WO2022039604A1 - Multiplex assay for johne's disease and pregnancy - Google Patents

Abstract

Johne's disease, an infection caused by Mycobacterium avium paratuberculosis (MAP), is a major production limiting disease of dairy cows. Calves can become infected with Johne's disease during birth. The present invention provides methods for detecting both Johne's disease and pregnancy associated glycoproteins in a single multiplex immunoassay.

Description

MULTIPLEX ASSAY FOR JOHNE’S DISEASE AND PREGNANCY

FIELD OF THE INVENTION

[0001] The present invention relates generally to multiplex immunoassays and specifically to a multiplex immunoassay (MIA) for Johne’s disease and pregnancy.

BACKGROUND INFORMATION

[0002] Johne’s disease, an infection caused by Mycobacterium avium paratuberculosis (MAP), is a major production limiting disease of dairy cows. There is no cure for this disease and the only way to mitigate losses is via whole herd testing for Johne’s disease and intervention. Further, the diagnosis of pregnancy is an essential component of sound reproductive management in the dairy industry, which is confirmed by the detection of pregnancy associated glycoproteins (PAGs) in milk and serum samples from cattle.

[0003] Cattle with signs of Johne’s disease shed billions of bacteria through their manure and serve as a major source of infection for future calves. In cattle, the main signs of Johne’s disease are diarrhea and wasting. Most cases are seen in 2- to 6-year-old animals. The initial signs can be subtle, and may be limited to weight loss, decreased milk production, or roughening of the hair coat. Several weeks after the onset of diarrhea, a soft swelling may occur under the jaw. Known as "bottle jaw" or intermandibular edema, this symptom is due to protein loss from the bloodstream into the digestive tract. Paratuberculosis is progressive; affected animals become increasingly emaciated and usually die as the result of dehydration and severe cachexia.

[0004] Signs are rarely evident until two or more years after the initial infection, which usually occurs shortly after birth. Animals are most susceptible to the infection in the first year of life. Newborns most often become infected by swallowing small amounts of infected manure from the birthing environment or udder of the mother. In addition, newborns may become infected while in the uterus or by swallowing bacteria passed in milk and colostrum. Animals exposed at an older age, or exposed to a very small dose of bacteria at a young age, are not likely to develop clinical disease until they are much older than two years.

[0005] It would be useful to diagnose Johne’s disease during pregnancy to try to prevent new calves from contracting the disease. Bovines have been found to express pregnancy related glycoproteins (PAGs) during pregnancy. PAGs belong to a large family of inactive aspartic proteinases expressed by the placenta of domestic ruminants including cows, ewes, and goats. In cattle, the PAG gene family comprises at least 22 transcribed genes as well as some variants (Telugu et al., 2009). Mean PAG concentrations in cattle increase from 15 to 35 days in gestation. [0006] PAGs are produced by mono- and binucleate trophoblast cells in the ruminant placenta. PAG appears in maternal blood and, from approximately 4 weeks after fertilization onward, may serve as a reliable means of diagnosing pregnancy. A range of factors are said to affect plasma PAG concentrations, such as number and sex of fetus, mass of calf and placenta, level of milk production and genetic constitution.

[0007] Johne’s disease and pregnancy diagnosis are currently performed during the same season by two separate ELISAs using milk samples from cattle. There is a need for a rapid and accurate diagnostic test for the simultaneous detection of Johne’s disease infection and pregnancy in bovines.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the seminal discovery of the use of a multiplex immunoassay for detection of Johne’s disease and pregnancy in bovines and other ruminants. Specifically, the invention provides immunoassays that simultaneously detect bovine pregnancy associated glycoproteins (PAGs) and antibodies produced in response to infection by Mycabactenuni avium subspecies paratuberculosis (MAP).

[0009] In one embodiment, the present invention provides a substrate with at least two capture elements specific for Johne’s disease and/or pregnancy on the substrate, each capture element corresponding to and being able to bind a target analyte, the substrate further optionally has a plurality of control elements including at least one fiduciary marker, at least one negative control to monitor background signal, at least one negative control to monitor assay specificity, at least one positive colorimetric control, at least one positive control to monitor assay performance and any combination thereof. In one aspect, the capture elements bind target analytes, wherein the target analytes are indicative of a Johne’s disease infection and/or pregnancy. In an additional aspect, the target analyte is a protein, a protein fragment, a peptide, a polypeptide, a polypeptide fragment, an antibody, an antibody fragment, an antibody binding domain, an antigen, an antigen fragment, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, epitope, or any combination thereof. In a further aspect, the target analyte is a bovine pregnancy associated glycoprotein (PAG) or epitope thereof. In certain aspects, the PAG is selected from PAG-4, PAG-6, PAG-9, PAG-20, PAG-21 or any combination thereof. In one aspect, the target analyte is a Mycobacterium avium subspecies paratuberculosis (MAP) antibody, fragment or binding domain thereof. In some aspects, the capture element is a protein, a protein fragment, a binding protein (BP), a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')2 antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, or any combination thereof. In an additional aspect, capture element is a Mycobacterium avium subspecies paratuberculosis (MAP) antigen. In a further aspect, the capture element is a bovine pregnancy associated glycoprotein (PAG) or epitope thereof, antibody, fragment or binding domain thereof. In various aspects, the PAG is selected from PAG-4, PAG-6, PAG-9, P AG-20, P AG-21 or any combination thereof. In one aspect, the substrate is a solid or porous substrate. In an additional aspect, the solid substrate is a paramagnetic bead, microtiter plate, microparticle, or a magnetic bead.

[0010] In an additional embodiment, the present invention provides a kit for detecting a plurality of target analytes in a sample, including a substrate and optionally one or both of a background reducing reagent, and a colorimetric detection system. In one aspect, the kit further has one or more items selected from a wash solution, one or more antibodies for detection of antigens, ligands or antibodies bound to the capture elements or for detection of the positive controls, software for analyzing captured target analytes, a protocol for measuring the presence of target analytes in samples, a sample diluent, blotting TMB (3,3',5,5’-tetramethylbenzidine; MW = 240.4) and a secondary antibody. In an additional aspect, the antibodies for detection comprise antibody-binding protein (BP) conjugates, antibody- enzyme label conjugates, or any combination thereof. In various aspects, the sample is a milk or a blood sample. In certain aspects, blood sample is serum or plasma. In an additional aspect, the substrate is a solid. In certain aspects, the solid substrate is a paramagnetic bead, microtiter plate, or microparticle. In one aspect, the background reducing reagent is, Mycobacterium phlei. In an additional aspect, the colorimetric detection system comprises HRP-labelled anti-PAGs Ab and HRP-labelled anti-bovine IgG Ab. In a further aspect, the secondary antibody comprises at least one bovine anti-PAG antibody.

[0011] In a further embodiment, the present invention provides a method for processing a microarray by providing a substrate, adding at least one sample to the substrate; and processing the substrate such that a detectable result is given by two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to monitor assay performance.

[0012] In one embodiment, the present invention provides a method for detecting an analyte in a sample comprising providing a substrate, adding at least one sample to the substrate, and processing the substrate such that a detectable result is provided. In one aspect, the detectable result includes two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to detect an analyte in the sample.

[0013] In one embodiment, the present invention provides an isolated peptide having the amino acid sequence DTVRIGDLVSTDQ (SEQ ID NO: 1). In an additional embodiment, the present invention provides an isolated peptide having the amino acid sequence GSWMFGGVDHRYYKGELNW (SEQ ID NO:2). In a further embodiment, the present invention provides an isolated peptide having the amino acid sequence ITIGTPPQEFQV (SEQ ID NO:3).

[0014] In one embodiment, the present invention provides an antibody that binds a peptide having the amino acid sequence DTVRIGDLVSTDQ (SEQ ID NO: 1). In one aspect, the antibody is monoclonal.

[0015] In an additional embodiment, the present invention provides an antibody that binds a peptide having the amino acid sequence GSVVMFGGVDHRYYKGELNW (SEQ ID NO:2). In one aspect, the antibody is monoclonal.

[0016] In a further embodiment, the present provides an antibody that binds a peptide having the amino acid sequence ITIGTPPQEFQV (SEQ ID NO:3). In one aspect, the antibody is monoclonal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows assay formats used for the detection of anti-MAP antibody and PAGs in the membrane-free multiplex immunoassay (MIA) for the diagnosis of Johne’s disease and pregnancy, respectively. The anti-MAP Ab in Mycobacterium paratuberculosis infected cow’ s milk sample in Johne’s disease are detected via an indirect ELISA (left) while PAGs in pregnant cow’s milk sample are detected by a sandwich ELISA (right). [0018] Figures 2A-E show a membrane-free MIA procedure. Fig 2A. Printing of MAP Ag and anti-PAGs Ab on the assay surface. Fig. 2B. Blocking of assay surface after protein/ Ab printing. Fig. 2C. Detection of target analytes (anti -MAP Abs and PAGs) in the cow’s milk sample. Fig. 2D. Detection of specifically bound analytes by binding with HRP-labelled detection Ab against the analytes. Fig. 2E. Generation of colorimetric array spots by the addition of HRP substrate (TMB).

