Method for detecting or monitoring the development of a chronic autoimmune disease by immunological assay

Abstract

The subject of the invention is an ex vivo method for detecting or monitoring the development of a chronic autoimmune disease, in a sample of human or animal biological fluid, by immunological assay of the presence of antibodies in the sample, including at least:- one or more antibodies directed against at least one enterobacterium, and- one or more antibodies directed against a product resulting from lipoperoxidation and / or one or more antibodies directed against a nitrated or nitrosylated product. The invention also relates to a kit for implementing such a method

Description

Title of the invention: Method for detecting or monitoring the development of a chronic autoimmune disease by immunological assay

[0001] The invention relates to the ex vivo detection and monitoring of the development of chronic autoimmune diseases in humans or animals.

[0002] Autoimmune diseases are pathologies that result from a dysfunction of the immune system leading it to attack the constituents of the body.

[0003] In addition to having the characteristic of being autoimmune, proliferative and / or degenerative processes can develop during the course of the disease. We then speak of co-morbidity which corresponds to the result of the association between autoimmune and degenerative and / or proliferative disease.

[0004] Therapeutic solutions provided depend on the type of autoimmune disease and often involve drugs that suppress the activity of the immune system. These drugs also have very limited or no effectiveness, are generally toxic and have significant side effects.

[0005] Furthermore, treatments are often ineffective in the very common cases where autoimmune diseases are diagnosed too late to be able to stop or slow down the progression of the disease in time. Indeed, there is no satisfactory diagnostic test. Several blood tests are generally carried out to look for a possible autoimmune disease. It is generally not known what triggers autoimmune diseases and the symptoms vary depending on the disorder that develops and the part of the body that is affected.

[0006] Furthermore, since these pathologies are chronic, they require regular monitoring of the progress of the disease and there is currently no simple and reliable solution that allows such monitoring.

[0007] The objective of the invention is to overcome the drawbacks of the prior art by proposing a method for both detecting and monitoring the development of a chronic autoimmune disease in humans or animals, which is simple to implement, minimally invasive, rapid, effective and reliable.

[0008] To this end, the invention relates to an ex vivo method for detecting or monitoring the development of a chronic autoimmune disease, in a sample of human or animal biological fluid, preferably of human or animal plasma and / or serum, by immunological assay of the presence of antibodies in the sample of which at least: - one or more antibodies directed against at least one enterobacteria, i.e. against at least one constituent of at least one enterobacterium, and - one or more antibodies directed against a product resulting from lipoperoxidation and / or one or more antibodies directed against a nitrated or nitrosylated product.

[0009] The method may also comprise immunoassaying the presence in the sample of: - at least one antibody directed against a tryptophan oxidation product, and / or - at least one antibody directed against a nitrated or nitrosylated product, and / or - at least one antibody directed against Mycobacterium tuberculosis.

[0010] Advantageously, this method is simple to implement since it is carried out on a sample of biological fluid and it uses conventional immunological assay techniques. The immunological assay method mimics ex vivo what happens in vivo where antigens are bound to immunoglobulins (antigens).

[0011] The method according to the invention can be implemented using a kit which constitutes another object of the invention.

[0012] Other characteristics and advantages of the invention will emerge from the detailed description which follows.

[0013] Definitions

[0014] For the purposes of the invention, the term "circulating antibodies" means antibodies found in human or animal biological fluids, in particular in the blood, serum and / or plasma of human beings or animals.

[0015] For the purposes of the invention, the term “reagent blank” means a solution or mixture containing all of the reagents used during the method according to the invention, but which does not contain the sample to be analyzed. It allows a basic correction to be made to the assay results. It defines the background noise of the assay method.

[0016] For the purposes of the invention, the term "biological fluid" means fluid from the body of a human being or an animal, in particular blood, serum and / or plasma, cerebrospinal fluid, pleural and intraperitoneal fluids, intra-articular fluids, saliva or urine.

[0017] By "neo-antigen" in the sense of the invention is meant the result of a covalent bond between a radical compound (NO, NO2, and...) or resulting from lipoperoxidation (Malondialdehyde, Azelaic Acid, etc.) or resulting from the hyperproduction of metabolic compounds (tryptophan derivatives) and an endogenous cellular or tissue constituent. A neo-antigen is also the result of the unmasking of a cellular constituent (Fatty acid, Phosphatidyl inositol, etc.) not physiologically accessible to the immune system, which, following active inflammatory processes, is exposed to the immune system.

[0018] By "product resulting from lipoperoxidation" within the meaning of the invention is meant a product which results from the peroxidation of lipids, i.e. the oxidation of unsaturated lipids or saturated, either by radical species of oxygen and nitrogen, or catalyzed by enzymes. Lipid peroxidation leads to their degradation into products known as lipopreoxidation products.

[0019] For the purposes of the invention, the term "nitrated or nitrosylated product" means neoantigens resulting from hyperproduction of NO and / or peroxynitrite. This hyperproduction results from the activation of inducible NO synthase mainly in the cells of the innate immune system by bacteria, viruses, pollutants, etc.

[0020] For the purposes of the invention, the term "tryptophan oxidation product" means a metabolite resulting from the degradation of tryptophan, after the enzymatic activation of the IDO (Indolamine Dioxygenase) and / or TDO (Tryptophan Dioxygenase) pathways. This enzymatic activation takes place in mono-macrophage cells under the action of bacterial, viral, mycotic, pollutant, cytokine and / or proinflammatory constituents.

[0021] By “bacterial constituent” is meant a degradation product of bacteria which results from bacterial lysis. Detailed description of the invention

[0022] The subject of the invention is therefore an ex vivo method for detecting or monitoring the development of a chronic autoimmune disease.

[0023] The disease may be any chronic autoimmune disease, and in particular it may be a disease chosen from multiple sclerosis, polyarthritis and rheumatic pelvis-pondylitis.

