Combinatorial MAP Antibody Assay for Detection and Diagnosis

Abstract

The present invention generally relates to kits for detecting Mycobacterium avium subsp. paratuberculosis (MAP) infection and biomarkers associated with a MAP-associated disease or disorder, as well as methods for diagnosing a subject with a MAP-associated autoimmune disease or disorder and / or determining a course of treatment for a subject diagnosed with a MAP-associated autoimmune disease or disorder.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 942,480, filed on December 2, 2019, the entire disclosure of which is incorporated herein by reference.

Background Art

[0002] Background of the Invention Mycobacterium avium subspecies paratuberculosis (MAP) is recognized as the cause of Johne's disease (JD), a chronic diarrheal wasting disease in cattle and a wasting disease in sheep and goats (Rathnaiah, G., et al., Front VetSci, 2017, 4: 187), and has long been suspected of being involved in the etiology of Crohn's disease (CD), an inflammatory bowel disease (IBD) in humans (Chacon, O., et al., Annu Rev Microbiol, 2004, 58: 329-363). MAP and / or CD are also associated with multiple sclerosis (Bo, M., et al, Microorganisms, 2020, 8 ; Cossu, D., et al., Future Microbiol, 2019, 14: 643-646; Slavin, Y.N., et al., J Neuroimmunol, 2018, 323: 49-52; Frau, J., et al., BMC Neurol, 2016, 16: 148; Cossu, D., et al., Sci Rep, 2016, 6: 29227; Mameli, G., et al., Sci Rep, 2016, 6: 22401; Mameli, G., et al., Eur J Neurol, 2016, 23: 140-147; Frau, J., et al., J Neurol Sci, 2015, 349: 249-250; Cossu, D., et al., Mult Scler, 2015, 21: 984-995), type I diabetes (Dow, C.T., et al., Microorganisms, 2019, 7; Bo, M., et al., Microorganisms, 2019, 7; Niegowska, M., et al., PLoS One, 2016, 11: e0157962; Hesam Shariah, S., et al, J Infect Dev Ctries, 2016, 10: 857-862; Niegowska, M., et al., Sci Rep, 2016, 6: 22266; Masala, S., et al., Pediatr Diabetes, 2015, 16: 189-195), rheumatoid arthritis (Bo, M., et al., J Inflamm Res, 2019, 12: 301-308; Naser, A., et al., Microorganisms, 2019, 7; Bo, M., et al., Clin Exp Rheumatol, 2018, 36: 376-381), Sjögren's syndrome (Zhang, P., et al., Microorganisms, 2019, 8), amyotrophic lateral sclerosis (Pierce, E.S., Med Flypotheses, 2018, 119: 1-5), celiac disease (Biet, F., et al., Dig Dis Sci, 2011, 56: 1794-1800), depression (Euesden, J., et al.. PLoS One, 2017, 12: e0173015), thyroiditis (Niegowska, M., et al., PLoS One, 2015, 10: e0133497), and neurodegenerative diseases including Alzheimer's disease (Lin, T.M., et al., PLoS One, 2018, 13: e0186475) and Parkinson's disease (Ami, G., et al., J Neuroimmunol, 2016, 293: 86-90). Diarrhea / wasting diseases associated with MAP infection have also been reported in non-human primates (McClure, H.M., et al., J Infect Dis, 1987, 155: 1011-1019).

[0003] Live MAP is present in our food and drinking water and can be isolated from commercially available pasteurized milk (Ellingson, J.L., et al, J Food Prot, 2005, 68: 966-972; Grant, I.R., et al., Appl Environ Microbiol, 2002, 68: 2428-2435), and MAP has been identified in 2.7% of retail pasteurized milk samples purchased in the states of Wisconsin, Minnesota, and California in the United States (Ellingson, J.L., et al., J Food Prot, 2005, 68: 966-972). Thus, humans are very likely to always be exposed to this animal pathogen. Screening of human sera by MAP-specific antibody assays has found evidence of MAP-specific immunorecognition in subjects with various underlying diseases, including CD patients and asymptomatic controls (Singh, S.H., et al., Journal of Biological Sciences, 2014, 14: 237-247). Previous meta-analyses that focused mainly on molecular detection methods also showed a tendency for studies to indicate that the proportion of MAP detection in samples from CD patients was significantly higher compared to non-IBD controls (Abubakar, I., et al., Inflamm Bowel Dis, 2008, 14: 401-410; Feller, M., et al., Lancet Infect Dis, 2007, 7: 607-613).However, problems and controversies in the proof of the association between MAP and any of these states (pages 498 of Lichtenstein GR, Loftus EV Jr, Isaacs, KL, Regueiro, MD, Gerson LB and Sands BE, ACG Clinical Guideline: Management of Crohn’s Disease in Adults. http: / / www.nature.com / ajgAm J Gastroenterol 2018; 113:481-517; doi:10.1038 / ajg.2018.27; published online in March 2018) have been hindered by a definitive and reproducible method for detecting and growing MAP from humans. Individual small-scale studies have cultured MAP from human blood and included significantly greater success in CD patients than in controls (Naser, S.A., et al., Lancet, 2004, 364: 1039-1044; Naser, S.A., et al., The Open Inflammation Journal, 2009, 2: 22-23). However, the method used (MGIT ParaTB culture tubes) was not optimal, required 3 to 6 months of incubation, and had relatively low reproducibility. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0004] Accordingly, there is a need in the art for improved systems and methods for detecting MAP and diagnosing MAP-related diseases or disorders. The present invention meets this unmet need. MEANS FOR SOLVING THE PROBLEMS

[0005] SUMMARY OF THE INVENTION In one embodiment, the present invention includes a kit for diagnosing one or more autoimmune diseases or disorders, the kit comprising: a) a first assay for detecting biomarkers that are positively correlated with the autoimmune disease or disorder; and b) a second assay for detecting biomarkers that are negatively correlated with the autoimmune disease or disorder.

[0006] In one embodiment, the autoimmune disease or disorder diagnosed by the kit is one or more selected from the group consisting of Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome (IBS), multiple sclerosis (MS), type 1 diabetes mellitus (T1DM), Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), depression, Parkinson's disease (PD), Alzheimer's disease (AD), celiac disease, thyroiditis, rheumatoid arthritis, psoriasis, Blau syndrome, lymphangioma, and complex regional pain syndrome (CRPS).

[0007] In one embodiment, the biomarker of the first assay in the kit is one or more selected from the group consisting of an antibody specific to heat shock protein 65 (HSP65), Hpp65, and a nucleic acid encoding Hpp65. In one embodiment, the first assay includes a diagnostic device comprising a solution containing: a) a first surface area; b) Hsp65 or an antigenic fragment thereof that is bound to the first surface area; and c) a labeled antibody against the antibody specific to Hsp65.

[0008] In one embodiment, the biomarker of the second assay in the kit is one or more selected from the group consisting of an antibody specific to protein kinase G (PknG), PknG, and a nucleic acid encoding PknG. In one embodiment, the second assay includes a diagnostic device comprising a solution containing: a) a first surface area; b) PknG or an antigenic fragment thereof that is bound to the first surface area; and c) a labeled antibody against the antibody specific to PknG.

[0009] In one embodiment, the kit further comprises a third assay for detecting biomarkers associated with MAP infection. In one embodiment, the biomarker of the third assay is one or more selected from the group consisting of an antibody specific for Mycobacterium avium subsp. paratuberculosis (MAP) lipopentapeptide (L5P), L5P, and a nucleic acid encoding L5P. In one embodiment, the third assay comprises a diagnostic device comprising a solution containing: a) a first surface area; b) L5P or an antigenic fragment thereof, wherein the L5P or the antigenic fragment thereof is bound to the first surface area; and c) a labeled antibody against the antibody specific for L5P.

[0010] In one embodiment, the present invention includes a method for diagnosing one or more autoimmune diseases or disorders in a subject, the method comprising: a) detecting MAP infection in the subject; b) detecting a first biomarker that is positively correlated with the autoimmune disease or disorder; c) detecting a second biomarker that is negatively correlated with the autoimmune disease or disorder; and d) diagnosing that the subject has the autoimmune disease or disorder when the MAP infection, the first biomarker, and the second biomarker are detected.

[0011] In one embodiment, the autoimmune disease or disorder diagnosed by the method is one or more selected from the group consisting of CD, UC, IBS, MS, T1DM, SS, SLE, depression, PD, AD, celiac disease, thyroiditis, rheumatoid arthritis, psoriasis, Blau syndrome, lymphangioma, and CRPS.

[0012] In one embodiment, the detection of MAP infection in this method further comprises measuring one or more selected from the group consisting of an antibody specific for L5P, L5P, and a nucleic acid encoding L5P.

[0013] In one embodiment, the first biomarker of the method is one or more selected from the group consisting of an antibody specific to Hsp65, Hpp65, and a nucleic acid encoding Hpp65. In one embodiment, the second biomarker of the method is one or more selected from the group consisting of an antibody specific to protein kinase G (PknG), PknG, and a nucleic acid encoding PknG.

[0014] In one embodiment, the method further comprises administering a therapeutic agent for treating the one or more autoimmune diseases or disorders to the subject diagnosed with the one or more autoimmune diseases or disorders.

[0015] In one embodiment, the method includes a method of selecting a subject diagnosed with one or more autoimmune diseases or disorders to be treated with one or more antibiotics, the method comprising: a) detecting MAP infection in the subject; b) detecting a first biomarker that is positively correlated with the autoimmune disease or disorder; c) detecting a second biomarker that is negatively correlated with the autoimmune disease or disorder; d) diagnosing the subject as having the autoimmune disease or disorder when the MAP infection, the first biomarker, and the second biomarker are detected; e) detecting the presence of MAP in an additional clinical assay; and f) treating the subject with one or more antibiotics specific for MAP infection when the subject is diagnosed with the autoimmune disease or disorder and MAP is detected in the clinical assay.

[0016] In one embodiment, the autoimmune disease or disorder of the method is one or more selected from the group consisting of CD, UC, IBS, MS, T1DM, SS, SLE, depression, PD, AD, celiac disease, thyroiditis, rheumatoid arthritis, psoriasis, Blau syndrome, lymphangioma, and CRPS.

[0017] In one embodiment, the detection of MAP infection in the method further comprises measuring one or more selected from the group consisting of an antibody specific to L5P, L5P, and a nucleic acid encoding L5P.

[0018] In one embodiment, the first biomarker of the method is one or more selected from the group consisting of an antibody specific to Hsp65, Hpp65, and a nucleic acid encoding Hpp65. In one embodiment, the second biomarker of the method is one or more selected from the group consisting of an antibody specific to protein kinase G (PknG), PknG, and a nucleic acid encoding PknG.

[0019] In one embodiment, the clinical assay of the method is one or more selected from the group consisting of a MAP phage amplification assay, a Pozzato culture assay, a TiKa culture assay, and a mycobacteria growth indicator tube (MGIT) culture assay.

Brief Description of the Drawings

[0020] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the present invention is not limited to the exact construction and means of the embodiments shown in the drawings.

[0021]

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DETAILED DESCRIPTION OF THE INVENTION

[0022] (Detailed Description) The present invention is based in part on the knowledge that MAP infection does not always manifest as a disease or disorder, and thus there is a need in the art for an improved method of diagnosing diseases or disorders associated with MAP infection, including autoimmune diseases or disorders. The present invention is also based in part on the novel discovery that certain antibodies are positively or negatively correlated with autoimmune diseases including Crohn's disease and ulcerative colitis, and that a model combining biomarkers positively and negatively correlated with the disease has an improved predictive power compared to the diagnosis of any single biomarker alone. Further, the present invention is based on the discovery that additional clinical assays for detecting MAP infection, used in combination with kits and methods for measuring the antibodies of the present invention, further improve the predictability and reliability of diagnosing an autoimmune disease or disorder.