[0019] Figures 3 shows a membrane-free MIA. The desired biomarkers, i.e. MAP Ag and anti- PAGs Ab, are printed as duplicate spots in each well of detachable 8-wells modules of a 96-well MTP.

[0020] Figure 4 shows leach-proof immobilization of biomolecules (antibody (Ab) or antigen (Ag)) to the solid substrate of MTP’s well using 3-aminopropyltriethoxysilane (APTES).

[0021] Figures 5 shows the steps involved in the MIA. Onto the printed and blocked assay surface, the initial step involves the addition of the sample and the detection of target analytes (anti-MAP Abs and PAGs) within it. Then, anti-PAG secondary antibody is added forming the sandwich reaction. Subsequently, HRP-labeled detection antibody is added, which binds to the bovine IgG of both assays. Finally, there is the generation of colorimetric array spots by the addition of HRP substrate (TMB).

[0022] Figure 6 shows the capture Ab against PAG printed in duplicate in each well of a strip while MAP antigen is printed in duplicate in each well of another strip. The detection addition and other internal control spots are printed in all the wells. The white circles signify that nothing has been printed at that specific position.

[0023] Figures 7A-B show alignments of bovine pregnancy associate glycoproteins (PAGs) and a surface model of PAG-20. Fig. 7A Sequence alignment of PAG-20, PAG-21, PAG-9, PAG- 6, and PAG-4. The three antigenic peptides that are common to all PAGs are highlighted. Three of the four antigenic sites, framed on squares, are the sequences selected to synthesize the peptides required for the Phage display technology based animal-free production of anti-PAGs Ab. Fig. 7B. An example of surface model of PAG-20 and its antigenic sites.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is based on the seminal discovery of the use of a multiplex immunoassay for detection of Johne’s disease and pregnancy in bovines and other ruminants. Specifically, the invention provides immunoassays that simultaneously detect bovine pregnancy associated glycoproteins (PAGs) and antibodies produced in response to infection by Mycobacterium avium subspecies paratuberculosis (MAP).

[0025] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0026] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein, which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0027] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0029] Johne’s disease, an infection caused by Mycobacterium avium paratuberculosis (MAP), is a major production limiting disease of dairy cows. There is no cure for this disease and the only way to mitigate losses is via whole herd testing for Johne's disease and intervention. Further, the diagnosis of pregnancy is an essential component of sound reproductive management in the dairy industry, which is confirmed by the detection of pregnancy associated glycoproteins (PAGs) in milk and serum samples from cattle. Johne’s disease and pregnancy diagnosis are currently performed during the same season by two separate ELISAs using milk samples from cattle. The present invention provides a multiplex immunoassay (MIA) procedure that detects both the antigens (Ags) and antibodies (Abs) in a single MIA. Moreover, the anti-PAGs Abs of the invention are custom-produced via an animal-free antibody production technology using the custom- synthesized multiple-epitope bearing PAGs peptides. Three epitopes have been identified that are present in PAG-4, PAG-6, PAG-9, PAG-20, and PAG-21 and thus, enable the early diagnosis of pregnancy in cattle.

[0030] The invention describes the multiplex immunoassay (MIA) procedures for the simultaneous detection of multiple analytes for the diagnosis of Johne’s disease and pregnancy using the milk samples from cattle and other farm animals. It involves the detection of two distinct bovine analytes, PAGs and IgG antibodies (Abs) towards MAP, in milk or serum via a multiplex immunoassay.

[0031] In one embodiment, the present invention provides a substrate with at least two capture elements specific for Johne’s disease on the substrate, each capture element corresponding to and being able to bind a target analyte, the substrate further optionally has a plurality of control elements including at least one fiduciary marker, at least one negative control to monitor background signal, at least one negative control to monitor assay specificity, at least one positive colorimetric control, at least one positive control to monitor assay performance and any combination thereof. In one aspect, the capture elements bind target analytes, wherein the target analytes are indicative of a Johne’s disease infection. In an additional aspect, the target analyte is a protein, a protein fragment, a peptide, a polypeptide, a polypeptide fragment, an antibody, an antibody fragment, an antibody binding domain, an antigen, an antigen fragment, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, epitope, or any combination thereof. In a further aspect, the target analyte is a bovine pregnancy associated glycoprotein (PAG) or epitope thereof. In certain aspects, the PAG is selected from PAG-4, PAG- 6, PAG-9, PAG-20, PAG-21 or any combination thereof. In one aspect, the target analyte is a Mycobacterium avium subspecies paratuberculosis (MAP) antibody, fragment or binding domain thereof. In some aspects, the capture element is a protein, a protein fragment, a binding protein (BP), a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')2 antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, or any combination thereof. In an additional aspect, capture element is a Mycobacterium avium subspecies parattihecculosis (MAP) antigen or epitope thereof. In a further aspect, the capture element is a bovine pregnancy associated glycoprotein (PAG) antibody, fragment or binding domain thereof. In various aspects, the PAG is selected from PAG-4, PAG-6, PAG-9, PAG-20, PAG-21 or any combination thereof. In one aspect, the substrate is a solid or porous substrate. In an additional aspect, the solid substrate is a paramagnetic bead, microtiter plate, microparticle, or a magnetic bead.

[0032] As used herein, the term “substrate” is any surface that supports an immunoassay. The substrate of the invention may be a solid substrate or a porous substrate, for example.

[0033] In certain aspects, the substrate is a solid substrate. Examples of solid substrates include, but are not limited to, 96 well microtiter plate, glass, microbeads, nano/micro- particles and magnetic beads. In one aspect, a 96 well microtiter plate is polystyrene, polydimethylsiloxane (PDMS), poly (methyl methacrylate) (PMMA), polycarbonate, cyclic polyolefins, Zeonor, Zeonex, or cellulose acetate. In various aspects, the solid substrate maybe glass beads, nano- /microparticles, magnetic beads or paramagnetic beads.

[0034] The assay elements (control and capture elements) are placed on the substrate surface, with or without an adapter molecule between the substrate and the element. Preferably, the assay elements bind to the substrate by covalent or non-covalent interaction. One of skill in the art will recognize that methods of placing assay elements on the substrate include printing, spotting or other techniques known in the art. For purposes of the present application, the term “printing” can be used to include any of the methods for placing the assay elements on a membrane.

[0035] The terms “array” or “microarray” as used herein refer to a collection of multiple assay elements on a substrate. Specifically, an array is a collection of capture elements and/or control elements on a substrate.

[0036] In various aspects, the elements on the array are placed on the substrate in discrete areas of between 100 pm to 500 pm in diameter. More preferably, the discrete areas are between 350pm to 400pm in diameter. In certain aspects, the discrete areas of the array are placed in a 5x5 grid. In one aspect, the array comprises up to nine control elements and two replicates of each of eight different capture elements. In one aspect, the capture elements are printed in two or more replicates of four different capture elements and multiples thereof. [0037] As used herein, the term “assay element” refers to any of a number of different elements for use in an array of the invention. Exemplary assay elements include, but are not limited to, capture elements and control elements.

[0038] The term “capture element” refers to a molecule that is able to bind to a target analyte. Examples of useful capture elements include proteins, protein fragments, polypeptides, polypeptide fragments, binding proteins, binding protein fragments, antibodies (polyclonal, monoclonal, or chimeric), antibody fragments, antibody heavy chains, antibody light chains, single chain antibodies, single-domain antibodies (a VHH for example), Fab antibody fragments, Fc antibody fragments, Fv antibody fragments, F(ab')2 antibody fragments, Fab' antibody fragments, single-chain Fv (scFv) antibody fragments, antibody binding domains, antigens, antigenic determinants, epitopes, haptens, immunogens, immunogen fragments, and binding domains. Useful capture elements will correspond to and be able to bind a specific target analyte, such as a molecule or class of molecules that are present in a sample to be tested.

[0039] In one embodiment, the capture element is selected from a protein, a protein fragment, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody (a VHH for example), a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')2 antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, and a binding domain.

[0040] In another aspect, the capture elements may comprise antibodies or fragments thereof that are immobilized on the substrate surface and are specific for different antigens or ligands that may be present in a sample. In certain aspects, the capture elements may comprise antigens or ligands and the assay involves the detection of specific antibodies that may be present in a sample. In various aspects, the capture elements may comprise of a receptor or a subunit of a receptor that binds a specific ligand.

[0041] Specifically, the capture element can be Mycobacterium avium subspecies paratuberculosis (MAP) antigen or epitope thereof or antibodies to PAGs including PAG-4, PAG- 6, PAG-9, PAG-20, P AG-21 or any combination thereof.