[0024] The method is carried out ex vivo, outside the human or animal body, in a sample of human or animal biological fluid, preferably human or animal serum or plasma, which has been taken before the implementation of the method. The sample of biological fluid is a sample which has been taken and stored according to the usual standards known to those skilled in the art.

[0025] The method according to the invention is carried out by immunological assay of the presence in the sample of circulating antibodies directed against antigens specific to chronic autoimmune diseases.

[0026] The method may be any type of immunological assay method. In particular, it may be an Elisa assay, strip tests, tests carried out using magnetic beads or even a Western Blot test. The implementation of these tests may be carried out according to the knowledge of a person skilled in the art.

[0027] In the implementation of the method according to the invention: - The antigens used for implementing the method are preferably antigens synthesized ex vivo. In the case where the antigens are bacteria, they can be obtained from bacterial cultures, these bacterial cultures being preferentially identified by an automated microbial identification system such as Vitek®. - If the antigens used are not bacteria, they are preferentially coupled (conjugated) to a protein, such as Bovine Serum Albumin for example, to facilitate the fixation of the antigen on the support (plate, strip, etc. depending on the assay method); thus an antibody directed against an antigen within the meaning of the present invention can mean (excluding bacteria) an antibody directed against a conjugated antigen whether the expression "conjugated" after the antigen is indicated or not. - The secondary antibodies, called anti-isotypes, used are manufactured according to conventional methods known to those skilled in the art. In particular, they can be obtained as follows: * purification of human antibodies (immunoglobulins) from control sera * these human immunoglobulins are purified by chromatography to separate the different isotopes A, M and G for humans, A, M, G and E for animals, * each electrophoresis peak (A, M, G or A, M, G, E) is injected into naive animals (rats, rabbits for example; non-immunized animals) * after three or four immunizations, the response is optimal and animal sera are collected. * these sera are purified; we thus obtain immunoglobulins (Ig) either anti-IgA, or anti-IgM, or anti-IgG; or anti-IgE (for animals only), * an enzyme such as peroxidase or phosphatase can then be bound to these purified immunoglobulins. * excess reagent is removed by chromatography.

[0028] The method according to the invention preferably comprises an enzymatic immunoassay based on the Elisa method. The immunological assay method may comprise the following steps: - the antigens against which the antibodies that we seek to detect in the sample are directed are adsorbed and dried on micro-titration plates; this step consists of sensitizing micro-titration plates with the antigen(s) against which the antibodies whose presence we seek to detect in the sample are directed. The plates are then either used directly for implementing the process, or dried and stored in a dry atmosphere and away from light. - the sample to be tested is then preferably diluted; this dilution can be between 150 and 1000 times; - also, preferably, test biological fluids, called internal standards, which are biological fluids from healthy subjects, preferably sera or plasma from healthy subjects (representative population and matched with that of patients at less for sex and age), are used and preferably diluted; this dilution can be between 150 and 1000 times, - the sample, a reactive blank and preferably also the internal standards, are distributed, preferably in duplicate, in wells of at least one sensitized micro-titration plate, - preferably the plate(s) are incubated; according to a particularly suitable embodiment they are incubated for between 1h30 and 2h00 at a temperature of 35 to 39°C, - preferably the plate(s) are washed, i.e. rinsed, preferably several times, with the aim of eliminating all non-specific proteins and immunoglobulins; - then add anti-human or animal immunoglobulin antibodies called secondary antibodies, directed against isotypes A, M or G for humans, A, M, G or E in animals; these secondary antibodies are antibodies directed against the immunoglobulins bound to the antigens present in the wells of the microtitration plates; the secondary antibodies are preferentially coupled to an enzyme, and preferably to peroxidase or alkaline phosphatase or biotinylated, to allow a colorimetric reaction to be carried out, - preferably the plates are incubated again for 1 to 2 hours at a temperature between 35 and 39°C, - preferably the plates are then washed again, that is to say several rinses are carried out in order to eliminate what has not been specifically recognized, - preferably, an enzyme substrate is then applied (H202 for peroxidase or for phosphatase) with a chromogen, this step aims to visualize the immunological reactions. The chromogen can be, for example, tetramethylbenzedine (TMB) or DAB Diaminobenzedine; a colorimetric reaction appears; this is all the more intense as there are human or animal immunoglobulins fixed on the antigens present in the wells; - possibly incubate again for 10 to 30 minutes at a temperature between 18 and 20°C; the reaction is then stopped, preferably in acid solution; - the optical density of each well of the micro-titration plates is then read, preferably using a spectrophotometer; the reading is preferably done at 450 nm with an alpha correction at 650 nm; these optical densities are then preferably recorded in software.

[0029] A Z ratio is then established for each antigen tested, relative to the optical density of the internal standards: Z = (OD of the sample to be tested - mean OD of the internal standards) / Control standard deviation of the internal standards. The test is positive (presence of the antibody sought in the sample, therefore disease confirmed) if Z is greater than or equal to +2 or less than or equal to -2: - if Z is less than or equal to -2, there is negativity compared to the controls (internal standards): there has been immune activation but the antibodies are now complexed with their antigens (immune complexes). - if Z is greater than or equal to +2, there is positivity compared to the controls (internal standards): there has been immune activation. If Z is between -2 and +2, the values are said to be normal because they correspond to the Gaussian dispersion of the control population: the antibody sought is not present in the sample.

[0030] It is thus possible to detect whether the human being or the animal to whom the tested biological fluid belongs is suffering from an autoimmune disease or not.

[0031] In addition, the method according to the invention also makes it possible to monitor the progression of the disease. The variation in the Z value for the antibody(ies) sought makes it possible to verify the variation, the progression of the disease: - the closer Z gets to the interval -2 to +2, the more the antibody level decreases and the disease progresses towards remission. This allows us to verify that the treatment is active on the pathogenic processes; - as long as Z is greater than +2 or less than -2 the disease is still active.

[0032] The dosage of one or more specific antibodies also makes it possible to propose a suitable treatment depending on the antibody(ies) tested present in the sample.