[0023] Definitions Unless otherwise indicated, 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.

[0024] The following terms used herein have the meanings associated with them in this section.

[0025] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element.

[0026] As used herein when referring to measurable values such as amounts, lengths of time, etc., "about" means encompassing a variation of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, such variations being appropriate for carrying out the disclosed methods.

[0027] A disease or disorder is "attenuated" if the severity of the signs or symptoms of the disease or disorder, the frequency with which such signs or symptoms are experienced by a patient, or both, are decreased.

[0028] As used herein, "accuracy" refers to the total ratio of true results (true positives + true negatives) as a measure of the overall reliability of a detection or diagnostic kit and / or method.

[0029] As used herein, "assay" refers to any composition, device, system, and / or method for qualitatively and / or quantitatively measuring a moiety or substance in a sample. For example, an immunoassay can be used to detect the presence or measure the amount of a biomarker (e.g., an antibody) in a biological sample (e.g., plasma).

[0030] As used herein, "antigen" refers to a molecule that elicits an immune response. This immune response can include antibody production, activation of specific immunocompetent cells, or both. One of ordinary skill in the art will understand that virtually any macromolecule, including all proteins or peptides, can function as an antigen. It should be readily apparent to those skilled in the art that an antigen can be generated, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0031] As used herein, "biological sample" refers to any biologically obtained tissue or fluid on which the biomarkers of the invention can be assayed. Examples of such samples include, but are not limited to, blood, sputum, lymph fluid, bronchoalveolar lavage fluid, urine, gynecological fluid, biopsy, amniotic fluid, smear specimens.

[0032] A sample that is essentially liquid can be referred to herein as a "body fluid". The sample can be obtained from a patient by various techniques, for example, by rubbing or swabbing an area, or by aspirating body fluid using a needle. Methods for collecting various body samples are well known in the art. In many cases, the sample is a "clinical sample", i.e., a sample obtained from a patient. Such samples include, but are not particularly limited to, body fluids that may or may not contain cells, such as blood (e.g., whole blood, serum, or plasma), urine, sputum, saliva, tissue, or fine needle biopsy samples, and stored samples with known diagnostic, treatment, and / or outcome histories. Biological samples can also include tissue sections such as frozen sections taken for histological examination purposes. The sample includes all materials obtained by processing a biological sample. The materials obtained include, but are not particularly limited to, cells isolated from the sample (or their progeny), proteins or nucleic acid molecules extracted from the sample. Processing of a biological sample can include one or more of filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc.

[0033] As used herein, "biomarker" refers to a moiety or substance that is a characteristic indicator of a biological process, biological event, and / or pathological condition. For example, an antibody can be a biomarker for a disease (such as an autoimmune disease or disorder like Crohn's disease).

[0034] As used herein, the term "diagnosis" refers to the determination of the presence of a disease or disorder. In some embodiments of the present invention, methods are provided for performing a diagnosis that enables the determination of the presence of a specific disease or disorder.

[0035] "Disease" refers to the health state of an animal where the animal cannot maintain homeostasis and, if the disease is not improved, the animal's health continues to deteriorate. In contrast, an "injury" to an animal is a health state where the animal can maintain homeostasis, but the animal's health state is less favorable than it would be without the injury. Left untreated, an injury does not necessarily further degrade the animal's health state.

[0036] Terms such as "patient", "subject", and "individual" are used interchangeably herein and refer to any animal or its cells suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is human.

[0037] "Instructions" as the term is used herein includes publications, records, diagrams, or any other medium of expression that can be used to convey the utility of the nucleic acids, peptides, and / or compounds of the present invention, which are included in kits for identifying, diagnosing, or alleviating, or treating various diseases or disorders described herein. Optionally, or alternatively, the instructions may describe one or more methods for identifying, diagnosing, or alleviating a disease or disorder in a subject's cells or tissues. The instructions for the kit can be, for example, affixed to a container containing one or more components of the present invention or shipped together with a container containing one or more components of the present invention. Alternatively, the instructions can be shipped separately from the container, with the intention that the recipient use the instructions in cooperation with the components.

[0038] As used herein, "immunoassay" refers to any binding assay that includes an antibody capable of specifically binding to a target molecule. The assay can be designed, for example, as a sandwich enzyme-linked immunosorbent assay (ELISA) to detect and / or quantify the target molecule, or as an indirect ELISA to detect and / or quantify an antibody specific for the target molecule.

[0039] "Isolated" means changed or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting substances in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or, for example, in a non-natural environment such as a host cell.

[0040] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to a probe to generate a "labeled" probe. A label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or in the case of an enzyme label, may catalyze a chemical change in a detectable substrate compound or composition (e.g., avidin-biotin). In some cases, primers can be labeled to detect PCR products.

[0041] As used herein, the terms "measure" or "measuring", or "detect" or "detecting", mean to evaluate the presence, absence, quantity or amount (which may be an effective amount) of a predetermined moiety or substance in a sample, and include obtaining a qualitative or quantitative concentration level of such moiety or substance, or otherwise evaluating the value or classification of a clinical parameter of a subject.

[0042] As used herein, the terms "normal", "healthy", and "control" are used interchangeably. They include individuals or groups of individuals not diagnosed as having the disease or disorder of interest (e.g., an autoimmune disease or disorder). These terms are also used herein to describe samples (e.g., biological samples such as plasma) obtained from normal, healthy, or control individuals.

[0043] "Nucleic acid" refers to polynucleotide and includes polyribonucleotide and polydeoxyribonucleotide. The nucleic acid according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (see Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982). This is hereby incorporated by reference in its entirety for all purposes). In fact, the present invention contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases. The polymer or oligomer may have a heterogeneous or homogeneous composition and may be isolated from natural sources or generated artificially or synthetically. Further, the nucleic acid may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded forms, including homoduplex, heteroduplex, and hybrid states.

[0044] As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, incorporated herein by reference) that describes a method of increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without performing cloning or purification. This process for amplifying a target sequence consists of introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by performing a precise sequence of thermal cycling in the presence of DNA polymerase. The two primers are complementary to each of the strands of the double-stranded target sequence. To perform the amplification, the mixture is denatured and then the primers are annealed to these complementary sequences within the target molecule. Following annealing, the primers are extended by polymerase to form new pairs of complementary strands. The steps of denaturation, primer annealing, and polymerase extension are repeated many times (i.e., denaturation, annealing, and extension constitute one "cycle" and there can be a number of "cycles") to obtain a highly concentrated amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers to each other and thus this length is a controllable parameter. Because of the iterative nature of the process, this method is called the "polymerase chain reaction" (hereinafter "PCR"). Since the desired amplified segments of the target sequence become the major sequences (with respect to concentration) in the mixture, they are said to be "PCR amplified". As used herein, the terms "PCR product", "PCR fragment", "amplification product", or "amplicon" refer to a mixture of compounds obtained after two or more cycles of the PCR steps of denaturation, annealing, and extension. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.

[0045] As used herein, the terms "peptide", "polypeptide", and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds.

[0046] As used herein, this term refers to both short chains, which are also generally referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, which are generally referred to in the art as proteins of many types. "Polypeptide" includes, in particular, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or any combination thereof.

[0047] As used herein, "polynucleotide" includes cDNA, RNA, DNA / RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic forms, and mixed polymers of both sense and antisense strands, and can be chemically or biochemically modified to include non-natural, or derivatized, synthetic, or semi-synthetic nucleotide bases. Also contemplated are modifications of wild-type or synthetic genes, including, but not limited to, deletions, insertions, substitutions, or fusions of one or more nucleotides to other polynucleotide sequences.

[0048] The term "primer" refers to an oligonucleotide that can act as a starting point for synthesis along a complementary strand when the conditions are suitable for the synthesis of a primer extension product. The synthesis conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization inducer, such as reverse transcriptase or DNA polymerase. These are present in a suitable buffer, which may contain cofactors or components that affect conditions such as pH at various appropriate temperatures. The primer is preferably a single-stranded sequence so that the amplification efficiency is optimized, but a double-stranded sequence can be utilized.

[0049] As used herein with respect to an antibody, the terms "specifically binds" or "specific for" refer to an antibody that recognizes a particular antigen in a sample but does not substantially recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species can also bind to that antigen from one or more species. However, such cross-reactivity does not itself change the specific classification of the antibody. In another example, an antibody that specifically binds to an antigen can also bind to different genotypes of that antigen. However, such cross-reactivity does not itself change the specific classification of the antibody. In some cases, the terms "specific binding" or "specifically binds" can be used in the context of the interaction between an antibody, protein, or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the chemical species. For example, an antibody generally recognizes and binds to a specific protein structure rather than a protein in general. If an antibody is specific for epitope "A", in a reaction containing labeled "A" and the antibody, the amount of labeled A that binds to the antibody decreases when a molecule containing epitope A (or free unlabeled A) is present.

[0050] A "therapeutic" treatment is a treatment administered to a subject showing signs or symptoms of a disease or disorder for the purpose of reducing or eliminating those signs or symptoms.

[0051] As used herein, "treating a disease or disorder" means reducing the severity and / or frequency of the symptoms or manifestations of the disease or disorder experienced by a patient.

[0052] Scope: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, a range description should be considered to specifically disclose all the possible sub-ranges as well as the individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to have the disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6, etc. This applies regardless of the width of the range.

[0053] As used herein, "sensitivity" refers to the ability (true positive rate) to correctly identify the presence of a particular marker or pathological condition when it is present. As used herein, "specificity" refers to the ability (true negative rate) to correctly exclude the presence of a particular marker or pathological condition when it is absent. As used herein, "analytical sensitivity" or "analytical specificity" is the ability to detect the presence or absence of an infectious agent in a subject, respectively, while "clinical sensitivity" or "clinical specificity" as used herein is the ability to diagnose a subject's disease or disorder as positive or negative, respectively.

[0054] Explanation The present invention generally relates to kits, systems, and methods for detecting MAP infection in a patient and / or diagnosing an autoimmune disease or disorder. The present invention also relates to a method for determining when a patient having a detectable MAP infection should be treated with anti-MAP therapy.

[0055] Biomarker In various embodiments, the invention generally relates to kits and / or methods for detecting or measuring one or more biomarkers in a biological sample.

[0056] In various embodiments, the biomarker of the invention is measured in a biological sample. The biological sample can be a sample from any source containing polypeptides or nucleic acids, such as a fluid, tissue, cell, cell component, or a combination thereof. The biological sample can be obtained, for example, by appropriate methods such as blood collection, body fluid extraction, or biopsy. The biological sample can be used as a test sample. Alternatively, the biological sample can be processed to enhance access to polypeptides or nucleic acids, or copies of nucleic acids, and then the processed biological sample can be used as a test sample.

[0057] In one embodiment, one or more of the biomarkers of the invention are one or more antibodies. In one embodiment, one or more of the antibodies are specific for MAP. In one embodiment, one or more of the antibodies are not specific for MAP. In one embodiment, one or more of the antibodies include an antibody specific for heat shock protein 65 (anti-HSP65). In one embodiment, one or more of the antibodies include an antibody specific for protein kinase G (anti-PknG). In one embodiment, one or more of the antibodies include an antibody specific for the MAP lipopentapeptide (anti-L5P).