[0042] As used herein, the terms “biomarker” refers to any substance used as an indicator of a biologic state. Thus, a biomarker can be any substance whose detection indicates a particular disease state (for example, the presence of an antibody may indicate an infection). Furthermore, a biomarker can be indicative of a change in expression or state of a protein that correlates with the risk or progression of a disease, or with the susceptibility of the disease to a given treatment. Once a proposed biomarker has been validated, it can be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (e.g., choices of drug treatment or administration regimes). In evaluating potential drug therapies, a biomarker may be used as a surrogate for a natural endpoint such as survival or irreversible morbidity. If a treatment alters the biomarker, which has a direct connection to improved health, the biomarker serves as a “surrogate endpoint” for evaluating clinical benefit. In one aspect, the target analyte is a biomarker.

[0043] In one embodiment, the target analyte is selected from a protein, a protein fragment, a peptide, a polypeptide, a polypeptide fragment, an antibody, an antibody fragment, an antibody binding domain, an antigen, an antigen fragment, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, or any combination of any two or more thereof.

[0044] In one aspect, the target analyte is PAG-4, PAG-6, PAG-9, PAG-20, PAG-21 or any combination thereof. In an additional aspect, the target analyte may also be a MAP antibody, antibody fragment or binding domain thereof.

[0045] Capture elements specific for a target analyte are used to detect the presence or absence of the analyte in a sample. A wide range of complementary binding or coupling partners are known, with the choice of capture elements determined by the analytes to be detected, the requirement for adapter molecules and the level of specificity required for the assay. In various aspects, the capture elements are specific for binding/ detecting PAGs (e.g. PAG-4, PAG-6, PAG-9, PAG-20, PAG-21 or any combination thereof.) and IgG antibodies produced by a Johne’s disease infection (e.g. antibodies to Mycobacterium avium subspeciesparatuberculosis (MAP)).

[0046] The term “control element” refers to an element that is used to provide information on the function of the assay, for example binding specificity, the level of non-specific background binding, the degree of binding cross-reactivity, and the performance of assay reagents and the detection system. Preferred controls useful herein include at least one negative control to monitor background signal, at least one negative control to monitor assay specificity, at least one positive colorimetric control, and at least one positive control to monitor assay performance. [0047] The substrate of the invention comprises at least one fiduciary marker that will always be detectable on the substrate, preferably detectable irrespective of the performance of the assay or processing of the substrate.

[0048] The term “fiduciary marker” refers to a colored marker or label that will always be detectable on the substrate, preferably irrespective of the performance of the assay or processing of the substrate. The use of at least one fiduciary marker will obviate the necessity of this element being detected based on successful array processing, in comparison to the positive colorimetric controls. The fiduciary marker is therefore a “true” positive control that would always be detectable regardless of array processing, and can be used to orient and help to grid the array.

[0049] In preferred aspects, the fiduciary marker is a dye, dye-conjugated protein or a chromogenic protein such as hemoglobin.

[0050] The term “negative control” refers to an element comprising print buffer or an unrelated protein to which no complementary binding partner is intended to be present in the assay. Any detectable signal from the negative control can be used to determine the background threshold of the assay and the accuracy of any positive results. In one aspect, the negative control to monitor background signal is print buffer. The print buffer is a solution used to carry and print the capture elements and control elements onto the substrate and may comprise buffered saline, APTES, glycerol and a surfactant, preferably a polysorbate surfactant such as Tween 20. The blocking solution is used to reduce non-specific protein binding to the substrate surface and preferably comprises skim milk, casein, bovine serum albumin, gelatins from fish, pigs or other species, dextran or any mixture of any two or more thereof, preferably in a solution of phosphate buffered saline and a surfactant such as Tween 20.

[0051] The term “control capture element” refers to a capture element that functions as a control, either a negative control that should not bind any analyte or a positive control that will bind a non-target analyte.

[0052] The substrate of the invention also comprises at least one control to monitor assay performance. The control is intended to provide information of the efficiency of the complementary binding interactions or the quality or performance of the reagents used.

[0053] The term “control to monitor assay performance” refers to an element that forms one part of a complementary binding interaction during an assay and is intended to provide information on the accuracy of the assay result. In one embodiment, the positive control to monitor assay performance comprises one binding partner of a complementary binding pair, where the other binding partner is a sample component or an assay reagent. The assay performance control is preferably selected from a target analyte, a binding partner corresponding to and able to bind a non-target analyte that will be present in the sample, a binding partner corresponding to and able to bind an assay reagent, and a colorimetric enzyme label, or any combination of any two or more thereof. An example of a binding partner corresponding to and able to bind a non-target analyte that will be present in the sample is an anti-Ig antibody that will bind an immunoglobulin present in a serum sample, therefore confirming a sample has been added. An example of a binding partner corresponding to and able to bind an assay reagent is an anti-Ig antibody that will bind a secondary immunoglobulin that is used to process the assay, such as biotinylated anti-target analyte antibody. Another example of a binding partner corresponding to and able to bind an assay reagent is a biotinylated antibody that will bind a streptavidin-per oxidase conjugate that is used to process the assay.

[0054] In one aspect, the assay performance control comprises one binding partner of a complementary binding pair, wherein the other binding partner is an assay reagent. The assay performance control is preferably selected from the list comprising the target analyte, a nonspecific binding partner or a colorimetric enzyme label.

[0055] In another aspect, the complementary binding partners comprise antibody-antigen interactions or antibody-ligand interactions.

[0056] The substrate of the invention also comprises at least one control to monitor assay specificity. The control is intended to provide information of the specificity of binding between the capture element and the target analyte, or between the binding partners of the assay detection steps.

[0057] The term “control to monitor assay specificity” refers to an element that is closely related to at least one binding partner of a complementary binding pair present in the assay and is intended to provide information of the specificity of the complementary binding. This control is a negative control that is not expected to generate a detectable result during normal assay processing. For example, in an antibody array for antigen detection, the assay specificity control would comprise an antibody that should not bind any antigen in the sample. Alternatively, in an antigen array for antibody detection, the assay specificity control would comprise an antigen that should not bind any antibody in the sample. [0058] In one aspect, the assay specificity control is Mycobacterium phlei protein extract. In another aspect, the assay specificity control Mycobacterium tuberculosis antigen.

[0059] The term “positive colorimetric control” as used herein refers to an enzyme or enzyme conjugate that provides a detectable signal upon addition of the enzyme substrate.

[0060] In one embodiment, the positive colorimetric control is an enzyme label conjugate capable of reacting with a colorimetric substrate, comprising an enzyme selected from the list comprising horseradish peroxidase, alkaline phosphatases, P-D-galactosidase or glucose oxidase. [0061] The identity of the assay controls will be dependent on the type of array, the identity of the target analyte, and the type of sample to be analyzed.

[0062] For example, either anti-bovine IgG-HRP or specific monoclonal IgG-HRP may be used in arrays printed with antigens and antibodies, respectively. The final detection antibody in antigen arrays will often be anti-bovine IgG-HRP, while for antibody arrays it will often be a HRP conjugated IgG antibody specific for the targeted analyte or a bovine IgG specific for the target analyte that is detected later on the next step with anti-bovine IgG-HRP. These controls can provide a positive control in addition to providing information on the performance or quality of the HRP substrate.

[0063] The target protein or antigen and mouse IgG, bovine IgG and anti-bovine IgG present on antigen or antibody arrays can act either as positive or negative controls depending on the array format, in addition to providing information of assay specificity. For example, antigen or mouse IgG spots should provide the positive signal in antibody arrays, while the latter two should provide a positive signal in antigen arrays. These controls can also serve as controls for overall assay performance.

[0064] The terms “sample” and “specimen” as used herein are used in their broadest sense to include any composition that is obtained and/or derived from biological or environmental source, as well as sampling devices (e.g., swabs) which are brought into contact with biological or environmental samples. “Biological samples” include body fluids such as milk, urine, blood, plasma, fecal matter, cerebrospinal fluid (CSF), and saliva. In one embodiment, the biological sample is fluid obtained from a mammal, including milk, blood, plasma, serum, and stool. These examples are illustrative, and are not to be construed as limiting the sample types applicable to the present invention. [0065] In various aspects of the present invention, the sample is a milk or blood sample, including a plasma or serum sample.

[0066] The assay techniques used in conjunction with the substrates of the present invention include any of a number of well-known colorimetric enzyme-linked assays. Examples of such systems are well known in the art. The assay techniques are based upon the formation of a complex between a complementary binding pair, followed by detection with a colorimetric detection system comprising an enzyme-conjugate label and a colorimetric substrate. The detection system will be described with reference to enzyme-linked immunosorbent assays (ELISA), though a skilled person would appreciate that such techniques are not restricted to the use of antibodies but are equally applicable to any colorimetric assay.

[0067] In one embodiment, the ELISA is in the “sandwich” assay format. In this format, the target analyte to be measured is bound between two antibodies — the capture antibody and the detection antibody. In another embodiment, the ELISA is a non-competitive assay, in which an antibody binds to the capture antigen and the amount of bound antibody is determined by a secondary detection antibody.