[0033] According to a particular embodiment, the method according to the invention comprises at least the implementation of the following steps: - manufacture at least one microtitration plate sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, or use at least one microtitration plate already sensitized with these antigens, - distribute the same quantity of internal standards, sample and reagent blank on the plate, - add the secondary antibody(ies) coupled to an enzyme, - add an enzyme substrate and a chromogen, wait for the wells to color, - stop the coloring reaction, - read the optical density of the wells using a spectrophotometer at an appropriate wavelength.

[0034] In particular, a particularly suitable embodiment of the invention is a method which comprises at least the implementation of the following steps: - manufacture at least one microtitration plate sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, or use at least one microtitration plate already sensitized with these antibodies, - dilute the sample to be tested and internal standards, - distribute in duplicate on the plate the same quantity of diluted internal standards, diluted sample and reagent blank, - incubate, - wash, - add the secondary antibody(ies) coupled to peroxidase or alkaline phosphatase or biotinylated, - incubate, - wash, - add a substrate and a chromogen, - stop the reaction in acid solution - read at 450 nm with a correction alpha at 650 nm.

[0035] The antibodies sought in the context of the method according to the invention, whatever the antigens against which they are directed, can be IgM (immunoglobulins of isotype M), IgA (immunoglobulins of isotype A), or IgG (immunoglobulins of isotype G) and in animals only IgE (immunoglobulins of isotype E): - IgA antibodies: are the result of mucosal immune activation (intestinal, ENT, skin, bladder), - IgM antibodies: are the result of an activation of the current immune system, - IgG antibodies: are the result of an activation of the old immune system (memory immunity), - IgE antibodies (in animals only) are the result of stimulation of mast cells which release histamine, that is to say they are the result of contact with allergenic antigens.

[0036] The method according to the invention is therefore carried out by immunological assay of the presence of antibodies in the sample, including at least: - one or more antibodies directed against at least one enterobacteria, and - one or more antibodies directed against a product resulting from lipoperoxidation and / or one or more antibodies directed against a nitrated or nitrosylated product.

[0037] Thus the method according to the invention is carried out by immunological assay of the presence of antibodies in the sample, including at least: - one or more antibodies directed against at least one enterobacterium, and one or more antibodies directed against a product resulting from lipoperoxidation, or - one or more antibodies directed against at least one enterobacterium, and one or more antibodies directed against a nitrated or nitrosylated product, or - one or more antibodies directed against at least one enterobacterium, and one or more antibodies directed against a product resulting from lipoperoxidation and one or more antibodies directed against a nitrated or nitrosylated product.

[0038] Indeed, according to the invention, if these antibodies are present in the sample of biological fluid tested, in particular in the serum and / or plasma tested, that is to say if the test is positive for at least these antibodies, then this means that the person is suffering from a chronic autoimmune disease.

[0039] Preferably, the method according to the invention comprises the immunological assay of the presence of at least one antibody directed against an endobacterium (against a bacterial constituent of an endobacterium) chosen from the following antibodies: - antibody(ies) directed against the bacterium Pseudomonas putida, - antibody(ies) directed against the bacterium Hafnia alveL - antibody(ies) directed against the bacterium Pseudomonas aeruginosa, - antibody(ies) directed against the bacterium Pseudomonas pneumonae, - antibody(ies) directed against the bacterium Morganella morganii, - antibody(ies) directed against the bacterium Proteus mirabilis - antibody(ies) directed against the bacterium Citrobacter koserii.

[0040] Enterobacteria, and in particular Gram-negative enterobacteria, have a wall with a specific three-layer structure. This wall is made up, from the outside to the inside, of: an external membrane, a thin layer of peptidoglycan, a periplasmic space which surrounds the cytoplasmic membrane. The external membrane is made up of a double lipid layer in which lipopolysaccharide molecules are included. The peptidoglycan constitutes a rigid layer, thinner and looser than in Gram-positive bacteria. It is composed of linear chains and polysaccharides linked together by peptides. The periplasmic space surrounds the cytoplasmic membrane.

[0041] These bacteria are non-pathogenic and can be present on the mucous membranes. However, if they pass through the mucous membranes they can cause chronic pathologies, in particular chronic autoimmune pathologies. Their transmucosal passage causes: - activation of the innate immune system, - activation of the adaptive immune system, - non-specific activation of self-reactive clones (super antigens), - direct toxicity (lipopolysaccharides, endotoxins) - activation of enzymatic systems: inducible NO synthase and indoleamine 2,3-dioxygenase (IDO-1).

[0042] According to the invention, the presence in the biological fluid tested, in particular in the serum and / or in the plasma of one or more antibodies directed against these bacteria signifies mucosal hyper permeability, which contributes to the development of autoimmune diseases.

[0043] Preferably, the method according to the invention comprises the immunological assay of the presence in the sample of: - IgA (and / or IgE for animals) directed against the bacterium Pseudomonas putida, and / or - IgA (and / or IgE for animals) directed against the bacterium Hafnia alvei, and / or - IgA (and / or IgE for animals) directed against the bacterium Pseudomonas ae-ruginosa, and / or - IgA (and / or IgE for animals) directed against the bacterium Pseudomonas pneumonae, and / or - IgA (and / or IgE for animals) directed against the bacteria Morganella morganii, and / or - IgA (and / or IgE for animals) directed against the bacterium Proteus mirabilis, and / or - IgA (and / or IgE for animals) directed against the bacteria Citrobacter koserii. and / or.

[0044] Preferably, the method according to the invention comprises the immunological assay of the presence of at least one antibody directed against a lipoperoxidation product chosen from the following antibodies: - antibody(ies) directed against palmitic acid, - antibody(ies) directed against myristic acid, - antibody(ies) directed against oleic acid, - antibody(ies) directed against malondialdehyde - antibody(ies) directed against acetylcholine - antibody(ies) directed against azelaic acid, - antibody(ies) directed against a phospholipid.