[0058] The methods described herein enable the specific detection of low concentrations of antibodies in fluid samples. Detection can be carried out at antibody concentrations of about 50, 40, 30, 20, 10, 5, or even down to 0.5 ng / ml, for example, at concentrations of about 1000, 750, 500, 400, 300, 200, 100, 75, 50, 40, 30, 20, 10, 5, or even less than 0.5 ng / ml.

[0059] The methods described herein related to the detection of antibodies are particularly relevant to diagnostic and / or prognostic assays related to the treatment and prevention of diseases or disorders such as autoimmune diseases or disorders. Many diagnostic, prognostic, and / or monitoring assays rely on the detection of biological markers of specific medical conditions or disease susceptibilities. Such biological markers are generally proteins or polypeptides that are characteristic of a particular disease or are related to susceptibility to a disease.

[0060] The detection of specific antibodies can be used diagnostically or prognostically to evaluate other factors such as the progression of a medical condition or disease. Antibodies typically function as biological markers of infection, disease, or disease susceptibility. For example, antibodies specific to the MAP antigen indicate MAP infection. MAP infection has been implicated in many autoimmune diseases such as Crohn's disease and ulcerative colitis. However, it should be noted that MAP infection alone does not necessarily predict the risk of developing a symptomatic disease. Thus, as contemplated herein, additional biomarkers that specifically correlate with autoimmune diseases or disorders that include antibodies are needed for rapid and accurate diagnosis.

[0061] In one embodiment, one or more of the biomarkers of the present invention are one or more proteins. In one embodiment, one or more of the proteins are antigenic to the host and induce an immune response. In one embodiment, the immune response includes antibodies specific to the one or more proteins.

[0062] In one embodiment, one or more of the biomarkers of the present invention are one or more nucleic acids. In one embodiment, the nucleic acid encodes a protein that is antigenic to the host and induces an immune response. In one embodiment, the immune response includes antibodies specific to the one or more proteins.

[0063] Assay In various embodiments, the present invention generally relates to kits and / or methods comprising one or more assays for detecting or measuring one or more biomarkers in a biological sample. In some embodiments, the present invention relates to kits and / or methods comprising one or more assays for determining whether the one or more biomarkers are differentially expressed in the biological sample.

[0064] Biomarkers can generally be measured and detected via a variety of assays, methods, and detection systems known to those of skill in the art. The various methods include, but are not particularly limited to, immunoassays, microarrays, PCR, RT-PCR, refractive index spectroscopy (RI), ultraviolet spectroscopy (UV), fluorescence analysis, electrochemical analysis, radiochemical analysis, near-infrared spectroscopy (near-IR), infrared (IR) spectroscopy, nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), mass spectrometry, thermal decomposition mass spectrometry, nephelometry, dispersive Raman spectroscopy, gas chromatography, liquid chromatography, combinations of gas chromatography and mass spectrometry, combinations of liquid chromatography and mass spectrometry, combinations of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and mass spectrometry, combinations of ion spray spectroscopy and mass spectrometry, capillary electrophoresis, colorimetric analysis, and surface plasmon resonance (according to the system provided by Biacore Life Sciences). See also PCT Publications WO / 2004 / 056456 and WO / 2004 / 088309. In this regard, biomarkers can be measured using the detection methods described above, or other methods known to those of skill in the art. Other biomarkers can be detected similarly using reagents specially designed or adapted for their detection.

[0065] In various embodiments, to determine whether the expression level of one or more biomarkers in a biological sample of a subject is increased or decreased, the expression level of at least one biomarker is compared to at least one comparative control, such as a positive control, a negative control, a past average, a past standard, or the level of another reference molecule within the biological sample. The results of the diagnostic assay can be used alone, or in combination with other information from the subject, or other information from the biological sample obtained from the subject.

[0066] In various embodiments of the assays of the invention, when compared to a comparative control, the expression level of a biomarker is determined to be elevated or increased when it has increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, at least 1000%, at least 1500%, at least 2000%, at least 2500%, at least 3000%, at least 4000%, or at least 5000%.

[0067] In various embodiments of the method of the present invention, when compared to a comparator, the expression level of a biomarker in a biological sample is at least 1-fold, at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 6.5-fold, at least 7-fold, at least 7.5-fold, at least 8-fold, at least 8.5-fold, at least 9-fold, at least 9.5-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1000-fold increased, the expression level of the biomarker is determined to be elevated or increased.

[0068] Different types of biomarkers and their measurements can be combined in the compositions and methods of the present invention. In various embodiments, one or more antibodies are biomarkers. In other embodiments, one or more proteins are biomarkers. In one embodiment, one or more of the proteins are antigenic and induce the production of antibodies. In various embodiments, one or more nucleic acids are biomarkers. In one embodiment, the nucleic acid encodes one or more antigenic proteins that induce antibodies.

[0069] In one embodiment, the biomarker of the present invention is an antibody. In one embodiment, the antibody is detected by measuring the formation of an antibody-antigen complex. To facilitate the detection of the antibody-antigen complex, a suitable detectable label can be utilized. There are many different labels and labeling methods known to those skilled in the art. Examples of the types of labels that can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds. Those skilled in the art will recognize a suitable label or will be able to identify such by using routine experimentation. The labeled moiety is observable by conventional immunohistochemical detection techniques that are, for example, fluorescent dyes such as fluorescein, chemiluminescent reagents, radioisotopes, colloidal labels such as colloidal gold or colored latex beads, enzyme labels, or other known labeled complexes.

[0070] As an example, when a label on an antibody against an antibody is involved in a chemical reaction, the product of the reaction can be quantified to quantify the binding. For example, the label may cause a color change that can be quantified with a spectrometer. The criterion in such a case can be the absorption of light at a specific wavelength. The reference substance may also be a color chip or a color scale. Further, when the label is luminescent, the binding can be quantified by measuring the intensity of light at a specific wavelength with a spectrometer. In such a case, the reference value can be an intensity value. Further, the binding can be quantified by observing the interaction between a label on an antibody specific to an antibody and a label bound to an antigen bound to various surface areas. For example, when a label on an antibody specific to an antibody contains a fluorescent substance and an antigen bound to the surface has the fluorescent substance bound thereto, the energy transfer between the fluorescent substances after exposure to light can be quantified. By exposing the antibody-antibody-antigen complex to light of a wavelength and intensity sufficient to excite one of the fluorescent substances, energy can be transferred between the fluorescent substances. If another fluorescent substance is close enough, the energy is transferred from the excited fluorescent substance to the other fluorescent substance, causing the other fluorescent substance to emit light at a wavelength different from the wavelength used to stimulate the first fluorescent substance. Next, the intensity of the light at the emitted wavelength can be quantified using various devices such as a spectrometer, and thus the binding between the antibody in the sample and the antigen in the solution is quantified.

[0071] As is known in the art, detectable labels can be used to tag any member of an antibody-antigen complex either directly (e.g., direct binding) or indirectly (e.g., secondary antibody) to facilitate detection. A variety of methods for detecting the binding of an antigen to an antibody via a suitable detectable label are known in the art. Detection can be by any method known in the art, such as immunological techniques including immunoassays. For example, detection of an antibody-antigen complex can be determined by techniques such as, but not limited to, Western blot analysis, flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive immunoassay, sandwich immunoassay, chemiluminescent assay, bioluminescent assay, fluorescent assay, and agglutination assay.

[0072] In one embodiment, the assay of the present invention includes a diagnostic device. In one embodiment, the diagnostic device includes: a first surface area; the protein or an antigenic fragment of the protein, wherein the protein or the antigenic fragment of the protein is bound to the first surface area; and a solution containing a labeled antibody against an antibody specific for the protein. In one embodiment, the immunoassay of the present invention includes an indirect ELISA. In one embodiment, the antibodies detected by the immunoassay include, but are not particularly limited to, Hsp65, PknG, and L5P.

[0073] In one embodiment of the present invention, the one or more biomarkers are proteins or polypeptides. Methods for measuring protein / polypeptide levels in a biological sample obtained from a subject are not particularly limited, but include immuno-chromatography assays, immunodot assays, Luminex assays, ELISA assays, ELISPOT assays, protein microarray assays, ligand-receptor binding assays, ligand displacement from receptor assays, ligand displacement from co-receptor assays, immunostaining assays, Western blot assays, mass spectrometry assays, radioimmunoassay (RIA), radioimmunodiffusion assays, liquid chromatography-tandem mass spectrometry assays, Ouchterlony immunodiffusion assays, reverse phase protein microarrays, rocket immunoelectrophoresis assays, immunohistochemical staining assays, immunoprecipitation assays, complement fixation assays, FACS, enzyme-substrate binding assays, enzyme assays, enzyme assays using detectable molecules such as chromogenic substances, fluorescent substances, or radioactive substrates, substrate binding assays substrates using such substrates, substrate displacement assays using such substrates, and protein chip assays (see also 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996, Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007).

[0074] In one embodiment of the present invention, one or more biomarkers are nucleic acids. Methods for detecting nucleic acids (e.g., mRNA), such as RT-PCR, real-time PCR, microarray, branched DNA, NASBA, etc., are well known in the art. Using the sequence information provided by the database entry of the biomarker sequence, the expression of the biomarker sequence can be detected (if present) and measured using techniques known to those skilled in the art. For example, using the sequence in the sequence database entry or the sequences disclosed herein, a probe for detecting the biomarker RNA sequence can be constructed, for example, in a Northern blot hybridization analysis or a method for specifically and preferably quantitatively amplifying a specific nucleic acid sequence. As another example, using the sequence, a primer for specifically amplifying the biomarker sequence can be constructed, for example, in a detection method for an amplification subject such as reverse transcription-mediated polymerase chain reaction (RT-PCR). When changes in gene expression are associated with gene amplification, deletion, polymorphism, and mutation, sequence comparison in the test and reference populations can be performed by comparing the relative amounts of the tested DNA sequences in the test population and the reference cell population. In addition to Northern blot and RT-PCR, RNA can also be measured using, for example, other target amplification methods (e.g., TMA, SDA, NASBA), signal amplification methods (e.g., bDNA), nuclease protection assay, in situ hybridization, etc.

[0075] In some embodiments, quantitative hybridization methods such as Southern analysis, Northern analysis, or in situ hybridization can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements). As used herein, a "nucleic acid probe" can be a DNA probe or an RNA probe. The probe can be, for example, a gene, a gene fragment (e.g., one or more exons), a vector containing the gene, a probe or primer, etc. For representative examples of the use of nucleic acid probes, see, for example, U.S. Pat. Nos. 5,288,611 and 4,851,330. The nucleic acid probe can be, for example, a full-length nucleic acid molecule or a portion thereof, e.g., an oligonucleotide that is at least 15, 30, 50, 100, 250, or 500 nucleotides in length and is sufficient to specifically hybridize to an appropriate target mRNA or cDNA under stringent conditions. The hybridization sample is maintained under conditions sufficient to allow specific hybridization of the nucleic acid probe to the mRNA or cDNA. Optionally, specific hybridization can be performed under high stringency conditions or medium stringency conditions. In a preferred embodiment, the hybridization conditions for specific hybridization are high stringency. Next, specific hybridization, if present, is detected using standard methods. If specific hybridization occurs between the nucleic acid probe having the mRNA or cDNA in the test sample, the level of mRNA or cDNA in the sample can be evaluated. In this method, multiple nucleic acid probes can also be used simultaneously. As described herein, specific hybridization of any one of the nucleic acid probes indicates the presence of the mRNA or cDNA of interest.