[0068] Either monoclonal or polyclonal antibodies may be used as the capture and detection antibodies in sandwich ELISA systems. Monoclonal antibodies have an inherent monospecificity toward a single epitope that allows fine detection and quantitation of small differences in antigen. A polyclonal antibody can also be used as the capture antibody to bind as much of the antigen as possible, followed by the use of a monoclonal antibody as the detecting antibody in the sandwich assay to provide improved specificity. A monoclonal antibody can also be used as the capture antibody to provide specific analyte capture, followed by the use of a polyclonal antibody as the detection antibody in the sandwich assay. Additionally, both the capture and the detection antibodies could be monoclonal.

[0069] The term “antibody” as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non- naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains (see Huse et al., Science 246: 1275- 1281, 1989, which is incorporated herein by reference). These and other methods of making, for example, chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known (Winter and Harris, Immunol. Today 14:243-246, 1993; Ward et al., Nature 341:544-546, 1989; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press, 1999); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995); each of which is incorporated herein by reference). In addition, modified or derivatized antibodies, or antigen binding fragments of antibodies, such as pegylated (polyethylene glycol modified) antibodies, can be useful for the present methods. As such, Fab, F(ab')2, Fd and Fv fragments of an antibody that retain specific binding activity are included within the definition of an antibody.

[0070] The term “secondary antibody” refers to an antibody that will bind a target analyte and that is conjugated with either an adaptor molecule such as biotin or an enzyme label such as horseradish peroxidase (HRP). Antibody-adaptor conjugates are processed to give a detectable result by contacting the antibody-adaptor conjugate with an adaptor- enzyme conjugate and then the enzyme substrate; for example, antibody-biotin conjugates will bind streptavidin-HRP conjugates. Antibody-enzyme label conjugates include antibody-HRP conjugates. Use of secondary antibodies is discussed and exemplified below.

[0071] The term “binds specifically” or “specific binding activity” or the like, means that two molecules form a complex that is relatively stable under physiologic conditions. The term is also applicable where, an antigen-binding domain is specific for a particular epitope, which is carried by a number of antigens, in which case the antibody carrying the antigen-binding domain will be able to bind to the various antigens carrying the epitope. Specific binding is characterized by a high affinity and a low to moderate capacity. Typically, the binding is considered specific when the affinity constant is about U I CF⁶ M, generally at least about U 1 CT⁷ M, usually at least about UK)^-8 M, and particularly at least about 1 xlO^-9 M or less.

[0072] After array manufacture and prior to sample addition, all available protein-binding sites on the substrate surface are blocked by addition and incubation with one or a combination of reagents. These reagents are called “Blockers” and serve to decrease or at best eliminate nonspecific protein binding from the sample on the substrate surface, thereby decreasing overall background signal. This increases the ratio of signal to noise, thereby increasing the overall sensitivity of the assay. Blockers play no active part in the subsequent reactions between the sample and other assay reagents and the immobilized proteins on the substrate. Exemplary blockers include, but are not limited to, bovine serum albumin, casein, non-fat dry milk, gelatin derived from fish, pigs and other sources, dextran, serum derived from sources other than the sample being analyzed such as from steelhead salmon, guinea pigs, hamsters, rabbit and other sources, polyethylene glycol, polyvinyl pyrrollidone, and commercial preparations including HeteroBlock (Omega Biologicals, Bozeman, Mont.), SuperBlock, StartingBlock, SEA BLOCK (Pierce, Rockford, Ill.). Typically, blockers are made up in buffer solutions such as, for example, phosphate buffer, phosphate buffered saline, Tris buffer, acetate buffer and others. The blockers may also be supplemented with detergents such as, for example, Tween 20, Tween 80, Nonidet P40, sodium dodecyl sulfate and others.

[0073] An important consideration in designing an array is that the capture and detection antibodies of each binding pair must recognize two non-overlapping epitopes so that when the antigen binds to the capture antibody, the epitope recognized by the detection antibody must not be obscured or altered. A large number of complementary binding pairs have already been developed for ELISA and can be used in the present invention.

[0074] For multiplexed assays, it is also important that there is no overlap between each of the binding pairs to eliminate cross-reactivity. A number of multiplexed ELIS As have been developed and it is anticipated other combinations of binding pairs could be configured through testing.

[0075] In one aspect, the enzyme-conjugate label comprising an enzyme selected from the list comprising horseradish peroxidase, alkaline phosphatase, P-D-galactosidase or glucose oxidase.

[0076] In an additional aspect, the enzyme label may be conjugated directly to a primary antibody or introduced through a secondary antibody that recognizes the primary antibody. It may also be conjugated to a protein such as streptavidin if the primary antibody is biotin labelled.

[0077] In a further aspect, the assay detection system comprises a detection colorimetric substrate selected from the list comprising 3,3', 5,5'-tetramethylbenzidine, diaminobenzidine, metal-enhanced diaminobenzidine, 4-chl oro-1 -naphthol, colloidal gold, nitro-blue tetrazolium chloride, 5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt and naphthol AS-MX phosphate+Fast Red TR Salt.

[0078] In certain aspects, the colorimetric reaction can be detected and optionally quantified and analyzed using an image capture device such as a digital camera or a desktop scanner attached to a computer. Known methods for image analysis may be used. For example, the concentration values of known standard elements can be used to generate standard curves. Concentration values for unknown analytes can be analyzed using the standard curve for each analyte to calculate actual concentrations. Values for each analyte can be identified based on the spotting position of each capture element within the array.

[0079] The present invention provides methods of in vitro diagnostic applications for the detection of Johne’s disease or pregnancy such as manual multiplex immune assay, automated multiplex immune assay (MIA), Automated MIA (e.g., PictArray™, U.S. Patent No. 9,625,453), multiple lateral flow immunoassay (LFIA), manual singleplex ELISA (solid phase), automated chemiluminescent immune assay (CLIA), wash-free immune assays (manual and automated), automated centrifugal microfluidics-based immunoassay, lab-on-a-chip based immunoassay, paramagnetic bead-based manual ELISA, point-of-care (POC) immunoassays, smart phone based immunoassay and other immunoassay formats. In one aspect, detection is by colorimetric imaging (e.g., PictArray™, U.S. Patent No. 9,625,453), absorbance (e.g., manual ELISA), chemiluminescence (e.g., automated CLIA, CRET), florescence (e.g., manual immunoassays, ELISA, FRET) and by the naked eye (e.g., lateral flow immunoassays).

[0080] In a manual multiplex immune assay (MIA), the PAGs are detected by sandwich immunoassay (IA) while anti-MAP antibodies (Abs) are determined via an indirect IA. Both the analytes would be simultaneously detected in the same well for the diagnosis of Johne’s disease and pregnancy a membrane-free MIA procedure will be used for the printing of microarray spots (e.g., PictArray™, U.S. Patent No. 9,625,453) directly on the solid surface of 96-well microtiter plate (MTP). The biorecognition elements responsible for the binding of PAGs and anti-MAP Abs will be printed onto the surface of the MTP well using a leach-proof biomolecular immobilization procedure that involves the addition of 1% aminopropyltri ethoxy silane (APTES) in the printing buffer.

[0081] The automated MIA automates all the steps in the manual MIA and employs an integrated colorimetric reader and image analysis software. The automated IAS are ideal for high- throughput tests for Johne’s disease and pregnancy in cattle.

[0082] For a multiplex lateral flow immunoassay (LFIA), biomarkers are immobilized in two different lines on a nitrocellulose membrane strip. The results from this assay can be interpreted by naked eye. Alternatively, the rapid LFIA tests could be performed using previously described MIA formats including manual and automated MIAs. The biorecognition elements responsible for the detection of PAGs and anti-MAP Abs would be immobilized in two control lines on a nitrocellulose membrane strip.

[0083] Manual singleplex ELISA could also be used to detect Johne’s disease and pregnancy and would use custom-synthesized anti -PAG Abs that would be specific for various PAGs (e.g. PAG-4, PAG-6, PAG-9, PAG-20, PAG-21).

[0084] Automated multiplex and singleplex CLIAs can also be developed for the diagnosis of Johne’s disease and pregnancy in cattle. The MAP and anti -PAG Ab could be bound covalently to paramagnetic beads (micron-sub-micron size) and used for the detection of anti-MAP Ab and PAGs in sample. Various formulations of paramagnetic beads could be coated with MAP and anti- PAG Ab and then mixed together for MIA. The detection signal in case of automated CLIAs could be generated by conjugating the detection antibody with acridinium or other chemiluminescent labels and providing the appropriate trigger solutions for the generation of chemiluminescent signal.

[0085] Manual and automated wash-free MIAs could be developed for the detection of anti- MAP Abs and PAGs. As an example, the sandwich IA for PAGs would involve the specific biomolecular interactions of anti-PAG Ab-coated donor beads with another anti-NP Ab-coated acceptor beads in the presence of PAGs in sample, thereby forming sandwich immune complexes and generating a chemiluminescent signal due to the proximity of donor and acceptor beads. Another wash-free MIA format, such as that based on electrochemiluminescent ELISA, could also be developed. It will involve the detection of analytes in sample using biomolecule-coated (antibody- or antigen-coated) carbon electrode surface-based microwell plates and SULFO-TAG- labeled detection Ab that emits light upon electrochemical stimulation.