[0045] NO is the origin of many toxic radical species called reactive oxygen species (ROS). Among these ROS, we can notably cite lipid peroxides and their decomposition products, aldehydes, but also signulet oxygen and peroxynitrites. ROS manifest their toxicity when they exceed the possibilities of cellular defense. These ROS have an aggressive role. They attack phospholipid membranes, proteins and DNA. When they attack phospholipid membranes, the radical attack can modify the lipid compounds constituting the cell and thus alter the messages coming from the environment to the interior of the cell, leading to a dysfunction which may be irreversible.

[0046] This lipoperoxidation is a cascade reaction that starts with the oxidation of the fatty acid chains constituting the phospholipids, preferentially unsaturated fatty acids having carbon-carbon double bonds. These unsaturated fatty acids best ensure the fluidity of the membranes. They become more unstable and more fragile. Lipoperoxidation is made possible, on the one hand, by the existence of these double bonds of polyunsaturated fatty acids which facilitate the delocalization of the free electron, and, on the other hand, by the presence of molecular oxygen which will easily pair one of its electrons with the delocalized free electron.

[0047] There are several types of lipoperoxidation: - a first radical cascade, very active, which will generate the production of free radicals, - the reaction of the superoxide radical O2 with NO which leads to the formation of very aggressive peroxynitrites which will cause the degradation of fatty acids into hydroperoxides then into malondialdehyde (small molecule, extremely reactive with amino residues, and not normally existing in the body). - another, milder type of lipoperoxidation causes, by hydrolysis, a breakage of the double bonds of polyunsaturated fatty acids with the formation of diacids, one of the main compounds of which is azelaic acid.

[0048] The presence of products resulting from lipoperoxidation in humans or animals causes a disruption of cellular and membrane constituents leading to the formation of neo-antigens and consequently immune and autoimmune activation at the origin of the autoimmune disease.

[0049] Preferably, the method according to the invention comprises the immunological assay of the presence in the sample of: - IgA (and / or IgE for animals) directed against myristic acid, and / or - IgA (and / or IgE for animals) directed against oleic acid, and / or - IgM (and / or IgE for animals) directed against malondialdehyde, and / or - IgM (and / or IgE for animals) directed against acetylcholine. - IgM (and / or IgE for animals) directed against azelaic acid, and / or - IgA and / or IgM and / or IgG (and / or IgE for animals) directed against a phospholipid. - IgM (and / or IgE for animals) directed against palmitic acid.

[0050] Preferably, the method according to the invention comprises the immunological assay of the presence of at least one antibody directed against a nitrated or nitrosylated product chosen from the following antibodies: - antibodies directed against NO-Cysteine, - antibodies directed against NO-asparagine, - antibodies directed against NO-histidine, - antibodies directed against NO-Tyrosine, - antibodies directed against NO2-Tyrosine, - antibodies directed against NO-citrulline, - antibodies directed against NO-arginine, - antibodies directed against NO-Tryptophan, - antibodies directed against NO-methionine, - antibodies directed against NO-histamine, - antibodies directed against NO-phenylalanine, - antibodies directed against NO-bovine serum albumin - antibodies directed against NO-proline.

[0051] NO (nitrogen monoxide) has a single electron which gives it a very rapid reactivity. It depends on its ability to share its electron with other radicals or metals. In biological environments, the synthesis of NO from L-arginine passes through an intermediate, hydroxy-L-arginine (HOA) under the action of a NOSynthase (NOS). Oxidation at the terminal nitrogen of the guanidine function leads to the formation of NO. Two types of NOS are present in most vertebrates: constitutive NOS (cNOS) and inducible NOS (iNOS).

[0052] Nitro or nitrosylated products are neoantigens resulting from hyper-production of NO and / or peroxynitrite following activation of inducible NO synthase mainly in cells of the innate immune system by bacteria, viruses, pollutants, etc. Excess NO and / or peroxynitrite leads to their binding to endogenous proteins which become immunogenic. Endogenous proteins thus modified in their structure lead to immune and autoimmune activation and consequently to an autoimmune disease.

[0053] Preferably, the method according to the invention comprises the immunological assay of the presence in the sample of: - IgA (and / or IgE for animals) directed against NO-Cysteine, and / or - IgA and / or IgG (and / or IgE for animals) directed against NO-asparagine, and / or - IgA and / or IgM (and / or IgE for animals) directed against NO-histidine - IgA and / or IgM (and / or IgE for animals) directed against NO-Tyrosine, and / or - IgA and / or IgM (and / or IgE for animals) directed against NO2-Tyrosine, and / or - IgA (and / or IgE for animals) directed against NO-citrulline, and / or - IgA (and / or IgE for animals) directed against NO-Tryptophan, and / or - IgA (and / or IgE for animals) directed against NO-methionine, and / or - IgA (and / or IgE for animals) directed against NO-histamine, and / or - IgA and / or IgG and / or IgM (and / or IgE for animals) directed against NO-phenylalanine, and / or - IgA and / or IgG (and / or IgE for animals) directed against NO-bovine serum albumin. - IgA and / or IgG (and / or IgE for animals) directed against NO-proline.

[0054] The method according to the invention, in addition (either at the same time or in a separate test) to the immunological assay of the presence in the sample of one or more antibodies directed against at least one enterobacterium and one or more antibodies directed against a tryptophan oxidation product, may also comprise the immunological assay of the presence in the sample of one or more other antibodies. This may preferably be: - at least one antibody directed against a tryptophan oxidation product, and / or - at least one antibody directed against Mycobacterium tuberculosis.

[0055] Preferably, the method according to the invention comprises the immunological assay of the presence of at least one antibody directed against a tryptophan oxidation product chosen from the following antibodies: - antibodies directed against kynuemic acid, - antibodies directed against 3-hydroxykynurenine, - antibodies directed against quinolinic acid, - antibody(ies) directed against quinaldic acid, - antibody(ies) directed against xanthurenic acid, - antibody(ies) directed against anthranilic acid, - antibody(ies) directed against 3-hydroxyanthranilic acid.

