g for 1 hour. The conjugate is resuspended by ultrasonication and washed x 2 in 50 mM MES buffer, pH = 8.4, and stored in a buffered protein solution @ 2-8 C.

2.4. Conjugation of Purified Ab's to MS

Polyclonal Ab's from chicken egg yolks, "IgY", are isolated and purified according to standard techniques. The Ab is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e-amino groups on the Ab with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1, is added to purified IgY in a cross-flow filtration apparatus. The MS, IgY, and buffer solution is mixed for 15 minutes and EDAC is added. The solution is mixed for 1 hour and the reaction is stopped by flushing excess reagents from the mixture in the cross-flow filtration apparatus with 50 mM MES buffer, pH = 8.4, and the conjugate is stored in a buffered protein solution @ 2-8 C. 2.5. Conjugation of Purified Ab's to MS

Polyclonal Ab's from chicken egg yolks, "IgY", are isolated and purified according to standard techniques. The Ab is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e-amino groups on the Ab with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1, is mixed with N-hydroxysuccinimide (NHS) and EDAC and added to purified IgY in a cross-flow filtration apparatus. The MS- NHS, IgY, and buffer solution is mixed for 1 hour and the reaction is stopped by flushing excess reagents from the mixture in the cross-flow filtration apparatus with 50 mM MES buffer, pH = 8.4, and the conjugate is stored in a buffered protein solution @ 2-8 C.

2.6. Conjugation of Purified Ag's to MS

The antigen to be used is isolated and purified according to standard techniques. The Ag is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e- amino groups on the Ag with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1 , is added to purified Ag. The MS, Ag, and buffer solution is mixed for 15 minutes and EDAC is added. The solution is mixed for 1 hour and the reaction is stopped by ultracentrifugation @ 40,000 x g for 1 hour. The conjugate is resuspended by ultrasonication and washed x 2 in 50 mM MES buffer, pH = 8.4, and stored in a buffered protein solution @ 2- 8 C.

2.7. Conjugation of Purified Ag's to MS

Antigen is isolated and purified according to standard techniques. The Ag is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e-amino groups on the Ag with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1 , is mixed with NHS and EDAC, then added to purified Ag. The solution is mixed for 1 hour and the reaction is stopped by ultracentrifugation @ 40,000 x g for 1 hour. The conjugate is resuspended by ultrasonication and washed x 2 in 50 mM MES buffer, pH = 8.4, and stored in a buffered protein solution @ 2-8 C.

2.8. Conjugation of Purified Ag's to MS

Antigen is isolated and purified according to standard techniques. The Ag is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e-amino groups on the Ag with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1 , is added to purified Ag in a cross- flow filtration apparatus. The MS, Ag, and buffer solution is mixed for 15 minutes and EDAC is added. The solution is mixed for 1 hour and the reaction is stopped by flushing excess reagents from the mixture in the cross-flow filtration apparatus with 50 mM MES buffer, pH = 8.4, and the conjugate is stored in a buffered protein solution @ 2-8 C.

2.9. Conjugation of Purified Ag's to MS Antigen is isolated and purified according to standard techniques. The Ag is chemically linked to 0.190 um polystyrene microspheres by formation of an amide bond between e-amino groups on the Ag with pendant carboxyl groups on the MS. A 1% solution of MS in 50 mM MES buffer, pH = 6.1, is mixed with NHS and EDAC, and added to purified Ag in a cross-flow filtration apparatus. The MS-NHS, Ag, and buffer solution is mixed for 1 hour and the reaction is stopped by flushing excess reagents from the mixture in the cross- flow filtration apparatus with 50 mM MES buffer, pH = 8.4, and the conjugate is stored in a buffered protein solution @ 2-8 C. 3. Assay Protocol Exemplified by D. immi tis Ag

Detection (Typically using an automated clinical analyzer)

.020 mL of serum or plasma specimen is mixed for 3 minutes with 0.25 mL of assay buffer, consisting of 150 mM sodium phosphate (dibasic) , 5g/L Polyethylene Glycol 8000 (PEG 8000) , 150 mM NaCl, pH = 8.0, or other suitable buffer. 0.075 mL of a 0.05%

MS-Ab solution specific for HW D . immi tis Ag is added, the solution is mixed for 5 minutes, and absorbance is read @ 700 nm. The increase in absorbance correlates with the concentration of Ag, for example HW D . immi tis Ag, in the specimens.

The results of this assay exemplifying HW D. immi tis Ag are shown in Fig. 5 with the absorbance of the heartworm antigen measured at 700 nm. As shown, the reaction of microsphere-antibody conjugate MS-Ab in which multiple Ab may be covalently or non- covalently bound to a microsphere form an agglutination complex with antigen (Ag) (D. immi tis) (MS-Ab-Ag) in which the Ag may be bound by multiple MS-Ab conjugates and different Ab residues, or the MS-Ab conjugate may be bound to multiple Ag molecules which causes the reaction mixture to become turbid in appearance, with greater turbidity associated with a greater degree of complex formation, in turn due to a greater concentration of Ag (or Ab) analyte in the physiological fluid specimen. Thus, as shown in this example, the concentration of D . immi tis in the specimen is directly proportional to the spectrophotometrically measured absorbance caused by the turbidity of the reaction mixture.