[0086] The centrifugal microfluidics-based automated immunoassay could also be performed to detect Johne’s disease and pregnancy. The MAP can be covalently bound to paramagnetic beads and used for the detection of anti-MAP antibodies. Similarly, the capture antibodies for PAGs can be coated covalently to the paramagnetic beads and used for the detection of PAGs via sandwich IA. The magnetic beads are transferred from one chamber to another in the apparatus by a magnet. The detection of analyte occurs in a reaction chamber, which is followed by washing the specific immune complexes formed on paramagnetic beads and their transfer to the detection chamber for the generation and reading of assay signal. The assay signal most commonly used in is the chemiluminescent, however, absorbance and fluorescence could also be used. All the microfluidic operations could be performed in a compact benchtop centrifugal microfluidic analyzer.

[0087] A lab-on-a-chip (LOC)-based immunoassay (IA) could also be developed that could use MIA procedures similar to those previously described. Such LOC-based IAS could use paramagnetic beads or solid surfaces for the covalent attachment of MAP and anti-PAGs Ab. The detection signal could be chemiluminescent, fluorescent, absorbance, electrochemical or colorimetric.

[0088] Manual singleplex ELISA can be used to detect Johne’s disease and pregnancy, employing paramagnetic beads that could be bound covalently to MAP or anti-PAGs Ab.

[0089] A point of care (POC) IA, such as that based on electrochemical detection using a disposable strip, can also be developed. The IA would be a label-free immunoassay using an electrochemical reader or a smartphone-based reader. Various smartphone-readout based IAs could also be developed where an optomechanical attachment would be developed enabling the readout of colorimetric detection signal via the smartphone back camera. Therefore, the smartphone will act as the POC colorimetric readout device.

[0090] In additional to the assay formats mentioned above, the invented assay formats for the detection of Johne’s disease and pregnancy diagnosis could also be used for the development of novel and emerging IA formats, which are based on advances in complementary technologies. Some examples of such prospective IA formats are wash-free IA based on FRET (fluorescence resonance energy transfer) or CRET (chemiluminescence resonance energy transfer); signal- enhanced IA based on the use of nanoparticle-based signal detection step or the use of micro- and subsmicro- beads for binding capture antibodies/antigens; rapid multiplex IAs based on Lab-in-a- tube technology, and vertical microfluidics.

[0091] The substrates of the present invention are particularly amenable to use in kits for the detection of target analytes. Such kits may comprise the substrates together with instructions and any assay consumables required. Different kits are envisaged for different target analytes and types of array. Accordingly, in an additional embodiment, the present invention provides a kit for detecting a plurality of target analytes in a sample, including a substrate and optionally one or both of a background reducing reagent, and a colorimetric detection system. In one aspect, the kit further has one or more items selected from a wash solution, one or more antibodies for detection of antigens, ligands or antibodies bound to the capture elements or for detection of the positive controls, software for analyzing captured target analytes, a protocol for measuring the presence of target analytes in samples, a sample diluent, blotting TMB (3,3',5,5’-tetramethylbenzidine; MW ^:::: 240.4) and a secondary antibody. In an additional aspect, the antibodies for detection comprise antibody-binding protein (BP) conjugates, antibody- enzyme label conjugates, or any combination thereof. In various aspects, the sample is a milk or a blood sample. In certain aspects, blood sample is serum or plasma. In an additional aspect, the substrate is a solid or porous substrate. In certain aspects, the solid substrate is a paramagnetic bead, microtiter plate, or microparticle. In one aspect, the background reducing reagent is Mycobacterium phlei protein extract. In an additional aspect, the colorimetric detection system comprises HRP-labelled anti-PAGs Ab and HRP-labelled anti- bovine IgG Ab. In a further aspect, the secondary antibody comprises at least one bovine anti-PAG antibody.

[0092] In another aspect, the invention also relates to a method of processing a substrate of the invention. Such a method comprises providing a substrate of the invention as described above, adding at least one sample to the substrate, and processing the substrate such that a detectable result is given by two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to monitor assay performance.

[0093] In another aspect, the present invention provides methods for processing a microarray by providing a substrate, adding at least one sample to the substrate, and processing the substrate such that a detectable result is given by two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to monitor assay performance.

[0094] In one aspect, the step of processing the substrate or microarray comprises a blocking step during which available protein-binding sites on the substrate or microarray are blocked with a blocker, an optional wash step, contacting the substrate or microarray with the sample containing the one or more analytes to be measured, a wash step to remove non-bound material from the substrate or microarray, contacting the substrate or microarray with one or more secondary antibodies that correspond to and will bind one or more target analytes and non-target analyte that is bound to an assay performance control, a wash step, and contacting the substrate or microarray with one or both of an enzyme conjugate or an enzyme substrate to generate a detectable result.

[0095] In one embodiment, the present invention provides methods for detecting an analyte in a sample comprising providing a substrate, adding at least one sample to the substrate, and processing the substrate such that a detectable result is provided. In one aspect, the detectable result includes two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to detect an analyte in the sample.

[0096] In another aspect, the substrate of the invention can be used for the simultaneous detection of at least one target analyte in a sample, and preferably a plurality of different target analytes in a sample, and have utility in diagnostic and screening assays.

[0097] Thus, the substrates of the invention provide the advantage that they can be adapted to high throughput (or ultra high throughput) analysis and, therefore, any number of samples (e.g., 96, 1024, 10,000, 100,000, or more) can be examined in parallel, depending on the particular support used. A particular advantage of adapting the substrates to high throughput analysis is that an automated system can be used for adding or removing reagents from one or more of the samples at various times, for adding different reagents to particular samples, or for subjecting the samples to various heating cycles.

[0098] For example, the automated system may consist of one or more temperature-controlled chambers and one or more robotic arms mounted on a deck that has platforms configured to hold 96-well plates. The movement of the robotic arms and the temperature in the chambers are controlled by a central computer unit. The array plates are stacked on the deck of the instrument. In one embodiment, the plates containing samples to be analyzed are stacked in a chamber with temperature of 4°C. One robotic arm then sequentially transfers each individual array plate on one platform while the other arm sequentially transfers each individual sample plate on the second platform. A nozzle containing 96 disposable tips then aspirates a predetermined volume of sample from each well of the sample plate and transfers the sample to the corresponding wells of the array plate. The array plate containing the sample is then transferred to a chamber with temperature of 37°C. This process is repeated until sample has been added to all the array plates stacked on the deck. The array plates are incubated for a predetermined time followed by transfer of each plate to the platform for addition of wash buffer with the nozzle containing 96 disposable tips. The wash buffer is aspirated after a predetermined time and this wash process is repeated multiple (i.e., two or more) times. Each array plate then receives the secondary antibody followed by transfer to a chamber with temperature of 37°C. The array plates are incubated for a predetermined time followed by transfer of each plate to the platform for addition of wash buffer with the nozzle containing 96 disposable tips. The wash buffer is aspirated after a predetermined time and this wash process is repeated multiple (i.e., two or more) times. Each array plate then receives the detection reagent followed by incubation for a predetermined time followed by transfer of each plate to the platform for addition of wash buffer with the nozzle containing 96 disposable tips. The wash buffer is aspirated after a predetermined time and the plate transferred to the 37° C chamber for drying. The plates are transferred back to the deck after a predetermined period and manually processed for analyses of data.

[0099] In addition to the convenience of examining multiple test agents and/or samples at the same time, such high throughput assays provide a means for examining duplicate, triplicate, or more aliquots of a single sample, thus increasing the validity of the results obtained, and for examining control samples under the same conditions as the test samples, thus providing an internal standard for comparing results from different assays.

[0100] In one embodiment, the present invention provides a method for detecting an analyte in a sample comprising providing a substrate, adding at least one sample to the substrate, and processing the substrate such that a detectable result is provided. In one aspect, the detectable result includes two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to detect an analyte in the sample.

[0101] The present invention also provides for antibodies to PAGs. In one aspect, the antibodies bind to antigenic sites common to all PAGs.

[0102] In one embodiment, the present invention provides an isolated peptide having the amino acid sequence DTVRIGDLVSTDQ (SEQ ID NO: 1). In an additional embodiment, the present invention provides an isolated peptide having the amino acid sequence GSWMFGGVDHRYYKGELNW (SEQ ID NO:2). In a further embodiment, the present invention provides an isolated peptide having the amino acid sequence ITIGTPPQEFQV (SEQ ID NO:3).

[0103] In one embodiment, the present invention provides an antibody that binds a peptide having the amino acid sequence DTVRIGDLVSTDQ (SEQ ID NO: 1). In one aspect, the antibody is monoclonal.

[0104] In an additional embodiment, the present invention provides an antibody that binds a peptide having the amino acid sequence GSVVMFGGVDHRYYKGELNW (SEQ ID NO:2). In one aspect, the antibody is monoclonal. [0105] In a further embodiment, the present provides an antibody that binds a peptide having the amino acid sequence ITIGTPPQEFQV (SEQ ID NO:3). In one aspect, the antibody is monoclonal.