[0076] Alternatively, in the quantitative hybridization method described herein, peptide nucleic acid (PNA) probes can be used instead of nucleic acid probes. PNA is a DNA mimic having a peptide-like inorganic backbone such as N-(2-aminoethyl)glycine units, with organic bases (A, G, C, T, or U) linked to the glycine nitrogen via methylene carbonyl linkers (see, e.g., 1994, Nielsen et al., Bioconjugate Chemistry 5:1). PNA probes can be designed to specifically hybridize to target nucleic acid sequences. Hybridization of PNA probes to nucleic acid sequences is used to determine the level of target nucleic acid in a biological sample.

[0077] In another embodiment, an array of oligonucleotide probes that are complementary to target nucleic acid sequences in a biological sample obtained from a subject can be used to determine the levels of one or more biomarkers in the biological sample obtained from the subject. The array of oligonucleotide probes can be used to determine the level of one or more biomarkers alone or the level of one or more biomarkers in relation to the level of one or more other nucleic acids in the biological sample. An oligonucleotide array typically comprises a plurality of different oligonucleotide probes attached to the surface of a substrate at different known positions. These oligonucleotide arrays, also known as "gene chips," are generally described in the art and are described, for example, in U.S. Patent No. 5,143,854 and PCT Patent Publications WO90 / 15070 and 92 / 10092. These arrays can generally be manufactured using a mechanical synthesis method or a light-directed synthesis method incorporating a combination of photolithography and solid-phase oligonucleotide synthesis methods. See Fodor et al., Science, 251:767-777 (1991), Pirrung et al., U.S. Patent No. 5,143,854 (see also PCT Application No. WO90 / 15070) and Fodor et al., PCT Publication WO92 / 10092 and U.S. Patent No. 5,424,186. Techniques for synthesizing these arrays using mechanical synthesis methods are described, for example, in U.S. Patent No. 5,384,261.

[0078] After the oligonucleotide array is prepared, the nucleic acid of interest is hybridized to the array and its level is quantified. Hybridization and quantification are generally carried out by the methods described herein and also, for example, by the methods described in published PCT application numbers WO92 / 10092 and WO95 / 1995, and U.S. Patent No. 5,424,186. Briefly, the target nucleic acid sequence is amplified by known amplification techniques such as PCR. Usually, this involves the use of primer sequences complementary to the target nucleic acid. Asymmetric PCR techniques can also be used. Next, the amplified target, generally incorporating a label, is hybridized to the array under appropriate conditions. When hybridization and washing of the array are complete, the array is scanned to determine the amount of hybridized nucleic acid. The hybridization data obtained from the scan is usually in the form of fluorescence intensity as a function of the amount or relative amount of the target nucleic acid in the biological sample. The target nucleic acid can be hybridized to the array in combination with one or more comparators (e.g., positive control, negative control, quantity control, etc.) to improve the quantification of the target nucleic acid in the sample.

[0079] The probes and primers according to the present invention can be labeled directly or indirectly by methods known to those skilled in the art using radioactive or non-radioactive compounds to obtain a detectable and / or quantifiable signal. Labeling of the primers or probes according to the present invention is carried out using radioactive elements or non-radioactive molecules. Among the radioisotopes used, 32P , 33 P, 35 S, or 3 H can be mentioned. Non-radioactive substances are selected from ligands such as biotin, avidin, streptavidin, or digoxigenin, haptens, dyes, and luminescent substances such as radio-luminescent substances, chemiluminescent substances, bioluminescent substances, fluorescent substances, or phosphorescent substances.

[0080] Nucleic acids can be obtained from cells using known techniques. The nucleic acids herein refer to RNA including mRNA and DNA including cDNA. The nucleic acids can be double-stranded or single-stranded (i.e., sense or antisense single-stranded) and can be complementary to nucleic acids encoding polypeptides. The nucleic acid content can also be an RNA or DNA extraction performed on biological samples including biological fluids and fresh or fixed tissue samples.

[0081] There are many methods known in the art for the detection and quantification of specific nucleic acid sequences, and new methods are continuously being reported. Most of the known specific nucleic acid detection and quantification methods utilize nucleic acid probes in specific hybridization reactions. Preferably, the detection of hybridization into a double-stranded form is the Southern blot technique. In the Southern blot technique, a nucleic acid sample is separated on an agarose gel based on size (molecular weight), affixed to a membrane, denatured, and exposed (mixed) to a labeled nucleic acid probe under hybridization conditions. When the labeled nucleic acid probe hybridizes with the nucleic acid on the blot, the label binds to the membrane.

[0082] In Southern blotting, the nucleic acid probe is preferably labeled with a tag. The tag can be a radioisotope, a fluorescent dye, or other well-known materials. Another type of process for specifically detecting nucleic acids in biological samples known in the art is the hybridization method as exemplified in U.S. Patents Nos. 6,159,693 and 6,270,974, and related patents. Briefly summarizing one of these methods, a nucleic acid probe of at least 10 nucleotides, preferably at least 15 nucleotides, more preferably at least 25 nucleotides having a sequence complementary to the nucleic acid of interest is hybridized in a sample, subjected to depolymerization conditions, and the sample is treated with an ATP / luciferase system, which emits light if the nucleic acid sequence is present. In quantitative Southern blotting, the level of the nucleic acid of interest can be compared to the level of a second nucleic acid of interest and / or one or more comparative nucleic acids (e.g., positive control, negative control, quantity control, etc.).

[0083] Many methods useful for the detection and quantification of nucleic acids utilize the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). Briefly, nucleic acid primers complementary to the opposite strands of the nucleic acid amplification target sequence are annealed to the denatured sample. A DNA polymerase (usually thermostable) extends the DNA duplex from the hybridized primers. This process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, there is no corresponding amplified PCR product. In this case, the PCR primers function as hybridization probes.

[0084] In PCR, as discussed elsewhere herein, nucleic acid probes can be labeled with tags. Most preferably, the detection of double-stranded nucleic acids is performed using at least one primer directed to the nucleic acid of interest. In yet another embodiment of PCR, the detection of hybridized double-stranded nucleic acids involves electrophoretic gel separation followed by visualization of the dye moiety.

[0085] Typical hybridization and wash stringency conditions depend in part on the size (i.e., the length in number of nucleotides), base composition, and monovalent and divalent cation concentrations of the oligonucleotide probe (Ausbel et al., 1994, eds Current Protocols in Molecular Biology).

[0086] In one embodiment, the process for determining the quantitative and qualitative profile of a nucleic acid of interest according to the present invention is characterized in that the amplification is real-time amplification performed using a labeled probe, preferably a labeled hydrolysis probe, that can specifically hybridize to a segment of the nucleic acid of interest under stringent conditions. The labeled probe can emit a detectable signal each time an amplification cycle occurs, and the signal obtained in each cycle can be measured.

[0087] Real-time amplification such as real-time PCR is well known in the art, and various known techniques will be used in the best mode for carrying out this process. These techniques are carried out using various groups of probes such as hydrolysis probes, hybridization adjacent probes, or molecular beacons. Techniques using hydrolysis probes or molecular beacons are based on the use of a fluorescence quencher / reporter system, and hybridization adjacent probes are based on the use of fluorescence acceptor / donor molecules.

[0088] Hydrolysis probes with a fluorescence quencher / reporter system are commercially available, for example, commercialized by the Applied Biosystems group (USA). Many fluorescent dyes such as FAM dye (6-carboxy-fluorescein) or any other dye phosphoramidite reagent can be used.

[0089] Among the stringency conditions applicable to any one of the hydrolysis probes of the present invention, there is a Tm in the range of about 65°C to 75°C. Preferably, the Tm of any one of the hydrolysis probes of the present invention is in the range of about 67°C to about 70°C. Most preferably, the Tm applicable to any one of the hydrolysis probes of the present invention is about 67°C.

[0090] In one embodiment, the invention includes a primer that is complementary to a nucleic acid of interest. More specifically, the primer includes a primer that contains 12 or more consecutive nucleotides that are substantially complementary to the nucleic acid of interest. Preferably, the primer characterized by the present invention includes a nucleotide sequence that is sufficiently complementary to hybridize to a nucleic acid sequence of about 12 to 25 nucleotides. More preferably, the primer differs from the target flanking nucleotide sequence by only 1, 2, or 3 nucleotides. In another embodiment, the length of the primer may vary, and preferably the length may be about 15 to 28 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length).

[0091] In one embodiment, the one or more assays of the present invention are culture assays. In one embodiment, the culture assay is specifically modified to culture MAP. Those skilled in the art will recognize that any known (or not yet developed) method of cell culture capable of growing MAP from a biological sample can be used in conjunction with the method of the present invention. In one embodiment, the culture assay is one or more selected from the group consisting of the Pozzato culture assay, the TiKa culture assay, and the Mycobacteria Growth Indicator Tube (MGIT) culture assay.

[0092] In one embodiment, the one or more assays are phage amplification assays. In one embodiment, the phage amplification assay is optimized for the detection of MAP. Those skilled in the art will recognize that any known (or not yet developed) phage amplification assay capable of detecting MAP infection from a biological sample can be used in the method of the present invention.

[0093] Kit The present invention also relates to kits useful in the methods of the present invention. Such kits include, for example, materials for quantitatively analyzing the biomarkers of the present invention (e.g., antibodies, polypeptides, and / or nucleic acids), materials for evaluating the activity of the biomarkers of the present invention (e.g., antibodies, polypeptides, and / or nucleic acids), and various combinations of compounds useful in any of the methods described elsewhere herein, including instructional materials. For example, in one embodiment, the kit includes components useful for quantifying a desired antibody in a biological sample. In a further embodiment, the kit includes components useful for evaluating the activity (e.g., binding activity, blocking activity, etc.) of a desired antibody in a biological sample.

[0094] In a further embodiment, the kit includes instructional materials and components for determining whether the level of a biomarker of the present invention in a biological sample obtained from a subject in need thereof is regulated during or after treatment, including components of an assay for tracking the effectiveness of a treatment administered to the subject. In various embodiments, to determine whether the level of a biomarker of the present invention is regulated in a biological sample obtained from a subject, the level of the biomarker is compared to at least one comparator included in the kit, such as a positive control, a negative control, a historical control, a historical standard, etc., or the level of another reference molecule within the biological sample. In certain embodiments, the ratio of the biomarker to the reference molecule is determined to assist in monitoring treatment.

[0095] Detection and Diagnosis In various embodiments, the present invention provides kits and / or methods for diagnosing a disease or disorder of a subject, assessing its prognosis, or assessing its risk of onset. In some embodiments, the present invention provides kits and / or methods related to biomarkers that can be used to diagnose, assess the prognosis of, or assess the risk of onset of a disease or disorder associated with MAP infection. In other embodiments, the methods of the present invention relate to kits and / or methods for diagnosing a subject having one or more autoimmune diseases or disorders.

[0096] In one embodiment, the method comprises detecting the level of at least one biomarker (e.g., an antibody) in a biological sample obtained from a subject, comparing the level of the at least one biomarker in the biological sample to a comparator of the at least one biomarker, and determining that the subject is at high risk of developing a disease or disorder if the at least one biomarker is differentially expressed in the biological sample compared to the comparator. In one embodiment, the biomarker is positively or negatively correlated with MAP infection. In one embodiment, the biomarker is positively or negatively correlated with one or more autoimmune diseases or disorders. In one embodiment, the biomarker comprises one or more antibodies. In one embodiment, the antibodies include, but are not particularly limited to, anti-Hsp65, anti-PknG, and anti-L5P.