[0056] L-tryptophan is metabolized into a large number of derivative products: neurotransmitters, neurohormonals active at the level of the nervous system. The best known are serotonin and melatonin via the tryptophan hydroxylase pathway. The IDO (Indolamine droxygenase) pathway also metabolizes tryptophan. It is an inducible enzymatic pathway. It is present in microbial neuronal cells and cells of the monocytomacrophage lineage. There are numerous molecules that activate the IDO pathway. The main ones are TNF-α, interleukins IL-12 and IL-18, interferon-γ, and microorganisms and their derivatives.

[0057] IDO degrades L-tryptophan to N-formylkynurenine. A complex enzyme system then converts N-formylkynurenine to L-kynurenine. The latter is metabolized into various compounds by numerous enzymes. Kynureninase converts L-kynurenine to anthranilic acid, which gives 3-hydroxyanthranilic acid. This acid can then be converted to 3-hydroxyanthranilic acid. L-kynurenine is also degraded to 3-hydroxykynurenine by kynurenine 3-hydroxylase. The hydroxylated form is converted to 3-hydroxyanthranilic acid by kynureninase. This latter compound is metabolized by 3-hydroxyanthranilic acid oxygenase into an intermediate product that originates in three distinct pathways. It gives either picolinic acid by picolinic carboxylase or quinolinic acid. L-kynurenine is converted to kynureamine by decarboxylases, or to kynurenic acid by kynurenine aminotransferase. This kynurenic acid is the origin of the synthesis of quinaldic acid.

[0058] The IDO pathway limits the pool of extracellular L-tryptophan, which promotes the replication of a large number of cells, and the oxidation products of tryptophan, i.e., the products resulting from the activation of the IDO pathway, induce either cellular apoptosis, immunotoxicity, or cellular and tissue protection. Their hyperproduction causes the formation of neoantigens and consequently an autoimmune disease.

[0059] Preferably, the method according to the invention comprises the immunological assay of the presence in the sample of: - IgA (and / or IgE for animals) directed against kynuemic acid, and / or - IgA (and / or IgE for animals) directed against 3-hydroxykynurenine, and / or - IgA (and / or IgE for animals) directed against quinolinic acid, and / or - IgA (and / or IgE for animals) directed against quinaldic acid, - IgG (and / or IgE for animals) directed against xanthurenic acid, and / or - IgA (and / or IgE for animals) directed against anthranilic acid, and / or - IgA (and / or IgE for animals) directed against 3-hydroxyanthranilic acid.

[0060] According to the invention, the presence in the biological fluid of at least one antibody directed against a tryptophan oxidation product, in particular IgA and / or Ig M and / or IgG (and / or IgE for animals), and the presence in the biological fluid of at least one antibody directed against an enterobacterium, in particular IgA and / or Ig M and / or IgG (and / or IgE for animals), necessarily means that the person or animal concerned is affected by an autoimmune disease.

[0061] The method according to the invention may also comprise the immunological assay of the presence of at least one antibody directed against at least one antibody directed against Mycobacterium tuberculosis.

[0062] Mycobacterium tuberculosis is a Gram-positive pathogenic bacterium responsible for tuberculosis. It also occurs in patients with other diseases such as people or animals with certain autoimmune diseases. It does not produce uncontrolled toxins in a favorable environment, but these membrane constituents are released and have a toxic effect and owes its pathogenic power to its ability to multiply. The lysis of the bacteria releases antigenic constituents which trigger an immune reaction inducing a state of hypersensitivity.

[0063] Their presence in humans or animals causes the release of mem constituents branes (mycolic acid), some of which have toxic activity and activate the innate immune system by binding to specific receptors, leading to immune and autoimmune activation and consequently autoimmune disease.

[0064] According to a particular embodiment, the method according to the invention is specifically intended for the detection or monitoring of the development of multiple sclerosis. In this case, the method is preferably carried out by immunological assay of the presence of antibodies in the sample, including at least: - one or more of the following antibodies: * antibodies directed against the bacterium Pseudomonas putida, * antibodies directed against the bacterium Hafnia alveL * antibodies directed against the bacterium Pseudomonas aeruginosa, * antibodies directed against the bacterium Pseudomonas pneumonae, * antibodies directed against the bacteria Morganella morganii, * antibodies directed against the bacterium Proteus mirabilis * antibodies directed against the bacterium Citrobacter koserii - and one or more of the following antibodies: * antibodies directed against palmitic acid, * antibodies directed against oleic acid, * antibodies directed against malondialdehyde * antibodies directed against acetylcholine * antibodies directed against azelaic acid, * antibody(ies) directed against a phospholipid * antibodies directed against NO-Cysteine, * antibodies directed against NO-asparagine, * antibodies directed against NO-histidine, * antibodies directed against NO-Tyrosine, * antibodies directed against NO2-Tyrosine, * antibodies directed against NO-citrulline, * antibodies directed against NO-arginine, * antibodies directed against NO-Tryptophan, * antibodies directed against NO-methionine, * antibodies directed against NO-histamine, * antibodies directed against NO-phenylalanine, * antibodies directed against NO-bovine serum albumin * antibodies directed against NO-proline. The method in this case may also include immunoassaying for the presence of one or more of the following antibodies: - antibodies directed against kynuemic acid, - antibodies directed against 3-hydroxykynurenine, - antibodies directed against quinolinic acid, - antibodies directed against quinaldic acid, - antibodies directed against xanthurenic acid, - antibodies directed against anthranilic acid, - antibodies directed against 3-hydroxyanthranilic acid

[0065] According to a particular embodiment, the method according to the invention is specifically intended for the detection or monitoring of the development of polyarthritis. In this case, the method is preferably carried out by immunological assay of the presence of antibodies in the sample, including at least: - one or more of the following antibodies: * antibodies directed against the bacterium Pseudomonas putida, * antibodies directed against the bacterium Hafnia alvei, * antibodies directed against the bacterium Pseudomonas aeruginosa, * antibodies directed against the bacteria Morganella morganii, * antibodies directed against the bacterium Proteus mirabilis * antibodies directed against the bacterium Citrobacter koserii - and one or more of the following antibodies: * antibodies directed against palmitic acid, * antibodies directed against myristic acid, * antibodies directed against oleic acid, * antibodies directed against malondialdehyde * antibodies directed against acetylcholine * antibodies directed against azelaic acid, * antibody(ies) directed against a phospholipid * antibodies directed against NO-Cysteine, * antibodies directed against NO-asparagine, * antibodies directed against NO-histidine, * antibodies directed against NO-proline.