[0106] The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. “Native antibodies” and “intact immunoglobulins”, or the like, are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. The light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (X), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, s, y, and p, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0107] Experimentally, antibodies can be cleaved with the proteolytic enzyme papain, which causes each of the heavy chains to break, producing three separate antibody fragments. The two units that consist of a light chain and a fragment of the heavy chain approximately equal in mass to the light chain are called the Fab fragments (i.e., the "antigen binding" fragments). The third unit, consisting of two equal segments of the heavy chain, is called the Fc fragment. The Fc fragment is typically not involved in antigen-antibody binding, but is important in later processes involved in ridding the body of the antigen. “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. “Single-chain Fv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Spring er- Verlag, New York, pp. 269-315 (1994).

[0108] An “antigen” according to the invention covers any substance that will elicit an immune response. In particular, an “antigen” relates to any substance, preferably a peptide or protein, that reacts specifically with antibodies. According to the present invention, the term “antigen” comprises any molecule which comprises at least one epitope. Preferably, an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction. According to the present invention, any suitable antigen may be used, which is a candidate for an immune reaction, wherein the immune reaction is preferably a cellular immune reaction. An antigen is preferably a product which corresponds to or is derived from a naturally occurring antigen.

[0109] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used

EXAMPLES

EXAMPLE 1

MEMBRANE-FREE BOVINE MIA

[0110] The membrane-free MIA format involves the simultaneous detection of anti-MAP Ab and desired PAGs (i.e., PAG-4, PAG-6, PAG-9, PAG-20, PAG-21) in a milk sample, which act as the diagnostic biomarkers of Johne’s disease and pregnancy in cattle.

[0111] The MIA is a membrane-free format that employs directly the polystyrene solid substrate of the 96- well microtiter plate’s (MTP) well for the spotting of microarrays. The raw materials to be used for the development of the assay to detect Johne’s disease have been identified and are shown in Table 1. Further, the selected raw materials for PAGs assay have also been identified. [0112] The generalized assay format of membrane-free MIA is summarized in Fig. 1. The antiMAP Abs would be detected by indirect immunoassay (IA) (Fig. 1A), while the PAGs would be detected by sandwich IA (Fig. IB). The multi-specific capture Ab against PAGs and the MAP antigen will be printed as spots onto the assay surface using a microarray printer either within the same well or in separate wells depending on the assay format. The subsequent steps in the MIA leads to the formation of colored spots if the analytes were present in the sample (Fig. 2). The intensities of colored spots are directly proportional to the concentration of analytes present in the sample.

[0113] The proof-of-concept of MIA was demonstrated on the detachable 8-well module of 96- well MTP, where the two bovine tests, i.e. Johne’s disease and pregnancy, were performed into a single well that also incorporated four IA controls for monitoring the performance of MIA (Fig. 3). The MIA could detect up to ten analytes in duplicate in a single well. A milk sample is the preferred sample for the analysis of Johne’s disease and pregnancy for non- invasive in vitro diagnosis. The MIA could also be performed using serum and plasma samples.

[0114] The Johne’s disease antigen, i.e. MAP, a protoplasmic cell extract of Mycobacterium ssp., was printed at a concentration of 5 mg/ml in 1% APTES using the carbonate buffer as the print buffer.

[0115] Table 1. Assay Materials

EXAMPLE 2

PRINTING OF BIOMOLECULES ONTO THE SOLID SUBSTRATE

[0116] The biomolecules, i.e. MAP Ag and anti -P AGs Ab, were prepared at their specific optimized concentrations in 1% APTES solution in deionized water (DIW) using the carbonate buffer as the print buffer (Fig. 4). The biomolecules were then printed onto the membrane-free substrate in the desired format using an array er, e.g. s PictArray™, U.S. Patent No. 9,625,453, as shown in Fig. 3. Subsequently, the printed 8-well detachable strips of a 96- well MTP were stored overnight and then blocked by incubating with 200 ul of a 2.5% casein blocking solution for 1 hour at 37°C. The excess unbound reagents were then washed away in three consecutive washings.

EXAMPLE 3

PRE-ABSORPTION STEP FOR SAMPLES BEFORE TESTING

[0117] Samples that are going to be tested for Johne’s disease (containing Abs towards Mycobacterium avium paratuberculosis) are pre-incubated with an extract of Mycobacterium phlei to eliminate all possible cross-reactive Abs to different mycobacteria from the sample.

[0118] Rehydrated product of lyophilized m. phlei whole cells: M. phlei bacterial culture is heat inactivated and lyophilized into dry pellets. The dry pellets are rehydrated in 0.85% saline water to form an insoluble suspension, which is used as absorbent at a concentration of 0.2-2.5 mg/ml.

[0119] Soluble bacterial protein extracts: Proteins are extracted from heat-killed M. phlei bacterial culture and mixed with homogenous buffer solution to form the absorbent for M.phlei.

[0120] There are three different ways of absorbing the cross-reactive Abs from the sample via the use of these absorbents:

[0121] Absorbent present in the sample dilution buffer: Diluted samples are incubated for 15- 60 mins before the MIA. Thereafter, the clear supernatant is taken as sample for the MIA. [0122] Absorbent-coated preincubation plate: Samples would first be diluted in absorbent- coated preincubation plate and incubated for 15-60 mins. Thereafter, the sample is transferred to the assay plate for the MIA.

[0123] Absorbent-coated paramagnetic beads: Absorbent-coated paramagnetic beads would be added to the sample and incubated for 5-15 mins. Thereafter, the clean sample is taken for the MIA while the paramagnetic beads are held together by a magnet at the bottom of the tube.

EXAMPLE 4

THREE STEP MIA

[0124] The protocol is shown in Fig. 2.

[0125] Sample: The milk sample is diluted 1:20 using sample diluent (PBS-T). 100 ul of the diluted milk sample is added to each MTP’s well. The MTP is incubated at 37°C for 30 min and subsequently washed 3 times with 300 pl of IX washing solution.

[0126] Detection solution: The HRP-labelled secondary Abs are then provided to detect the formation of specific immune complexes on the printed spots. 100 ul of mixed anti-bovine IgG conjugate and anti-PAG conjugate is added to each MTP’s well. The MTP is incubated at 37°C for 30 min and then washed 3 times with 300 pl of IX washing solution.

[0127] TMB: The precipitating TMB is used as a substrate for HRP to develop the colorimetric spots. 100 ul of TMB is added to the MTP’s well and incubated for 15 minutes at room temperature, where the MTP is covered to protect it from light. Afterwards, the liquid is discarded, and MTP is inverted and tapped onto an absorbent paper towel to eliminate any remaining liquid. [0128] Readout: MTP is placed into a commercial reader for the determination of colorimetric intensities of spots. The results of the MIA are generated by software

EXAMPLE 5

FOUR STEP MIA

[0129] The four step MIA is shown in Fig. 5.

[0130] Sample: The milk sample is diluted 1:20 using sample diluent (PBS-T). 100 ul of the diluted milk sample is added to each MTP’s well. The MTP is incubated at 37°C for 30 min and subsequently washed 3 times with 300 pl of IX washing solution. [0131] Secondary antibody: 100 ul of specific bovine anti-PAG Ab is added to each well of MTP. The MTP is incubated at 37°C for 30 min and then washed 3 times with 300 pl of IX washing solution.

[0132] Detection solution: 100 ul of anti-bovine IgG antibody conjugated to HRP is added to each well of MTP. The plate is incubated at 37°C for 30 min and subsequently washed 3 times with 300 pl of IX washing solution.

[0133] TMB: The precipitating TMB is used as a substrate for HRP to develop the colorimetric spots. 100 ul of TMB is added to the MTP’s well and incubated for 15 minutes at room temperature, where the MTP is covered to protect it from light Afterwards, the liquid is discarded, and MTP is inverted and tapped onto an absorbent paper towel to eliminate any remaining liquid.

[0134] Readout: MTP is placed into a multiplex colorimetric reader for the determination of colorimetric intensities of spots. The results of the MIA are generated by software.

EXAMPLE 6

COLORIMETRIC READOUT

[0135] The Bovine MIA results in the formation of colorimetric spots, the intensity of which is directly proportional to the concentration of analytes, anti -MAP IgG and PAGs, present in the milk sample. The colorimetric arrays are imaged by either using a handheld colorimetric reader device or commercially available colorimetric microarray readers.

EXAMPLE 7

IMAGE ANALYSIS

[0136] Image analysis software will be used and is based on an image analysis algorithm. The software will analyze the colorimetric images of microarray spots in each well, which are captured by commercial readers and generate the desired results. The software will identify the array wells followed by the detection of positive control spots within each well. The positive control spots act as alignment anchors and are used by the software to place a microarray grid for all spots in a well. The image analysis software then determines the pixel intensity for each colorimetric spot based on image analysis algorithm. The data generated from each spot is then collated.