[0097] In some embodiments, one or more autoimmune diseases or disorders are associated with MAP infection. In some embodiments, the one or more autoimmune diseases include, but are not particularly limited to, Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome (IBS), multiple sclerosis (MS), type 1 diabetes mellitus (T1DM), Sjogren's syndrome (SS), systemic lupus erythematosus (SLE), depression, Parkinson's disease (PD), Alzheimer's disease (AD), celiac disease, thyroiditis, rheumatoid arthritis, Blau syndrome, psoriasis, and complex regional pain syndrome (CRPS).

[0098] In various embodiments, the subject is a human subject and can be of any race, gender, and age. Representative subjects include those at risk of developing a MAP infection, those suspected of having a MAP infection, those diagnosed with a MAP infection, and those diagnosed with one or more autoimmune diseases associated with MAP infection.

[0099] In some embodiments, the diagnosing of the subject comprises determining whether the subject has differentially expressed levels of one or more biomarkers. In some embodiments, at least two biomarkers are required to diagnose the subject. In some embodiments, at least three biomarkers are required to diagnose the subject. In some embodiments, at least one biomarker and at least one additional clinical assay are required to diagnose the subject. In some embodiments, at least two biomarkers and at least one additional clinical assay are required to diagnose the subject. In some embodiments, at least three biomarkers and at least one additional clinical assay are required to diagnose the subject.

[0100] In one embodiment, the method comprises using a multidimensional non-linear algorithm to determine whether the levels (e.g., antibodies) of a series of biomarkers in a biological sample are statistically different from the levels in a comparator sample. In some embodiments, the algorithm is essentially drawn from the group consisting of: linear or non-linear regression algorithms; linear or non-linear classification algorithms; ANOVA; neural network algorithms; genetic algorithms; support vector machine algorithms; hierarchical analysis or clustering algorithms; hierarchical algorithms using decision trees; kernel-based machine algorithms, such as kernel partial least squares algorithms, kernel matching pursuit algorithms, kernel fisher discriminant analysis algorithms, or kernel principal component analysis algorithms; Bayesian probability function algorithms; Markov blanket algorithms; multiple algorithms arranged in a committee network; and forward floating search or backward floating search algorithms.

[0101] In one embodiment, the method includes detecting one or more markers in a biological sample of a subject. In some embodiments, the level of one or more markers of the invention in a biological test sample of a subject is compared to the level of a biomarker in a comparator. Non-limiting examples of comparators include, but are not limited to, negative controls, positive controls, standard controls, standard values, expected normal background values of the subject, past normal background values of the subject, reference standards, reference levels, expected normal background values of a population of which the subject is a member, or past normal background values of a population of which the subject is a member. In one embodiment, the comparator is the level of one or more biomarkers in a sample obtained from a subject without a disease or disorder such as an autoimmune disease or disorder. In one embodiment, the comparator is the level of one or more biomarkers in a sample obtained from a subject known to be free of a disease or disorder.

[0102] In various embodiments, the kits and / or methods of the invention can detect biomarkers having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sensitivity; at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% specificity; and / or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% accuracy.

[0103] In various embodiments, the kits and / or methods of the present invention can detect the presence of infectious agents with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sensitivity; at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% specificity; and / or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% accuracy.

[0104] In various embodiments, the kits and / or methods of the present invention can diagnose a disease or disorder with at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sensitivity; at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% specificity; and / or at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% accuracy.

[0105] Treatment In one embodiment, the present invention includes a method for detecting MAP infection in a subject. In one embodiment, the present invention provides a method for determining whether a subject having a detectable MAP infection should be diagnosed with an autoimmune disease. In one embodiment, the method of the present invention determines whether a subject diagnosed with an autoimmune disease should be treated with a therapeutic agent specific for MAP infection. In one embodiment, the method of the present invention determines whether a subject diagnosed with an autoimmune disease should be treated with a therapeutic agent specific for said autoimmune disease. In one embodiment, said therapeutic agent is an antibiotic.

[0106] In one embodiment, the method includes administering to a subject an effective amount of one or more therapeutic agents. In one embodiment, the therapeutic agent is an antibiotic. Those skilled in the art should recognize that any antibiotic capable of treating MAP infections can be used in the method of the present invention.In some embodiments, the one or more antibiotics are not particularly limited, but include the following: Cefradine, cefradine, cefuroxime, cefoxitin, cefotaxime, ceftazidime, ceftezole, cefibuten, cefthiofur, cefthiolene, ceftobiprole, ceftriaxone, cefuroxime axetil, cefoxitin, cefozopran, cephalosporin, chloramphenicol, cilastatin, ciprofloxacin, clarithromycin, clinafloxacin, clindamycin, clofazimine, cloxacillin, demeclocycline, dicloxacillin, dirithromycin, doripenem, doxycycline, enoxacin, ertapenem, erythromycin, flumequine, fluoroquinolone, gemifloxacin, gentamicin, grepafloxacin, imipenem, kanamycin, ketolide, levofloxacin, lincomycin, linezolid, lomefloxacin, meropenem, metronidazole, mezlocillin, minocycline, moxifloxacin, mycobutin, nadifloxacin, nafcillin, nalidixic acid, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin, oxacillin, oxolinic acid, oxytetracycline, paromomycin, pazufloxacin, pefloxacin, penicillin g, penicillin v, piperacillin, piromidic acid, pipemidic acid, pivampicillin, pivmecillinam, primaxin, prulifloxacin, rifampin, rosoxacin, rokitamycin, rufloxacin, sitafloxacin, sparfloxacin, streptomycin, sulfamethizole, sulfamethoxazole, sulfisoxazole, teicoplanin, telavancin, telithromycin, temafloxacin, tetracycline, ticarcillin, tobramycin, tosufloxacin, trimethoprim-sulfamethoxazole, trovafloxacin, vancomycin, vancomycin, and lipopeptide.

[0107] In other embodiments, the one or more antibiotics are not particularly limited, but may include: amoxicillin, ampicillin, cloxacillin, dicloxacillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, cefadroxyl, cephalexin, cephalothin, cephapirin, cefazolin, cephradine, cefaclor, cefotetan, cefoxitin, cefprozil, cefuroxime, cefdinir, cefixime, cefotaxime, cefpodoxime, ceftizoxime, ceftriaxone, ceftazidime, cefepime, ceftobiprole, ceftaroline, aztreonam, imipenem, cilastatin, doripenem, meropenem, ertapenem, azithromycin, erythromycin, clarithromycin, dirithromycin, roxithromycin, clindamycin, lincomycin, amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, trimethoprim-sulfamethoxazole, doxycycline, tetracycline, vancomycin, teicoplanin, telavancin, and linezolid.

[0108] Administration of a therapeutic agent by the methods of the present invention can be continuous or intermittent, for example, depending on the physiological state of the recipient and other factors known to those of skill in the art, whether the purpose of administration is therapeutic or prophylactic. Administration of the agents or modified cells of the present invention can be essentially continuous over a preselected period of time or can be a series of spaced administrations. Both local and systemic administrations are contemplated. The dosage will vary depending upon a variety of factors, including but not limited to the selected composition, the particular disease, the weight, condition, age of the mammal, and whether prophylaxis or treatment is being effected. Such factors can be readily determined by a clinician using well-known animal models or other test systems in the art.

[0109] As discussed below, one or more suitable unit dosage dosage forms having a therapeutic agent, which can optionally be formulated for sustained release (e.g., using microencapsulation; see WO94 / 07529, and U.S. Patent No. 4,962,091; the disclosures of which are incorporated herein by reference), can be administered by a variety of routes including parenteral including intravenous and intramuscular routes. The formulations can, where appropriate, be conveniently presented in discrete unit dosage forms and can be prepared by any of the methods well known in the pharmaceutical art. Such methods can include incorporating the therapeutic agent with a liquid carrier, solid matrix, semi-solid carrier, finely divided solid carrier, or combinations thereof and then, if necessary, introducing or shaping the product into the desired delivery system.

[0110] When the therapeutic agents used in the methods of the present invention are prepared for administration, they can be combined with a pharmaceutically acceptable carrier, diluent, or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredient in such formulations can comprise from 0.1 to 99.9% by weight of the formulation. "Pharmaceutically acceptable" refers to a carrier, diluent, excipient, and / or salt that is compatible with the other ingredients of the formulation and not harmful to its recipient. The active ingredient for administration can be present as a powder or granule, as a solution, suspension, or emulsion.

[0111] Formulations containing the therapeutic agent can be prepared by procedures known in the art using known and readily available ingredients. The therapeutic agent of the present invention can also be formulated as a solution suitable for parenteral administration, for example, by intramuscular, subcutaneous or intravenous routes.

[0112] Formulations of the therapeutic agent can also be in the form of an aqueous or anhydrous solution, or dispersion, or in the form of an emulsion or suspension.

[0113] Accordingly, the therapeutic agent can be formulated for parenteral administration (e.g., by injection, e.g., by bolus injection or continuous infusion) and provided in unit dosage form in ampoules, prefilled syringes, small volume infusion containers, or multi-dose containers with added preservatives. The active ingredient can be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle and can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient can be in powder form obtained by aseptic isolation of a sterile solid or by lyophilization from a solution for reconstitution prior to use with a suitable vehicle, e.g., sterile pyrogen-free water.

[0114] Since the required effective amount is achieved by administration of multiple dosage units, the unit content of the active ingredient or ingredients contained in each individual aerosol administration of each dosage form need not itself constitute an effective amount for treating a particular indication or disease. Furthermore, the effective amount can be achieved individually or in any of a series of administrations using a dosage less than the dosage in the dosage form.

[0115] Formulations used in the method of the present invention can contain, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the types known in the art. Specific non-limiting examples of carriers and / or diluents useful in the formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline (pH 7.0 - 8.0).

[0116] The therapeutic agent can be formulated and administered by any conventional means available for use in combination with a pharmaceutical by either as an individual therapeutic active ingredient or as a combination of therapeutic active ingredients. These can be administered alone, but generally are administered with a pharmaceutical carrier selected based on the chosen route of administration and standard pharmaceutical practice.

[0117] Generally, water, suitable oils, physiological saline, aqueous dextrose (glucose) solutions, and related sugar solutions, as well as glycols such as propylene glycol or polyethylene glycol, are carriers suitable for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizers, and, if necessary, buffering substances. Antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid alone or in combination are suitable stabilizers. Also, citric acid and its salts, and sodium ethylenediaminetetraacetate (EDTA) are used. Additionally, parenteral solutions can include preservatives such as benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington’s Pharmaceutical Sciences, a standard reference in this field.

[0118] The active ingredient of the therapeutic agent can be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals, particularly humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs thereof described in U.S. Patent Nos. 4,082,735, 4,082,736, 4,101,536, 4,185,089, 4,235,771, and 4,406,890. Other useful adjuvants include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate, and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12. Other components may include polyoxypropylene-polyoxyethylene block polymers (Pluronic®), nonionic surfactants, and metabolic oils such as squalene (U.S. Patent No. 4,606,918).