[0066] To implement the ex vivo method of detecting and / or monitoring the development of a disease, the invention also relates to diagnostic kits.

[0067] In particular, the subject of the invention is a kit for its use in the detection or monitoring of the development of a chronic autoimmune disease, in a sample of biological fluid, in particular in a sample of human or animal biological fluid comprising at least the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, namely preferably one or more antibodies chosen from: - antibodies directed against the bacterium Pseudomonas putida, - antibodies directed against the bacterium Hafnia alvei, - antibodies directed against the bacterium Pseudomonas aeruginosa, - antibodies directed against the bacterium Pseudomonas pneumonae, - antibodies directed against the bacteria Morganella morganii, - antibodies directed against the bacterium Proteus mirabilis - antibodies directed against the bacterium Citrobacter koserii - antibodies directed against palmitic acid, - antibodies directed against myristic acid, - antibodies directed against oleic acid, - antibodies directed against malondialdehyde - antibodies directed against acetylcholine - antibodies directed against azelaic acid, - antibodies directed against a phospholipid - antibodies directed against NO-Cysteine, - antibodies directed against NO-asparagine, - antibodies directed against NO-histidine, - antibodies directed against NO-Tyrosine, - antibodies directed against NO2-Tyrosine, - antibodies directed against NO-citrulline, - antibodies directed against NO-arginine, - antibodies directed against NO-Tryptophan, - antibodies directed against NO-methionine, - antibodies directed against NO-histamine, - antibodies directed against NO-phenylalanine, - antibodies directed against NO-bovine serum albumin - antibodies directed against NO-proline - antibodies directed against kynuernic acid, - antibodies directed against 3-hydroxykynurenine, - antibodies directed against quinolinic acid, - antibodies directed against quinaldic acid, - antibodies directed against xanthurenic acid, - antibodies directed against anthranilic acid, - antibodies directed against 3-hydroxyanthranilic acid - antibodies directed against Mycobacterium tuberculosis.

[0068] Preferably, the kit according to the invention comprises at least: - the antigen(s) against which the antibodies are directed whose presence in the sample is to be detected, preferably coupled to a protein when it is not bacteria, and a microtitration plate intended to be sens sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, or a microtitration plate already sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, or a strip or bar already sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, and - standards for assessing the quality of the test and calculating the z, and / or - buffers and solutions suitable for carrying out an Elisa test, and / or - the secondary antibody(ies) in solution, said secondary antibody(ies) corresponding to the primary antibodies whose presence in the sample is to be detected and with the same isotype, and / or - dilution and washing buffers, and / or - revealing stamps, and / or - a stopping solution.

[0069] The invention is now illustrated by examples and results of implementing methods according to the invention.

[0070] For each example, the protocol was as follows: - collection of the patient's plasma - implementation of the method according to the invention - summary of the results obtained in a table to visualize the detection of circulating antibodies and monitor their evolution. If the value is < -2 or > +2 the disease is developing and if it goes between -2 and +2 the disease is stabilizing or even regressing.

[0071] Each example makes it possible to make the link between the antibody level and the patient's state in their pathology.

[0072] Example 1: Results of the implementation of a method according to the invention on a person with Multiple Sclerosis under treatment

[0073] The results of the detection and evolution of circulating antibodies during the course of the disease are presented in Table 1. [Tables 1] Markers / Ig 30 / 08 / 2 016 29 / 12 / 2 016 27 / 03 / 2 017 08 / 08 / 2 017 13 / 01 / 2 018 14 / 05 / 2 018 17 / 09 / 2 018 anti-oleic acid conjugate / M 2.03 2.62 3.16 2.41 2.70 3.54 1.78 anti-malondialdehyde conjugate / M 0.79 0.99 0.94 0.72 2.31 0.93 0.97 anti-phospholipid conjugate / M 1.32 2.58 1.36 0.77 1.55 2.83 0.54 anti-pseudomonas ae-ruginosa / M 2.65 1.78 1.25 1.48 2.24 3.48 2.57 anti-pseudomonas ae-ruginosa / A 1.57 2.70 1.60 1.41 2.12 2.54 3.50 anti-mycobacterium tu-berculosis / A -0.82 -0.81 -2.25 -0.94 -0.61 -0.96 -1.28

[0074]

[0075] Example 2: Results of the implementation of a method according to the invention on a person with multiple sclerosis, under treatment

[0076] The results of the detection and evolution of circulating antibodies during the course of the disease are presented in Table 2.