EXAMPLE 8

AUTOMATED BOVINE MIA (MEMBRANE-FREE, 96-WELL MTP) [0137] The automated MIA will be performed inside an analyzer, where all the steps in the manual MIA will be automated. The dispensing and aspiration of reagents is done by a needle attached to the robotic arm. The analyzer would have a dedicated compartment for putting the patient sample vials, and dedicated spaces for putting the wash buffer, TMB substrate and other buffers. The washing of the MTP wells will be done by the robotic needle using specific washing programs. Similarly, the needle will be washed after each dispensing step. However, disposable tips could also be used, which would obviate the cleaning of the needle after each dispensing step. All the steps of the IA will be optimized for the automated MIA. The readout of the colorimetric array spots in the processed 96-well MTP will be performed using an integrated colorimetric reader and an image analysis software.

EXAMPLE 9

AUTOMATED CHEMILUMINESCENT IMMUNOASSAY (CLIA)

[0138] The assay formats used for development of MIA could be further employed for the development of automated CLIAs, both multiplex as well as singleplex, for the diagnosis of Johne’s disease and pregnancy in cattle. The MAP antigen could be bound covalently to paramagnetic beads (micron-sub-micron size) and used for the detection of bovine IgG against MAP via indirect immunoassay. The detection of PAGs could also be done by sandwich immunoassay, where the capture Abs against PAG would be coated covalently onto the paramagnetic beads. The detection signal in case of automated CLIAs could be generated by conjugating the detection Ab with acridinium or other chemiluminescent labels and providing the appropriate trigger solutions for the generation of chemiluminescent signal. All the automated CLIAs are performed using a high- throughput analyzer. The assay reagents are stored in the form of assay cartridges that can used for up to 100 tests. The buffers, wash solution and trigger solutions are stored at the respective places in the analyzer.

EXAMPLE 10

BOVINE ARRAY IN TWO SEPARATE WELLS

[0139] The Bovine MIA (Membrane-free, Two wells) format utilizes the 96-well microtiter plate as the assay surface. For each 96-well plate, anti -PAG capture Ab is immobilized as duplicate spots in wells of every odd 8-well strip while MAP antigen is immobilized in duplicate in wells of every even numbered 8-well strip. [0140] The assay protocol follows the general procedure described above. The milk sample is diluted and added to a well from an odd and even numbered 8-well strip and incubated at 37°C then washed with wash buffer. HRP labelled anti-PAG detection Ab is added to all odd numbered wells and HRP labelled anti-bovine IgG detection Ab is added to all even numbered wells of the 96-well plate. The wells are incubated at 37°C and then washed followed by the addition of TMB substrate. After a short incubation, the wells are washed once and then analyzed.

EXAMPLE 11

JOHNE’S DISEASE AND BOVINE PREGNANCY ELISA

[0141] Manual ELISA can be developed for the detection of anti -MAP IgG and PAGs separately using the developed MIA procedure with customization of some steps for ELISA. The 96-well MTP would be coated with a mixture MAP antigen either passively or using a leach-proof biomolecular immobilization procedure based on silane chemistry. All the immunoassay steps for the detection of anti-MAP IgG by ELISA would then be performed exactly as specified in the bovine MIA except the last step. In the case of ELISA, the signal would be generated by enzyme-substrate reaction by providing TMB and H2O2 to the HRP-labeled detection Ab. The enzyme-substrate reaction is stopped by providing a stop solution comprising of IN H2SO4. The optical density of the colorimetric solution is then read at 450 nm with reference at 650 nm. The detection of anti-MAP IgG is done by indirect assay while the detection of PAGs is could done by sandwich assay.

EXAMPLE 12

PREGNANCY ELISA

[0142] Ab immobilization and Casein blocking: Mix anti-PAG capture Ab (optimal concentration, in pg mL'¹) with 0.5-3 % (v/v) APTES in the ratio of 1 : 1 (v/v). Incubate each of the desired wells of a 96-well MTP with 100 pL of the freshly prepared anti-PAG capture Ab solution for 30-60 min at room temperature. Wash five times with 300 pL of 0.1M PBS, pH 7.4. Washing can also be performed with an automatic plate washer. Passive Ab immobilization, by incubating with the Ab overnight at 4 deg C, could also done. Block the Ab-bound wells with 300 pL of 0.5- 5% (w/v) casein or other appropriate blocking buffer for 30 min-2 h at 37°C followed by extensive PBS washing.

[0143] Sandwich ELISA: Add 100 pL of the milk/serum/plasma sample to different casein or other buffer blocked wells. Incubate for 30 min-1 h at 37°C and wash extensively with PBS. Add 100 pL of HRP-labeled anti-PAG detection Ab in each of the PAG-captured wells. Incubate for 30 min-1 h at 37 °C and wash extensively with PBS. Add 100 pL of TMB-H2O2 mixture to each of these wells and incubate at room temperature to develop color for 5-30 min. Stop the enzymesubstrate reaction by adding 50 pL of 1 N H2SO4 to each well. Determine the absorbance at a primary wavelength of 450 nm taking 540 nm as the reference wavelength a microplate reader.

EXAMPLE 13

JOHNE’S DISEASE (ANTI-MAP IgG ELISA)

[0144] Ab immobilization and Casein blocking: MAP antigen solution with 0.5-3% (v/v) is mixed with APTES in the ratio of 1 : 1 (v/v). Desired wells of a 96-well MTP are incubated with 100 pL of the freshly prepared antigen solution for 30 min at room temperature. Wells are washed five times with 300 pL of 0.1M PBS, pH 7.4. Washing can also be performed with an automatic plate washer. Passive Ag immobilization, by incubating with the Ag overnight at 4 deg C, could also be done. MAP Ag-bound wells are blocked with 300 pL of 0.5-5% (w/v) casein or other blocking buffer for 30 min - 2 h at 37°C followed by extensive PBS washing.

[0145] ELISA: 100 pL of the milk/serum/plasma sample is added to different casein-blocked wells. Wells or plate are incubated for 30 min-2 h at 37°C and washed extensively with PBS. 100 pL of HRP-labeled anti-bovine IgG detection Ab is added in each of the wells. Wells are incubated for 30 min-1 h at 37 °C and washed extensively with PBS. 100 pL of TMB-H2O2 mixture is added to each of these wells and incubated at room temperature to develop color for 5-30 min. The enzyme-substrate reaction is stopped by adding 50 pL of 1 N H2SO4 to each well. The absorbance at a primary wavelength of 450 nm taking 540 nm as the reference wavelength is determined in a microplate reader.

EXAMPLE 14

RAPID ONE STEP KINETICS-BASED ELISA

[0146] A customized rapid one step kinetics-based ELISA procedure will be used for the detection of PAGs and anti-MAP IgG, as described by Vashist et al. in Biosensors and Bioelectronics 67, 73-78, 2015.

EXAMPLE 15

RAPID ONE STEP KINETICS-BASED ELISA USING PARAMAGNETIC BEADS [0147] A customized rapid one step kinetics-based ELISA procedure using paramagnetic beads will be used for the detection of PAGs and anti-MAP IgG, as described by Vashist et al. in Analytical Biochemistry 456, 32-37, 2014.

EXAMPLE 16

CENTIFUGATION MICROFLUIDIC S-BASED AUTOMATED POINT OF CARE IMMUNOASSAY

[0148] A customized centrifugal microfluidics-based automated point-of-care immunoassay procedure using paramagnetic beads will be used for the detection of PAGs and anti-MAP IgG, as described by Czilwik et al. in RSC Advances 5(76), 61906-61912, 2015.

EXAMPLE 17

WASH-FREE IMMUNOASSAY

[0149] Manual and automated wash-free MIAs could be developed for the detection of anti- MAP IgG and PAGs. As an example, the sandwich IA for PAG would involve the specific biomolecular interactions of anti-PAG Ab coated donor beads with another anti-PAG Ab-coated acceptor beads in the presence of PAG in sample, which form sandwich immune complexes and generate a chemiluminescent signal as the donor and acceptor beads are in proximity.

[0150] Another wash- free MIA format, such as that based on electrochemiluminescent ELISA, could also be developed. It will involve the detection of analytes in sample using biomolecule- coated (antibody- or antigen-coated) carbon electrode surface- based microwell plates and SULFO- TAG-labeled detection Ab that emits light upon electrochemical stimulation.

EXAMPLE 18

CUSTOM SYNTHESIS OF ANTI-PAGs Ab FOR PREGNANCY TESTING

[0151] Identification of common antigenic sites in desired PAGs. The published sequences of the desired early PAGs (i.e. PAG-4, PAG-6, PAG-9, PAG-20, and PAG-21) were studied and the common antigenic sites were identified.