In similar manner to the protocol set forth above in Example 3, microsphere-enhanced immunoassays may be carried out with the employ of antibodies or antigens, competitively or noncompetitively, for the detection of any of the several veterinary disease states known and which several are listed hereinabove.

Claims

- 19 -Claims

1. Method for diagnosing the presence of veterinary pathogens characterized by pathogen specific Ag in physiological media of suspected infected mammals and humans comprising contacting a conjugate of a pathogen-specific Ab and a solid support with said physiological media suspected of containing antigen specific to conjugated pathogen-specific Ab to produce a bound agglutinated conjugate-antigen reaction complex, and then determining the presence of antigen in said fluid by measurement of the turbidity associated with the conjugate-antigen reaction complex.

2. The methods of claim 1 wherein the pathogen-specific Ab is either a monoclonal or polyclonal Ab .

3. The method of claim 1 wherein the solid support is microspheres .

4. The method of claim 1 wherein said microspheres range in size from about 0.010 to about 2.000 um.

5. The method of claim 4 wherein said microspheres range in size from about 0.01 um to about 0.3 um.

6. The method of claim 1 wherein the pathogen-specific Ab is covalently linked to a microsphere.

7. Method for diagnosing the presence of veterinary pathogens characterized by pathogen-specific Ab in physiological fluid of suspected infected mammals and humans comprising contacting a conjugate of a pathogen-specific Ag and a solid support with said physiological fluid suspected of containing antibodies specific to conjugated pathogen specific Ag to produce a bound agglutinated conjugate-antibody reaction complex, and then, determining the presence of antibody in said fluid by measurement of the turbidity associated with the conjugate antibody reaction complex.

8. The method of claims 1 or 7 wherein said pathogen specific Ab and/or Ag is selected from the group consisting of: heartworm antigens (D. immi tis excretory/secretory products) ; heartworm- specific IgE Ab; heartworm-specific antibodies; simian immunodeficiency virus; feline immunodificiency virus; feline leukemia; feline lymphosarcoma; feline panleukopenia virus; feline infectious anemia; feline thrombocytopenia; feline infectious peritonitis; feline enteric coronavirus; feline respiratory disease complex; equine infectious anemia; equine ehrlichial colitis (equine monocytic ehrlichiosis, potomac horse fever); equine uveitis equine encephalomyelitis; contagious equine metritis; equine herpesvirus 1 (EHV-1); bovine leukemia; bovine immunodeficiency- like virus; bovine spongiform encephalopathy (mad cow disease) ; bovine thrombocytopenia; virus -induced bovine pneumonia; bovine respiratory syncytial virus (BRSV) ; sporadic bovine encephalomyelitis; infectious bovine rhinotracheitis (IBR) ; contagious bovine pleuropneumonia; bovine herpevirus 1 (BHV-1) ,- progesterone in cow's milk; brucella abortus; brucella suis; brucella melitensis; brucella canis; bovine genital campylobacteriosis; chlamydial abortion; enzootic abortion; canine rheumatoid arthritis; canine systemic lupus erythematosus ; canine malignant lymphoma; canine thrombocytopenia; canine hepatitis; canine coronavirus; canine parvovirus; canine distemper; canine ehrlichiosis; porcine thrombocytopenia; porcine streptococcal lymphadenitis; porcine epidemic diarrhea; african swine fever; hog cholera; swine vesicular disease; swine influenza; trichinosis; glasser's disease; rabies; pseudorabies; anaplasmosis ; babesiosis; trypanosomiases ; campylobacteriosis; giardiasis; enteric colibacillosis; rotaviral enteritis; transmissible gastroenteritis; thryroxine (T4) , 3,5,3'- triiodothyronine (T3), and thyroid stimulating hormone (TSH) in animals; chlamydial conjunctivitis; chlamydial pneumonia; bacillary hemoglobinuria,- blackleg (clostridium chauvoei) ; akabane disease ,- erysipelas; foot-and-mouth disease; aspergillosis; Lyme disease (borreliosis) (in animals); melioidosis; neosporosis; Q-fever; toxoplasmosis; vesicular stomatitis; bluetongue; crimean-congo hemorrhagic fever; heartwater (cowdriosis) ; malignant catarrhal fever; paratuberculosis ; rift valley fever; Rocky Mountain spotted fever; scrapie pneumonic pasteurellosis; hemorrhagic septicemia; infectious pustular vulvovaginitis (IPV) ; mycoplasmal pneumonia; pasteurellosis; tracheobronchitis ; allergy testing in general (allergen-specific reaginic antibody (IgE)); chlamydiosis ; corynebacterium kutscheri infection; murine respiratory mycoplasmosis ; tyzzer's disease; sendai virus infection; tuberculosis; viral hemorrhagic disease; chich anemia virus; hemorrhagic enteritis of turkeys; avian paramyxovirus type 3 (PMV- 3); avian spirochetosis; fowl cholera; goose viral hepatitis; avian leukosis; mycoplasma gallisepticum (MG) infection; mycoplasma synoviae (MS) infection; Marek' s disease; reticuloendotheliosis ; Newcastle disease; viral arthritis; egg drop syndrome (EDS); infectious bronchitis; infectious coryza; fowl plague; turkey bordetellosis; and, turkey rhinotracheitis .