[0119] Furthermore, the duration of action can be controlled using standard formulation methods. These are known in the art and include controlled release preparations and can include suitable polymers such as polymers, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulfate. The concentration and method of incorporation of the polymer can be controlled to adjust the release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene vinyl acetate copolymers. In addition to being incorporated, these agents can also be used to encapsulate the compound in microcapsules.

[0120] Accordingly, the pharmaceutical compositions used in the method of the present invention can be delivered via various routes and to various sites within a mammalian body to achieve a specific effect (see, for example, Rosenfeld et al., 1991; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). Multiple routes can be used for administration, and one of ordinary skill in the art will recognize that a particular route may provide a more rapid and effective response than another. Local or systemic delivery can be achieved by administration including application or instillation of the formulation into a body cavity, inhalation of an aerosol, or insufflation, or by parenteral introduction including intramuscular, intravenous, peritoneal, subcutaneous, intradermal, and topical administration.

[0121] The active ingredient of the therapeutic agent can be provided in unit dosage form, and each dosage unit, for example, a tablespoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, either alone or in suitable combination with other active agents. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit dosages for human and mammalian subjects, each unit containing, either alone or in combination with other active agents, a predetermined amount of the composition of the present invention calculated to produce the desired effect in a sufficient amount, together with a pharmaceutically acceptable diluent, carrier, or vehicle as appropriate. The specifications for the unit dosage forms of the present invention depend on the specific effect to be achieved and the specific pharmacokinetics of the pharmaceutical composition in combination with the specific host.

[0122] Experimental Examples The present invention will be described in more detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting, particularly unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all modifications that become apparent as a result of the teachings provided herein.

[0123] Even without further elaboration, those skilled in the art will be able to create and utilize the present invention and implement the claimed method using the foregoing description and the following exemplary examples. Accordingly, the following examples should not be construed as in any way limiting the remainder of the disclosure.

[0124] Example 1: Culture methods, antibody-based methods, and phage amplification methods variably detect MAP In accordance with the consensus recommendations from the MAP Conference held at Temple University in March 2017, this example of a study was conducted to explore alternative approaches for detecting, isolating, and / or culturing MAP from human blood samples (Kuenstner, J.T., et al., Front Public Health, 2017, 5: 208). The primary objective of this study was to assess the extent of viable culturable MAP bacteremia in the study human population, to definitively identify the cultured bacteria, and to determine the relative preponderance of MAP in CD patients compared to controls including several non-CD subjects with various autoimmune diseases. Secondary objectives included the parallel evaluation of a rapid phage amplification detection method, which is one of two culture-based approaches (TiKa culture and Pozzato culture) developed in different laboratories, the existing MGIT culture method, and a series of MAP-specific antibody tests developed in several other laboratories. Finally, to establish the degree of reliability of the results, selected subjects who initially showed a positive MAP detection were followed up with repeat testing one year later.

[0125] The materials and methods of this example are described herein.

[0126] Study design and participants The test protocol was reviewed by the Temple University IRB (IRB Protocol #24790) on October 20, 2017. This case-control study included 201 subjects, 61 with CD and 140 non-CD controls. The non-CD control group included 16 patients with UC and various other autoimmune diseases. The first 159 subjects of this study were recruited from the practice of Dr. Ira Shafran, a gastroenterologist in Winter Park, Florida. Additionally, 42 subjects were recruited from the website of the Human Paratuberculosis Foundation (www.humanpara.org), and blood draws for the second group were performed in New York City. From May to August 2018, a single blood draw was performed on 192 subjects. For 9 subjects, a second blood sample was taken in August 2019 to examine whether there was transient or persistent MAP infection. The selection of these subjects was based on being positive in the initial MAP culture or phage assay and being able to provide a second sample in Philadelphia one year later. One of these subjects had chronic thyroiditis and irritable bowel syndrome (IBS), 3 had chronic thyroiditis, 4 subjects were asymptomatic and healthy, and 1 had IBS.

[0127] Diagnosis and diagnostic classification Subjects completed a consent form and a questionnaire regarding their medical history and BCG status. Patients with CD or UC also completed an additional questionnaire during enrollment to be evaluated for the modified Harvey Bradshaw index (HBI). All subject information was kept confidential in accordance with standard medical practice.

[0128] Procedure Blood samples were collected into EDTA tubes from all 201 registered participants. Peripheral blood leukocyte (PBL) / buffy coat specimens were prepared from the whole blood samples and then shipped by courier to each of the laboratories performing the cultures (the Bull and Grant laboratories in London and Belfast, UK, and the Naser laboratory in Florida, USA). Courier delivery to the Bull and Grant laboratories took 3 - 7 days as the application did not comply with international customs regulations, while delivery to the Naser laboratory took 1 day. Plasma was harvested from the blood samples and frozen at -80°C until serological testing was performed later. All samples were identified by test number, and laboratory scientists were not informed of any clinical information, diagnosis, or personal information.

[0129] MGIT culture Peripheral blood leukocytes (PBLs) from the EDTA buffy coat were inoculated into BACTEC MGIT ParaTB medium containing supplements (OADC and mycobactin J detailed above) and incubated at 37°C for 6 months. After incubation, the MGIT cultures were centrifuged, DNA was extracted from the pellet, and nested IS900 PCR was performed as described (Naser, S.A., et al., Lancet, 2004, 364: 1039 - 1044). Subcultures were made from all PCR-positive MGIT cultures and attempts were made to recover MAP in pure culture.

[0130] TiKa culture The buffy coat transported in Middlebrook 7H9 transport medium was centrifuged at 800 g for 10 minutes at room temperature, and the pellet was resuspended in 10 ml of freshly prepared TiKa-KiC (Bull, T.J., et al., Front Microbiol, 2016, 7: 2112) decontamination cocktail and then incubated with shaking (150 rpm) at 37 °C for 20 - 24 hours. The sample was centrifuged at 2500 g for 15 minutes at room temperature, and the pellet was resuspended in 1 ml of recovery medium (Pozzato culture) + TiKa supplement A (1 μg / ml), M1, M2, and M3 (1 μl / ml) and incubated at 37 °C for 2 days. Next, the sample was added to MGIT culture tubes supplemented with PANTA + mycobactin J (2 μg / ml) and TiKa supplement A (1 μg / ml) and incubated at 37 °C for up to 4 months. Cultures showing visible growth were centrifuged at 2500 g for 10 minutes at room temperature, and the pellet was subcultured onto solid Pozzato culture (1.5% agar) in a 24-well plate. Next, 0.75% agar semi-solid Pozzato culture containing TiKa supplement A (1 μg / ml) was overlaid, sealed with a gas-permeable membrane, and incubated at 37 °C in 5% CO2. MAP DNA was extracted from the colonies as previously described (Bull, T.J., et al., J Clin Microbiol, 2003, 41: 2915 - 2923). Samples were considered MAP positive if IS900 and F57 PCR-positive biomass was obtained.

[0131] Pozzato culture and phage amplification assay The PBL samples were centrifuged (2500 g for 15 min) and resuspended in 1 ml of Middlebrook 7H9 broth (Difco) supplemented with 10% OADC (Difco) and 2 mM CaCl2 (Sigma). Phage amplification assays and Pozzato cultures were performed as previously described (Swift, B.M., et al., Virulence, 2016, 7: 779-788; Pozzato, N., et al., J Microbiol Methods, 2011, 84: 413-417; Grant, I.R., et al., J Dairy Sci, 2017, 100: 9723-9735; Foddai, A., et al., Appl Environ Microbiol, 2009, 75: 3896-3902). Briefly, as described by Pozzato et al. (Pozzato, N., et al., J Microbiol Methods, 2011, 84: 413-417), after incubating for 15 min at room temperature and mixing thoroughly by vortexing, 500 μl of each PBL sample was inoculated into screw-cap glass culture tubes containing 4 ml of modified 7H9 medium, PANTA antibiotic supplement, and mycobactin J, without the addition of egg yolk (referred to as "Pozzato culture") (Grant, I.R., et al., J Dairy Sci, 2017, 100: 9723-9735). A second 500 μl of each PBL sample was subjected to an optimized phage amplification assay (Foddai, A., et al., Appl Environ Microbiol, 2009, 75: 3896-3902) and performed as follows: 108 D29 mycobacteriophages were added to each 1 ml of test sample to infect the MAP cells present, and the samples were incubated at 37°C. After 2 h, the external seed phages were inactivated by treatment with virucide (ammonium ferrous sulfate at a final concentration of 10 mM) for 10 min, and then the samples were diluted with 5 ml of 7H9 / OADC / CaCl2 (2 mM) broth.

[0132] Incubate the sample until a total of 3.5 hours have elapsed since the addition of the phage. At that time, the entire sample was seeded onto 155 sensor cells and 5 ml of molten Middlebrook 7H9 agar medium in a petri dish with Mycobacterium smegmatis mc 2 After solidification, the agar plates were incubated overnight at 37°C and the next day, examined for evidence of clear zones ("plaques") that would indicate viable mycobacteria in the sample. If 1S900 and F57 PCR-positive biomass (broth pellets or colonies on Herrold's egg yolk - mycobactin J (HEYM)) were obtained, the PML sample was considered MAP culture positive.

[0133] MAP antibody assay Plasma samples from each patient were assayed using an IDEXX Mycobacterium paratuberculosis antibody test kit to detect antibodies against MAP in bovine serum, plasma, and milk. This kit was modified to be suitable for use in humans as previously described (Bernstein, C.N., et al., J Clin Microbiol, 2004, 42: 1129-1135). The optical density (OD) values of human plasma controls were used to calculate the sample / positive (S / P) ratio to interpret the assay.

[0134] PipA and PknG ELISA tests Antibodies against pathogenicity factors secreted by MAP during infection were measured in plasma specimens as previously described (Bach, H., et al., Scand J Gastroenterol, 2011, 46: 30-39; Bach, H., et al., Biomed Res Int, 2018, 2018: 1450828). These antigens included protein tyrosine phosphatase (PtpA) and protein kinase G (PknG) (Bach, H., et al., Biomed Res Int, 2018, 2018: 1450828; Bach, H., et al., Cell Host Microbe, 2008, 3: 316-322). Recombinant PtpA and PknG were produced in Mycobacterium smegmatis according to published procedures (Bach, H., et al., Biomed Res Int, 2018, 2018: 1450828; Bach, H., et al., Cell Host Microbe, 2008, 3: 316- 322).

[0135] Hsp65 antibody assay Hsp65 antibodies from blood were measured by a previously described direct ELISA assay (Zhang, P., et al Microorganisms, 2019, 8). Recombinant Hsp65 of Mycobacterium avium hominissuis subspecies (MAH) was produced by contract research at GenScript Corp (https: / / www.genscript.com / ) and used for coating 96-well plates.