[0152] Two of the recognized antigenic sites are mentioned at the Green et al. publication (J. A. Green et al. Theriogenology 63 (2005) 1481-1503). The author had generated monoclonal antibodies towards the entire PAGs (group of early pregnancy PAGs). A selection of monoclonal antibodies would be used to identify specific PAGs within the early pregnancy cotyledonary extracts by affinity chromatography. The eluted proteins were subjected to SDS-PAGE followed by in-gel trypsin digestion giving a range of different peptides per PAG. The peptides were identified through mass fingerprinting by MALDI-TOF mass spectrometry, and the matching sequence for each peptide is detailed in a table within the publication. The specific peptides were not identified by affinity to the antibodies, i.e. they were not identified as antigenic, so they were just a product of the in-gel trypsin digestion of the entire PAGs that they isolated.

[0153] Peptide antigen synthesis. Three peptides, designed from the antigenic sites that are common to all the PAGs, were synthesized via “solid-phase peptide synthesis” that involves stepwise amino-acid coupling, which leads to the desired peptide chain (Table 2). Peptides will be used individually as antigen (Peptide 1, Peptide 2, Peptide 3).

[0154] Table 2 PAG peptides

Name Peptide Sequence Purity

Peptide 1 DTVRIGDLVSTDQ (SEQ ID

NO:1 )

Peptide 2 GSWMFGGVDHRYYKGEL 90.62%

NW (SEQ ID NO:2)

ITIGTPPQEFQV (SEQ ID

Peptide 3 90% NO:3)

[0155] Peptide conjugation. The synthesized PAGs peptides will be conjugated by Proteogenix, France to KLH, OVA and/or BSA for the phage display technology based custom synthesis of anti-PAGs Ab. The peptides (containing the immunogenic epitopes) bound to carriers would be used as baits for the phage display.

[0156] Phage display screening. A mixture of the 3 custom synthesized PAG peptides bound to carriers in equimolar ratio would be used as bait for the screening of specific responders in a high diverse rabbit Ab phage display library, which consists of 1.09 xlO¹⁰ clones. .

[0157] Biopanning: 4-6 rounds of library screening against peptide conjugated to carriers, with specific strategy to achieve efficient (1) enrichment in binders to peptide, and (2) depletion in binders to carriers: - KLH, BSA and OVA conjugates to be alternately used: e.g. round 1 with KLH conjugate, round 2 with BSA-conjugate, round 3 with OVA-conjugate.

- Preliminary library depletion round against carrier alone to be performed before each round against carrier-conjugate.

[0158] Screening of unique binders for PAG peptides via ELISA. The multiple phage binders (more than 96) selected in the previous step would then be screened based on their performance in ELISA targeted at the binding of specific PAG peptides. This will lead to identification of at least 3-10 unique binders for our PAG peptides.

[0159] Phage DNA extraction + antibody sequencing. The phage DNA of the screened unique binders is then extracted and sequenced. The sequences of phage display binders will be owned and filed for patent protection in this application, as clearly specified by our contract manufacturer, Proteogenix, France.

[0160] Additional ELISA for individual peptide. Positive binders against the peptide mixture would be screened against each PAG peptide individually also so that the targeted peptide (epitope) of each binder could be identified.

[0161] Recombinant antibody production in CHO system. The recombinant anti-PAGs Abs would then be produced in Chinese Hamster Ovary (CHO) cells via the standard Ab production procedure being followed by the custom manufacturer (Proteogenix, France). .

[0162] Affinity mapping of anti-PAGs Abs. The anti-PAGs Abs corresponding to the screened unique binders would be analyzed for their binding kinetics (KON, KOFF, KD) and then graded on the basis of their affinity. The affinity mapping would be done by surface plasmon resonance based BiaCore instruments or bilayer interferometry-based Octet systems. The anti-PAGs Abs with the highest affinity would then be used for the development of sandwich IA for PAGs in the bovine MIA.

[0163] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A substrate comprising at least two capture elements specific for Johne’s disease and/or pregnancy on the substrate, each capture element corresponding to and being able to bind a target analyte, the substrate further optionally comprising a plurality of control elements comprising: a) at least one fiduciary marker, b) at least one negative control to monitor background signal, c) at least one negative control to monitor assay specificity, d) at least one positive colorimetric control, e) at least one positive control to monitor assay performance and any combination thereof.

2. The substrate of claim 1, wherein the capture elements bind target analytes, wherein the target analytes are indicative of a Johne’s disease infection and/or pregnancy.

3. The substrate of claim 1, wherein the target analyte is selected from a protein, a protein fragment, a peptide, a polypeptide, a polypeptide fragment, an antibody, an antibody fragment, an antibody binding domain, an antigen, an antigen fragment, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, epitope, or any combination thereof.

4. The substrate of claim 3, wherein the target analyte is a bovine pregnancy associated glycoprotein (PAG) or epitope thereof.

5. The substrate of claim 4, wherein the PAG is selected from PAG-4, PAG-6, PAG-9, PAG-20, PAG-21 or any combination thereof.

6. The substrate of claim 3, wherein the target analyte is a Mycobacterium avium subspecies paratuberculosis (MAP) antibody, fragment or binding domain thereof.

7. The substrate of claim 1, wherein the capture element is selected from a protein, a protein fragment, a binding protein (BP), a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, a single chain antibody, a single-domain antibody, a Fab antibody fragment, an Fc antibody fragment, an Fv antibody fragment, a F(ab')2 antibody fragment, a Fab' antibody fragment, a single-chain Fv (scFv) antibody fragment, an antibody binding domain, an antigen, an antigenic determinant, an epitope, a hapten, an immunogen, an immunogen fragment, or any combination thereof.

8. The substrate of claim 7, wherein the capture element is a Mycobacterium avium subspecies paratuberculosis (MAP) antigen.

9. The substrate of claim 6, wherein the capture element is a bovine pregnancy associated glycoprotein (PAG) or epitope thereof antibody, fragment or binding domain thereof.

35

10. The substrate of claim 8, wherein the PAG is selected from PAG-4, PAG-6, PAG-9, PAG-20, PAG-21 or any combination thereof.

11. The substrate of claim 1, wherein the substrate is a solid or porous substrate.

12. The substrate of claim 11, wherein the solid substrate is a paramagnetic bead, microtiter plate, microparticle, or a magnetic bead.

13. A kit for detecting a plurality of target analytes in a sample, comprising a) a substrate of claim 1 and optionally one or both of b) a background reducing reagent, and c) a colorimetric detection system.

14. The kit of claim 13, further comprising one or more items selected from the group consisting of: a) a wash solution, b) one or more antibodies for detection of antigens, ligands or antibodies bound to the capture elements or for detection of the positive controls, c) software for analyzing captured target analytes, d) a protocol for measuring the presence of target analytes in samples, e) a sample diluent, f) blotting TMB (3,3',5,5'-tetramethylbeiizidine; MW = 240.4) and g) a secondary antibody.

15. The kit of claim 14, wherein the antibodies for detection comprise antibody-binding protein (BP) conjugates, antibody-enzyme label conjugates, or any combination thereof.

16. The kit of claim 13, wherein the sample is a milk or a blood sample.

17. The kit of claim 16, wherein the blood sample is serum or plasma.

18. The kit of claim 13, wherein the substrate is a solid or porous substrate.

19. The kit of claim 18, wherein the solid substrate is a paramagnetic bead, microtiter plate, or microparticle.

20. The kit of claim 13, wherein the background reducing reagent is Mycobacterium phlei protein extract.

21. The kit of claim 13, wherein the colorimetric detection system comprises HRP-labelled anti-PAGs Ab and HRP-labelled anti-bovine IgG Ab.

22. The kit of claim 14, wherein the secondary antibody comprises at least one bovine anti- PAG antibody.

23. A method for processing a microarray comprising: a) providing a substrate of claim 1 ; b) adding at least one sample to the substrate; and

36 c) processing the substrate such that a detectable result is given by two or more of i) at least one fiduciary marker, ii) at least one positive colorimetric control, and iii) at least one positive control to monitor assay performance.

24 A method for detecting an analyte in a sample comprising providing a substrate of claim 1, adding at least one sample to the substrate, and processing the substrate such that a detectable result is provided.

25. The method of claim 24, wherein the detectable result includes two or more of at least one fiduciary marker, at least one positive colorimetric control, and at least one positive control to detect an analyte in the sample.

26. An isolated peptide comprising the amino acid sequence DTVRIGDLVSTDQ (SEQ ID NO:1).

27. An isolated peptide comprising the amino acid sequence GSVVMFGGVDHRYYKGELNW (SEQ ID NO:2).

28. An isolated peptide comprising the amino acid sequence ITIGTPPQEFQV (SEQ ID NO:3).

29. An antibody that binds a peptide comprising the amino acid sequence DTVRIGDLVSTDQ (SEQ ID NO: 1).

30. The antibody of claim 29, wherein the antibody is monoclonal.

31. An antibody that binds a peptide comprising the amino acid sequence GSVVMFGGVDHRYYKGELNW (SEQ ID NO:2).

32. The antibody of claim 30, wherein the antibody is monoclonal.

33. An antibody that binds a peptide comprising the amino acid sequence ITIGTPPQEFQV (SEQ ID NO: 3).

34. The antibody of claim 33, wherein the antibody is monoclonal.