[0077] [Tables 2] Markers / Ig 08 / 15 / 201 6 02 / 27 / 201 7 08 / 28 / 201 7 04 / 04 / 201 8 09 / 24 / 201 8 anti-hafnia alvei / M 1.12 2.69 2.82 -0.70 1.53 anti-pseudomonas ae-ruginosa / M 2.16 5.06 4.65 -0.01 2.99 anti-morganella morganii / M 1.75 5.07 3.91 -0.30 2.34 anti-pseudomonas putida / M 3.26 6.41 7.08 0.01 4.12 anti-citrobacter koseri / M 1.71 3.84 3.87 -1.03 2.12 anti-klebsiella pneumoniae / M 2.11 5.64 6.42 2.19 2.47 anti-pseudomonas ae-ruginosa / A 1.51 1.34 0.77 2.56 1.23 anti-pseudomonas putida / A 1.42 1.66 1.19 3.18 0.40 anti-klebsiella pneumoniae / A -0.27 5.93 0.72 7.60 3.33 anti-conjugated oleic acid / M 2.38 3.89 6.67 4.26 3.41 anti-conjugated malondialdehyde / M 1.47 5.17 3.87 3.83 1.41 anti-conjugated azelaic acid / M 2.99 4.65 3.64 2.32 1.12 anti-phospholipid conjugate / M 1.86 5.86 3.63 3.00 1.39 anti-NO-cysteine conjugate / M 0.57 4.70 2.64 2.92 1.32 anti-NO2-tyrosine conjugate / M 1.43 2.65 1.94 2.90 1.47 anti-NO-tryptophan conjugate / M 0.66 2.20 1.91 2.69 1.16 anti-NO-serum albumin / M 2.98 5.21 5.09 6.39 1.66 anti-mycobacterium tu-berculosis / G 3.03 3.00 4.78 3.91 2.99,

[0078]

[0079] Example 3: Results of the implementation of a method according to the invention on a person suffering from rheumatoid arthritis under treatment.

[0080] The results of the detection and evolution of circulating antibodies during the course of the disease are presented in Table 3.

[0081] [Tables3] Markers / Ig 07 / 01 / 2017 12 / 09 / 201 7 17 / 02 / 201 8 02 / 06 / 201 8 anti-pseudomonas aeruginosa / A 3.46 5.46 2.37 3.20 anti-pseudomonas putida / A -0.17 -0.85 -1.15 -1.12 anti-citrobacter koseri / A -0.38 -1.11 0.18 0.76 anti-oleic acid conjugate / M 12.04 10.21 12.02 16.56 anti-malondialdehyde conjugate / M 19.84 10.82 14.46 13.59 anti-azelaic acid conjugate / M 6.19 3.38 4.87 5.96 anti-phospholipid conjugate / M 9.60 11.55 12.49 12.95 anti-NO-cysteine conjugate / M 36.52 15.74 17.57 17.72 anti-NO2-tyrosine conjugate / M 3.45 2.92 2.87 3.05 anti-NO-tryptophan conjugate / M 27.15 13.00 35.52 20.01 anti-NO-serum albumin / M 41.31 23.70 26.08 36.13 anti-phospholipid conjugate / A 2.00 -0.77 -0.32 -0.45

[0082]

[0083] Example 4: Results of the implementation of a method according to the invention on a person suffering from rheumatoid arthritis, under treatment.

[0084] The results of the detection and evolution of circulating antibodies during the course of the disease are presented in Table 4.

[0085] [Tables 4] Marqueurs 04 / 07 / 201 6 17 / 10 / 201 6 23 / 01 / 201 7 09 / 05 / 201 7 06 / 09 / 201 7 anti-pseudomonas ae-ruginosa / M 1.73 2.44 2.09 1.14 2.06 anti-morganella morganii / M 3.08 2.78 2.07 2.41 4.18 anti-pseudomonas putida / M 2.46 2.07 3.50 2.56 2.18 anti-klebsiella pneumoniae / M 1.31 1.62 1.36 2.63 2.62 anti-hafnia alvei / A 2.90 2.70 3.29 4.72 3.82 anti-pseudomonas ae-ruginosa / A 9.12 5.79 6.09 4.19 7.64 anti-morganella morganii / A 5.22 9.57 6.16 9.53 15.25 anti-pseudomonas putida / A 9.48 6.33 4.47 8.37 4.72 anti-citrobacter koseri / A 2.16 2.57 4.01 2.92 5.75 anti-klebsiella pneumoniae / A 1.13 3.48 3.14 2.64 2.71 anti-acide oléique conjugué / M 3.64 -0.75 3.29 4.48 2.97 anti-malondialdéhyde conjugué / M 2.15 2.54 2.02 3.85 1.12 anti-acid azélaïque conjugué / M 3.01 2.66 5.10 5.35 2.69 anti-phospholipide conjugué / M 3.53 1.59 3.02 3.67 3.08 anti-NO-cystéine conjuguée / M -0.33 -0.05 1.47 2.11 -0.13 anti-NO-serum albumin / M 0.84 0.40 2.71 2.67 1.78 anti-mycobacterium tu-berculosis / A -0.79 0.86 0.64 0.03 2.09

Claims

1. Claims Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease, in a sample of human or animal biological fluid, by immunological assay of the presence of antibodies in the sample, including at least: - one or more antibodies directed against at least one enterobacteria, chosen from the following antibodies: * antibodies directed against the bacterium Pseudomonas putida, * antibodies directed against the bacterium Hafnia alvei, * antibodies directed against the bacterium Pseudomonas aeruginosa, * antibodies directed against the bacterium Pseudomonas pneumonae, * antibodies directed against the bacteria Morganella morganii, * antibodies directed against the bacterium Proteus mirabilis * antibodies directed against the bacteria Citrobacter koseriL and - one or more antibodies directed against a product resulting from lipoperoxidation and / or one or more antibodies directed against a nitrated or nitrosylated product, the antibody(ies) directed against a product resulting from lipoperoxidation being chosen from the following antibodies: * antibodies directed against palmitic acid, * antibodies directed against myristic acid, * antibodies directed against oleic acid, * antibodies directed against malondialdehyde * antibodies directed against acetylcholine * antibodies directed against azelaic acid, * antibody(ies) directed against a phospholipid, the antibody(ies) directed against a nitrated or nitrosylated product being chosen from the following antibodies: * antibodies directed against NO-Cysteine, * antibodies directed against NO-asparagine, * antibodies directed against NO-histidine, * antibodies directed against NO-Tyrosine, * antibodies directed against NO2-Tyrosine, * antibodies directed against NO-citrulline, * antibodies directed against NO-arginine, * antibodies directed against NO-Tryptophan, * antibodies directed against NO-methionine, * antibodies directed against NO-histamine, * antibodies directed against NO-phenylalanine, * antibodies directed against NO-bovine serum albumin * antibodies directed against NO-proline, and - one or more antibodies directed against a tryptophan oxidation product.

2. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to claim 1, characterized in that the sample is a sample of human or animal serum or plasma.

3. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that it also comprises the immunological assay in the sample of the presence of at least one antibody directed against a tryptophan oxidation product, the antibody(ies) directed against a tryptophan oxidation product being chosen from the following antibodies: - antibody(ies) directed against kynuemic acid, - antibody(ies) directed against 3-hydroxykynurenine, - antibody(ies) directed against quinolinic acid, - antibody(ies) directed against quinaldic acid, - antibody(ies) directed against xanthurenic acid, - antibody(ies) directed against anthranilic acid, - antibody(ies) directed against 3-hydroxyanthranilic acid.

4. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that it also comprises the immunological assay of the presence in the sample of at least one antibody directed against Mycobacterium tuberculosis.

5. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that the antibodies are immunoglobulins A and / or immunoglobulins G and / or immunoglobulins M and / or for animals only immunoglobulins E.

6. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that said disease is chosen from multiple sclerosis, polyarthritis and rheumatic pelvispondylitis.

7.

8. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that said disease is multiple sclerosis and in that the method is carried out by immunological assay of the presence of antibodies in the sample, including at least: - one or more of the following antibodies: * antibodies directed against the bacterium Pseudomonas putida, * antibodies directed against the bacterium Hafnia alvei, * antibodies directed against the bacterium Pseudomonas aeruginosa, * antibodies directed against the bacterium Pseudomonas pneumonae, * antibodies directed against the bacteria Morganella morganii, * antibodies directed against the bacterium Proteus mirabilis * antibodies directed against the bacterium Citrobacter koserii - and one or more of the following antibodies: * antibodies directed against palmitic acid, * antibodies directed against oleic acid, * antibodies directed against malondialdehyde * antibodies directed against acetylcholine * antibodies directed against azelaic acid, * antibody(ies) directed against a phospholipid * antibodies directed against NO-Cysteine, * antibodies directed against NO-asparagine, * antibodies directed against NO-histidine, * antibodies directed against NO-Tyrosine, * antibodies directed against NO2-Tyrosine, * antibodies directed against NO-citrulline, * antibodies directed against NO-arginine, * antibodies directed against NO-Tryptophan, * antibodies directed against NO-methionine, * antibodies directed against NO-histamine, * antibodies directed against NO-phenylalanine, * antibodies directed against NO-bovine serum albumin * antibodies directed against NO-proline. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to the preceding claim, characterized in that the method also comprises the immunological assay of the presence of one or more of the following antibodies: - antibody(ies) directed against kynuemic acid, - antibody(ies) directed against 3-hydroxykynurenine, - antibody(ies) directed against quinolinic acid, - antibody(ies) directed against quinaldic acid, - antibody(ies) directed against xanthurenic acid, - antibody(ies) directed against anthranilic acid, - antibody(ies) directed against 3-hydroxyanthranilic acid.

9. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of claims 1 to 6, characterized in that said disease is polyarthritis and in that the method is carried out by immunological assay of the presence of antibodies in the sample, including at least: - one or more of the following antibodies: * antibodies directed against the bacterium Pseudomonas putida, * antibodies directed against the bacterium Hafnia alvei, * antibodies directed against the bacterium Pseudomonas aeruginosa, * antibodies directed against the bacterium Morganella morganii, * antibodies directed against the bacterium Proteus mirabilis * antibodies directed against the bacterium Citrobacter koserii - and one or more of the following antibodies: * antibodies directed against palmitic acid, * antibodies directed against myristic acid, * antibodies directed against oleic acid,* antibodies to malondialdehyde * antibodies to acetylcholine * antibodies to azelaic acid, * antibodies to a phospholipid * antibodies to NO-cysteine, * antibodies to NO-asparagine, * antibodies to NO-histidine, * antibodies to NO-proline.,

10. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that the immunological assay is carried out by implementing an immunoenzymatic method.

11. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that it comprises at least the implementation of the following steps:

12.

13.

14. - manufacture at least one microtitration plate sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, or use at least one microtitration plate already sensitized with these antigens, - distribute the same quantity of internal standards, sample and reagent blank on the plate, - add the secondary antibody(ies) coupled to an enzyme, - add an enzyme substrate and a chromogen, wait for the wells to color - stop the coloring reaction, - read the optical density of the wells using a spectrophotometer at an appropriate wavelength. Ex vivo method for detecting or monitoring the development of a chronic autoimmune disease according to the preceding claim, characterized in that it comprises at least the implementation of the following steps: - manufacturing at least one microtitration plate sensitized with the antigen(s) against which the antibodies whose presence in the sample is to be detected are directed, or using at least one microtitration plate already sensitized with these antigens, - diluting the sample to be tested and internal standards, - distribute in duplicate on the plate the same quantity of diluted internal standards, diluted sample and reagent blank, - incubate, - wash, - add the secondary antibody(ies) coupled to peroxidase or alkaline phosphatase or biotinylated, - incubate, - wash, - add a substrate and a chromogen, - Stop the reaction in acid solution - read at 450 nm with a correction alpha at 650 nm. Kit for use in a method for detecting or monitoring the development of a chronic autoimmune disease according to one of the preceding claims, characterized in that it comprises at least the antigen(s) against which the antibodies are directed whose presence in the sample is to be detected. Kit according to claim 13, characterized in that it comprises at least: - a microtitration plate sensitized with the antigen(s) against which the antibodies are directed whose presence in the sample is to be detected, - buffers and solutions, - the secondary antibody(ies) in solution, secondary antibody(ies) corresponding to the antibodies whose presence in the sample is to be detected.