[0136] Whole genome sequencing (WGS) The whole genome sequence of one MAP isolate from a single subject, isolated mainly using the TiKa culture, was determined. Briefly, three colonies from the Pozzato semi-solid culture were washed once with TExl buffer (Tris-HCl - EDTA, pH 8, Sigma, UK), heated at 98 °C for 30 minutes in TExl to kill them, and then gDNA was extracted using the QiaPrep DNA kit (Qiagen, UK) according to the manufacturer's instructions. The whole genome sequencing method was as previously described (Witney, A.A., et al., BMC Med, 2016, 14: 46). DNA concentration and integrity were determined using the Qubit High Sensitivity DNA assay (Life Technologies, UK) using a genomic screen tape and the Agilent Tapestation 2100, respectively. Library preparation and sample indexing were performed using NexteraXT DNA according to the manufacturer's instructions, and then sequenced by bead-based normalization and pooling with other libraries. Sequencing was performed using paired-end 2×300bp reads with Illumina v3 chemistry, sequenced on an Illumina MiSeq Sequence, and the reads were mapped to the MAP reference genome (RefSeq accession number: NC_002944.2) using bwa mem v0.7.3a-r367 (Li, H., arXiv, 2013, 1303.3997v2 [q-bio.GN]), the alignments were classified, duplicates were removed with samtools vO.1.19 (Li, H., et al., Bioinformatics, 2009, 25: 2078-2079), and site statistics were generated using samtools mpileup. The assembled genome was mapped against other mycobacteria using the NCBI Sequence Viewer (Version 3.36.0, NCBI, USA) and Snapgene (Version 4.3.11, GSL Biotech, USA).

[0137] Data analysis and statistical methods Data were presented as frequencies and percentages of categorical variables and means ± standard deviation (SD) and / or medians (range or interquartile range) of continuous variables. The association between potential risk factors or the assay method of interest and the selected disease state (i.e., CD or CD+UC) was evaluated using Fisher's exact test for two groups and the Wald test for continuous variables. When other potential risk factors or confounding variables such as age and gender were adjusted in the regression model, multivariate logistic regression analysis was performed to investigate the association with or the predictability of the disease using different assay methods (culture, antibody, or both) for the selected disease. To define the cut-off values of continuous covariates or assay variables to be included in the logistic regression model for predicting patient outcomes, cut-offs were selected to achieve the optimal classification criteria based on the Euclidean distance method. Both the continuous and dichotomized versions of continuous variables were included in the logistic regression model as candidate variables for the stepwise variable selection procedure. The final regression model was reached using a stay probability of 0.10 and an entry probability of 0.25. Thus, variables with non-significant predictive ability for the disease state were removed from the multivariate logistic regression model to keep the model parsimonious. Age and gender were included in all regression models a priori. Both the raw and adjusted odds ratios for CD or CD+UC and their 95% confidence intervals (CIs) were reported as appropriate. P-values less than 0.05 were considered statistically significant. SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used for all data analyses.

[0138] The results of this example are described herein.

[0139] Data for the subject groups for each subgroup of the trial are shown in Figure 1. Since the data are asymmetric, in addition to the mean age and standard deviation, the median patient age and age range are displayed. This approach is suitable for asymmetric distributions such as age data. In this trial, non-CD patients were slightly older than CD patients (median: 57.5 years vs. 47 years) and appeared to have a similar gender composition (59% vs. 54% female).

[0140] The order from the highest to the lowest analytical sensitivity of the culture methods for live MAP bacteremia (the ability of the method to detect the organism, not the clinical sensitivity which is the ability of the test to detect the disease) is as follows: 1) Pozzato culture (124 / 201, i.e., 62% of all subjects, 35 / 61, i.e., 57% of CD patients), 2) phage assay (113 / 201, i.e., 56% of all subjects, 28 / 61, i.e., 46% of CD patients), 3) TiKa culture (64 / 201, i.e., 32% of all subjects, 22 / 61, i.e., 36% of CD patients), and 4) MGIT culture (36 / 201, i.e., 18% of all subjects, 15 / 61, i.e., 25% of CD patients). These results are summarized in Figure 2. Results are reported as the number and % of subjects in each category, and the number of subjects for CD and UC-only patients is shown in parentheses. A single asterisk (*) indicates that 2 CD patients were diagnosed with UC. A double asterisk (**) indicates that the non-CD category included 14 UC-only subjects. A dagger (†) indicates that the results of the TiKa culture were missing for 2 subjects.

[0141] The correlation of the results of the three liquid culture methods (MGIT, TiKa, Pozzato) is shown in Figure 3. An asterisk (*) indicates that there were 2 samples with "contaminated" results from the Bull laboratory. Additional data show the number of plaques observed in CD patients and non-CD controls (Figures 4 and 5).

[0142] Figure 6 shows the associations of clinically diagnosed CD patients with various categorical variables of interest. Notably, the younger age group (≤52 years) appeared to have a higher likelihood of having CD compared to the older age group (>52 years) (OR (95%Cl): 2.66 (1.42, 4.99); p = 0.003). Among all assay methods, when the Hsp65 antibody assay was less than 0.74 negative, it seemed to distinguish between CD and non-CD cases (p = 0.06 and 0.02, see Table 3). Figure 7 shows the comparison of MAP detected by all culture and serological methods among the subjects.

[0143] To examine the independent relationship of the presence of MAP in the subjects with clinical diagnosis, i.e., CD or CD+UC vs. (non-CD, or neither CD nor UC), with culture and serological methods, a multivariate logistic regression model was used for the correlation analysis. Important findings regarding the association with MAP culture and / or antibody data are provided using the odds ratio (OR) and its 95% confidence interval (Cl) and p-value for having CD or having either CD or UC. The OR data for all culture methods are shown in Figure 8 adjusted for age and gender, which is more robust than the unadjusted / marginal odds ratio data included in Figure 6. The results are reported from six logistic regression models (3 inclusion settings of assay method variables × 2 selected diseases (CD or CD+UC)), all adjusted for age (≤52 vs. >52) and gender. NS indicates not significant, and the double dagger (‡) indicates the OR and 95%Cl for a 0.2 unit increment of the Hsp65 antibody.

[0144] The associated p-value was 0.037, and for MAP-positive MGIT culture corresponding to CD patients, the OR (95%Cl) was 2.36 (1.06, 5.28), and this associated p-value supports the predictive power of this culture method. This also holds when UC subjects were included in the analysis together with CD subjects, and the result of this analysis showed that for having CD+UC, the p-value was 0.006 and the adjusted OR (95%Cl) was 3.19 (1.40, 7.23) when subjects with positive MGIT culture were compared with negative subjects.

[0145] The TiKa culture showed a significant association (data not included) for positive MAP cultures, but the TiKa, phage assay, and Pozzato culture methods were 0.10 and did not reach statistical significance. Therefore, with the methods described above, the final model to predict the presence of CD in our study was not created. Nevertheless, the phage assay and Pozzato culture method detected a high rate of MAP infection in the non-CD / UC cohort, while the TiKa culture showed the opposite trend similar to that shown by the MGIT culture method (see Figure 2).

[0146] Among several serological tests, the highest and only significant correlation with the presence of CD occurred with the Hsp65 antibody. At a cut-off value of 0.74, this method had the highest ability to distinguish CD patients from non-CD subjects (adjusted OR (95%Cl) for having CD comparing Hsp65Ab>0.74 and Hsp65Ab≦0.74: 2.40 (1.25, 4.61); p-value 0.009; Figure 8). The PknG antibody also had a significant negative correlation with the presence of CD / UC in our patients compared to non-CD / UC subjects (adjusted OR (95%Cl) for having CD / UC comparing PknG negative and positive: 2.18 (1.12, 4.23); p-value: 0.022). The Spearman correlation showed that the Hsp65 antibody had a weak correlation with the HBI (Spearman coefficient = 0.28, p = 0.03), and among 60 CD patients who had such data, the phage plaque count showed a very weak correlation with the HBI (Spearman coefficient = 0.12, p = 0.37). It is noteworthy that the range of HBI in the study population was limited and there were few patients with an HBI exceeding 5.

[0147] After adjusting for age and gender (and for the latter, mutually), the MGIT culture and the Hsp65 antibody assay were each, independently, in their own (culture or antibody) category and in the set combining culture and antibody, the best discriminators between CD patients and non-CD subjects in the study population (see Figure 8). When all three culture methods were combined into one category, i.e., the culture was positive if any of the three culture methods was positive and negative otherwise, the overall agreement between this category and the phage assay achieved a rate of 75% with an exact 95% Cl (68%, 80%). The kappa statistic (SE) for agreement between the two diagnostic methods was 0.26 (0.08) and the 95% Cl was (0.11, 0.42), indicating that the strength of agreement was fair (Figure 9).

[0148] Viable MAP bacteremia was detected even in patients with an autoimmune condition who had neither CD nor UC: 1) Pozzato culture (33 / 57, i.e., 58%), 2) phage assay (36 / 57, i.e., 63%), 3) TiKa culture (19 / 56, i.e., 34%), and 4) MGIT culture (9 / 57, i.e., 16%). All nine subjects who were initially positive for MAP culture or phage assay were positive on the second phage assay of the second blood sample obtained one year later.

[0149] The definitive identification of one viable isolate recovered from the blood of a subject with IBS was confirmed as MAP by whole-genome sequencing (WGS) and by the assembled contigs compared to MAP isolates in a large genomic bank. The isolate phage, TiKa culture isolate, and WGS dendrograms are shown in Figures 10, 11, and 12, respectively.

[0150] The conclusions of this example are described here.

[0151] This example clearly shows that viable MAP bacteria are present in the blood of a very large number of people, and this infectious state may persist. Bacteremia with live MAP bacteria was not exclusive to any of the tested groups, including patients with CD, UC, CD with UC, various other autoimmune diseases, or asymptomatic subjects.

[0152] In this study, three culture methods and a phage-based method + culture method were compared, and aliquots of buffy coat processed at various laboratories with expertise in the use of each method were blindly and concurrently tested. All MAP culture positives were identified and confirmed by a validated specific molecular identification method using nested IS900 PCR (Naser, S.A., et al., Lancet, 2004, 364: 1039-1044), or IS900 plus MAP gene F57 PCR (Bull, T.J., et al., J Clin Microbiol, 2003, 41: 2915-2923). Aliquots of the same samples were tested by each method, but there was not enough funding to perform the same method in all labs. The methods selected included the MGIT culture method previously used for MAP in human PBL as proposed by Naser (Naser, S.A., et al., The Open Inflammation Journal, 2009, 2: 22-23), and two new culture methods [TiKa decontamination and culture (Bull, T.J., et al., Front Microbiol, 2016, 7: 2112) and culture in 7H9 + broth (herein referred to as Pozzato culture; Pozzato, N., et al., J Microbiol Methods, 2011, 84: 413-417; Grant, I.R., et al., J Dairy Sci, 2017, 100: 9723-9735)]. Culture of MAP alone from buffy coat leukocyte fractions using the MGIT method showed an increased correlation with CD patients compared to non-CD controls. With the other two methods (TiKa culture and Pozzato culture), MAP was cultured from a significant number of patients, but there was no significant difference between any of the groups. A previous meta-analysis (Feller and Abubakar, 2007) showed a high rate of MAP detection using molecular detection methods on samples from CD patients compared to controls.

[0153] The results of the culture do not entirely support this conclusion overall. However, while these previous analyses were mainly based on data from randomly targeted gastrointestinal mucosal biopsies, in this study, blood samples that could potentially explain the discrepancies were examined. Additionally and in parallel, an optimized phage amplification assay designed to detect viable MAP was included for aliquots of blinded samples in another laboratory (Foddai, A., et al., Appl Environ Microbiol, 2009, 75: 3896-3902). This rapid culture-based / phage amplification assay can capture and amplify mycobacteria-specific phages, which then burst to release progeny phages, detecting only viable mycobacterial cells present in the sample. Once released, these are plaque assayed, and the plaques (including the original lysed mycobacteria) are subjected to species-specific PCR, enabling the detection and quantification of the presence of MAP within 48 hours. In this study, aliquots of the samples examined by the phage were further incubated in Pozzato medium and re-examined to confirm positivity after a certain period of growth. This method was able to detect viable mycobacteria in the samples within 2 days, and importantly, it was able to show that all of the 9 patients who were initially MAP positive and could be followed up 1 year later remained positive for MAP in their blood after this time.

[0154] These results indicate that both alternative culture approaches (TiKa culture and Pozzato culture) were able to culture viable MAP more effectively than the reference MGIT method for all subject groups. These findings suggest that the composition of the MGIT ParaTB medium is not optimal for the isolation of MAP from human PBL. The MGIT culture approach may have yielded more MAP-positive cultures from the PBL of CD patients compared to non-CD controls (results predicted from previous human PBL tests in CD patients (Naser, S.A., et al., Lancet, 2004, 364: 1039-1044; Naser, S.A., et al., The Open Inflammation Journal, 2009, 2: 22-23)), but TiKa culture and Pozzato culture tested more non-CD controls positive for viable MAP than CD patients, or equivalently. One explanation for this observation is that the MAP phenotypes persisting in the blood are not in a fully growth-competent physiological state, or have entered a dormant state as occurs with other mycobacterial species. This may be more involved in the induction of these phenotypes. Therefore, some form of MAP resuscitation may be required before growth can occur. The different compositions of the TiKa broth and Pozzato broth may account for this discrepancy in MAP resuscitation ability. Both are based on Middlebrook 7H9, but the TiKa system involves TiKa-supplemented Pozzato medium followed by long-term culture in TiKa-supplemented MGIT culture, while the Pozzato medium used in this study did not add egg yolk as previously described for the isolation of MAP from bovine feces (Pozzato, N., et al., J Microbiol Methods, 2011, 84: 413-417) and enabled monitoring of the optical density of the culture during incubation. In this case, further work is needed to determine the essential components.

[0155] A further difference between the sample tests at the various research institutes was the age of the PBLs at the time of culturing. The sample transport times were different, which could have affected the conditions to which they were subjected. The longer transit time may have reduced the survival of MAP cells in the PBL samples. This is demonstrated by the significant difference in the number of plaques obtained by the phage assay for the older PBL samples at the time of testing (Figure 13). Perhaps it would be beneficial to delay the PBL testing for several days to resuscitate the MAP cells in transport medium (MGIT medium with added OADC) or to lyse the PBL cells to make the MAP cells available for culturing.

[0156] The results of this example show that a significant proportion of subjects have MAP bacteremia and are positive by multiple culture methods, regardless of the underlying disease. Three explanations for this finding are possible: 1) Live MAP is passively acquired from ingested food and not eliminated by the host immediately after ingestion. Since other organisms consumed in the diet that survive the digestive process and enter the bloodstream through the "leaky gut" of IBD do not cause persistent bacteremia in these hosts, this explanation seems unlikely; 2) MAP is present, persistent, and alive but does not cause disease in the human host. Since there are no known examples of persistent bacteremia by known pathogens without some effect, this explanation also seems unlikely; 3) MAP persistently infects some hosts, and the pathogen may affect the disease process in some highly susceptible human hosts. Without being bound by a particular scientific theory, the third explanation is said to be the most likely and suggests that MAP is a zoonotic pathogen. In this case, human infection resembles the known pathogenic state of cattle, where only a small number of animals (10%) develop clinical JD that progresses, and most show subclinical persistent infection (Magombedze, G., et al., PLoS One, 2013, 8: e76636).

[0157] Previous studies have shown the proportion of apparently healthy individuals who may have asymptomatic mycobacterial infections. In an immunological recognition test (Zhang, P., et al., Microorganisms, 2019, 8), 2.8% of 288 healthy blood donors were positive for antibodies against mycobacterial heat shock protein 65 (anti-Hsp65 antibodies), while 67.9% of 109 CD patients and 85.7% of Sjogren's syndrome patients were positive for anti-Hsp65 antibodies, suggesting that MAP infection and immune presentation and processing are common.

[0158] This example indicates that more detailed MAP research is urgently needed to fully investigate the role of this organism in humans. An obvious further research area is the discovery of better therapies applicable to controlled clinical trials aimed at targeting and eliminating MAP (Graham, D.Y., In Proceedings of Annual Scientific Meeting and Postgraduate Course (ACG), San Antonio, Texas, October 25 - 30, 2019). Research on MAP culture and phage assays should be carried out by experts in these methods for other diseases of unknown etiology. Finally, as a minimal means of best practice, the possibility that MAP is a zoonotic pathogen should prompt governments around the world to promote public health measures for more appropriate management of JD and MAP spreading in food and the environment.

[0159] Example 2: Antibody biomarkers reliably predict CD when compared to healthy donors In the previous example, all donors were recruited from a gastroenterology facility. Therefore, regardless of the diagnosis of one or more inflammatory bowel diseases, the overall health status of the donors was not very good. To better understand the predictive power of the Hsp65 antibody as a biomarker for CD, samples from 61 CD donors in Example 1 were compared with samples from 288 healthy Red Cross blood donors (controls). As shown in Figure 14A, there was a large significant difference in the levels of Hsp65 antibody between CD donors and controls (p-value < 0.00001). Furthermore, a C statistic of 0.9912341 was obtained by logistic regression (Figure 14B). The range of this measurement is 0.5 - 1.0, and a value of 1.0 indicates that the model perfectly discriminates between them within the group, suggesting that the Hsp65 antibody is an excellent predictor of CD. Additionally, the area under the receiver operating characteristic (ROC) curve (AUC) was 0.99123 (Figure 14C; p-value < 0.00001). The ROC summarizes the performance of the model by evaluating the trade-off between the true positive rate (sensitivity) and the false positive rate (1 - specificity), and a maximum value of 1.0 indicates a perfect prediction, again showing that the logistic regression model accurately predicts the classification of CD vs. non-CD based on the Hsp65 antibody signal. Comparison. Finally, an analysis showing 51 true positives (TP; predicted CD, actual CD) and 281 true negatives (TN; predicted non-CD; actual non-CD) suggested the following point estimates (95% Cl): sensitivity: 0.84 (0.72, 0.92), specificity: 0.98 (0.95, 0.99), positive predictive value: 0.88 (0.77, 0.95), and negative predictive value: 0.97 (0.94, 0.98). Therefore, the Hsp65 antibody functions as a robust marker for predicting CD.

[0160] The predictive power of the Hsp65 antibody alone appears robust, but Pinsky and Zhu showed that combining positively correlated primary markers with negatively correlated markers always increased the AUC and thus improved the predictive power of the resulting biomarker panel (Biomarker Insights, 2011:6 83-93). Thus, without being bound by any particular theory, it is believed that combining markers positively correlated with CD, such as the Hsp65 antibody, with markers negatively correlated with CD, such as the PknG antibody described in Example 1 above, can further enhance the ability to predict MAP infection and related CD. Additionally, MAP-specific biomarkers, such as antibodies against MAP lipopentapeptide (L5P), would be useful for detecting the presence (active or remote) of MAP infection, regardless of the diagnosed disease.

[0161] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Although the invention has been disclosed with reference to particular embodiments, it is apparent that other embodiments and modifications of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent modifications.

Claims

1. A kit for use in diagnosing one or more autoimmune diseases or disorders, the kit comprising: a) a first assay for detecting a biomarker that positively correlates with the autoimmune disease or disorder, wherein the biomarker of the first assay is one or more selected from the group consisting of an antibody specific for heat shock protein 65 (Hsp65), Hsp65, and a nucleic acid encoding Hsp65; and b) a second assay for detecting a biomarker that negatively correlates with the autoimmune disease or disorder, wherein the biomarker of the second assay is one or more selected from the group consisting of an antibody specific for protein kinase G (PknG), PknG, and a nucleic acid encoding PknG; c) a third assay for detecting a biomarker associated with MAP infection, wherein the biomarker of the third assay is one or more selected from the group consisting of an antibody specific for Mycobacterium avium subspecies paratuberculosis (MAP) lipopentapeptide (L5P), L5P, and a nucleic acid encoding L5P; comprising: The kit, wherein the autoimmune disease or disorder is Crohn's disease (CD) and / or ulcerative colitis (UC).

2. The kit according to claim 1, wherein the first assay comprises: a) a first surface area; b) the Hsp65 or an antigenic fragment thereof, wherein the Hsp65 or an antigenic fragment thereof is bound to the first surface area; and c) a diagnostic device comprising a solution containing a labeled antibody against the antibody specific for Hsp65.

3. The second assay comprises: a) a first surface area; b) the PknG or an antigenic fragment thereof, wherein the PknG or an antigenic fragment thereof is bound to the first surface area; and c) a diagnostic device comprising a solution containing a labeled antibody against the antibody specific for PknG, the kit according to claim 1.

4. The third assay comprises: a) a first surface area; b) the L5P or an antigenic fragment thereof, wherein the L5P or an antigenic fragment thereof is bound to the first surface area; and c) a diagnostic device comprising a solution containing a labeled antibody against the antibody specific for L5P, the kit according to claim 1.

5. A method for diagnosing one or more autoimmune diseases or disorders in a subject, the method comprising the following steps: a) detecting MAP infection in the subject; b) detecting a first biomarker that positively correlates with said autoimmune disease or disorder, wherein said first biomarker is selected from the group consisting of an antibody specific for Hsp65, Hsp65, and a nucleic acid encoding Hsp65; c) detecting a second biomarker that negatively correlates with said autoimmune disease or disorder, wherein said second biomarker is selected from the group consisting of an antibody specific for protein kinase G (PknG), PknG, and a nucleic acid encoding PknG; and d) the detection of said MAP infection, said first biomarker, and said second biomarker indicates that the subject has said autoimmune disease or disorder, comprising: wherein said autoimmune disease or disorder is Crohn's disease (CD) and / or ulcerative colitis (UC). **Claim 6** The method according to claim 5, wherein said detection of MAP infection further comprises measuring one or more selected from the group consisting of an antibody specific for L5P, L5P, and a nucleic acid encoding L5P. **Claim 7** A method for selecting a subject diagnosed with one or more autoimmune diseases or disorders to be treated with one or more antibiotics, said method comprising the following steps: a) detecting MAP infection in said subject; b) detecting a first biomarker that positively correlates with said autoimmune disease or disorder, wherein said first biomarker is selected from the group consisting of an antibody specific for Hsp65, Hsp65, and a nucleic acid encoding Hsp65; c) detecting a second biomarker that negatively correlates with said autoimmune disease or disorder, wherein said second biomarker is selected from the group consisting of an antibody specific for protein kinase G (PknG), PknG, and a nucleic acid encoding PknG; d) the detection of said MAP infection, said first biomarker, and said second biomarker indicates that the subject has said autoimmune disease or disorder; e) detecting the presence of MAP in an additional clinical assay; and f) the indication that the subject has said autoimmune disease or disorder and the detection of MAP in said clinical assay indicates that treatment of the subject with one or more antibiotics specific for MAP infection is recommended, comprising: A method wherein the autoimmune disease or disorder is Crohn's disease (CD) and / or ulcerative colitis (UC). **Claim 8** The method according to claim 7, further comprising measuring one or more selected from the group consisting of an antibody specific for L5P, L5P, and a nucleic acid encoding L5P for detection of the MAP infection. **Claim 9** The method according to claim 7, wherein the clinical assay is one or more selected from the group consisting of a MAP phage amplification assay, a Pozzato culture assay, a TiKa culture assay, and a mycobacteria growth indicator tube (MGIT) culture assay.

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