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EP1399743A2 - Method and kit for following neurodegenerative diseases - Google Patents

Method and kit for following neurodegenerative diseases

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Publication number
EP1399743A2
EP1399743A2 EP02713027A EP02713027A EP1399743A2 EP 1399743 A2 EP1399743 A2 EP 1399743A2 EP 02713027 A EP02713027 A EP 02713027A EP 02713027 A EP02713027 A EP 02713027A EP 1399743 A2 EP1399743 A2 EP 1399743A2
Authority
EP
European Patent Office
Prior art keywords
antigens
isotype
tyr
following
cys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02713027A
Other languages
German (de)
French (fr)
Inventor
Michel Geffard
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GEMAC SA
Original Assignee
GEMAC SA
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Filing date
Publication date
Application filed by GEMAC SA filed Critical GEMAC SA
Publication of EP1399743A2 publication Critical patent/EP1399743A2/en
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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention relates to a method and a kit for monitoring neurodegenerative diseases.
  • the invention finds particular application in the medical field and in the immunological field.
  • rheumatoid arthritis Despite the immense medical progress of the past fifty years, a certain number of diseases, formerly known or of recent appearance, remain strictly speaking incurable, despite a significant cost for Public Health.
  • MS affects 1 in 1000 individuals in full maturity (40 to 60 cases per 100,000 inhabitants), or 50,000 French, and there are 2,000 new cases each year. Worldwide, there are 2 million cases.
  • the disease begins in young adults, between 15 and 50 years old (especially 20 and 40 years old). Its incidence is twice greater in women than in men.
  • MS includes three main distinct clinical forms: relapsing, progressive and relapsing progressive.
  • Amyotrophic Lateral Sclerosis (ALS) or Charcot's disease is a rapidly evolving neurological disorder of unknown etiology. It is characterized by degenerative damage to the motor neurons of the brain, brainstem and anterior horn of the spinal cord, while the sensory neurons are not affected and the intellectual functions remain intact, leaving patients fully aware of the progression of their illness and the deterioration of their functional capacities.
  • ALS Amyotrophic Lateral Sclerosis
  • Charcot's disease is a rapidly evolving neurological disorder of unknown etiology. It is characterized by degenerative damage to the motor neurons of the brain, brainstem and anterior horn of the spinal cord, while the sensory neurons are not affected and the intellectual functions remain intact, leaving patients fully aware of the progression of their illness and the deterioration of their functional capacities.
  • ALS a progressive neurodegenerative disease
  • ALS most often occurs between the ages of 45 and 70, reaching 1.5 to 2 times more men than women. It concerns increasingly young people: there are cases with early onset before 40 years of age.
  • ALS includes an evolving clinical heterogeneity that suggests biological heterogeneity.
  • RA Rheumatoid arthritis
  • active chronic arthritis is a chronic polyarticular inflammatory disease responsible for osteocartilaginous damage causing painful functional impotence by deformation and ankylosis.
  • Spondyloarthritis is a chronic rheumatic disease manifested by an attack on the lumbosacral level resulting in progressive ankylosis. This immunological and inflammatory condition is linked to an overexpression of the B27 histocompatibility antigen (found in 90% of cases).
  • the detection of these antibodies can therefore be part of a general diagnosis of neurodegenerative diseases, and in the monitoring of these diseases.
  • Such a method for detecting these antibodies in a biological fluid can comprise the following steps:
  • this antibody when the presence of an antibody directed against a bacterial "Ig" antigen is detected, this antibody can be isotype A and / or isotype M.
  • non-bacterial antigens other than possibly nitrosylated bovine serum albumin are advantageously conjugated.
  • conjugated antigen is meant within the meaning of the present invention an antigen (such as Pal, FarCys, NO-Cys, etc.) coupled to a carrier molecule, preferably bovine serum albumin, either directly or via glutaraldehyde or glutaric anhydride.
  • a carrier molecule preferably bovine serum albumin
  • glutaraldehyde or glutaric anhydride Such coupling is carried out in a manner well known to those skilled in the art, for example as described in Boulleme et al., 1995, 1996; Geff ard et al., 1998.
  • the bacterial antigens are obtained by sonication of cultures of the bacteria indicated in Table 1.
  • the biological fluid can be, in particular: a serum, a plasma, whole blood, urine, a cerebrospinal fluid.
  • the process used can advantageously be of the "sandwich” type.
  • the complexes possibly formed can be demonstrated using an anti-human immunoglobulin A or M antibody, coupled to a marker, for example a fluorescent marker, the biotin / streptavidin system, a (radio) isotopic element or a enzyme.
  • the process according to the invention is advantageously carried out using a suitable solid support.
  • any device suitable for handling cell suspensions can be used, preferably tubes, particulate magnetic supports or rigid or flexible microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose comprising microwells.
  • an antibody coupled to an (radio) isotopic element means that the antibody carries, either on a specific element of its structure, for example the tyrosine residues constituting, or on an appropriate radical which has been attached to it, a radioactive isotope allowing it to be determined by counting the radioactivity associated with it.
  • the antibody When the antibody is coupled to an enzyme, this, associated with the use of appropriate reagents, allows a quantitative measurement of this antibody.
  • the substrate and the reagents are chosen so that the final product of the reaction or of the sequence of reactions caused by the enzyme and using these substances is:
  • radioactivity associated with the sample is counted in a gamma counter according to any appropriate method.
  • the appearance of a colored or fluorescent product is obtained by adding to the solid support a solution containing the substrate of the enzyme and one or more auxiliary reagents making it possible finally to obtain as reaction product, either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as has been explained previously.
  • the light signal from the samples thus treated is then measured, using the apparatus adapted to each case: transmission or reflection photometer or fluorimeter respectively.
  • the solid support is a microtiter plate, the reading of the light signal can be carried out sequentially in all the wells of the same plate by the use of automated readers commonly used in biology laboratories.
  • alkaline phosphatase can be used, the preferred substrates of which are paranitrophenylphosphate for a spectrophotometric final reading or methyl-4-umbelliferylphosphate for fluorometric reading or bromo-5-chloro-4-indolyl-3-phosphate for obtaining a insoluble colored reaction product. It is likewise possible to use the enzyme ⁇ - galactosidase whose preferential substrate is orthonitrophenyl- ⁇ -D- galactopyranoside.
  • anti-Ig antibodies can be coupled in a conventional manner to peroxidase.
  • Reagents used to reveal peroxidase conjugated to anti-Ig antibodies contain hydrogen peroxide, enzyme substrate, and a suitable chromogen such as orthophenylenediamine, 3,3-diamino benzidine or TMB (3 , 3 ', 5,5'-tetramethylbenzidine) to obtain a final insoluble reaction product, or else parahydroxyphenylpropionic acid to obtain a fluorescent reaction product soluble in the medium.
  • the colorimetric reaction is stopped with sulfuric acid.
  • Another preferred embodiment of the invention is the use of anti-immunoglobulin antibodies coupled to acetylcholinesterase.
  • the acetylcholinesterase is coupled to the antibody, preferably using a process derived from that described in French Patent No. 2,550,799 or a process which schematically comprises the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate heterobifunctional agent and finally the coupling of the products thus obtained.
  • the revelation of the enzymatic activity is preferably carried out according to the well-known technique which employs acetylthiocholine as substrate of the enzyme and the reagent of Ellman, or dithio-5,5 '2-nitro-benzoic acid as chromogen, according to any variant adapted to the case under consideration, for example that described in Anal. Chem. 57 (1985) 1170-
  • the invention also relates to a kit for implementing the method described above.
  • This kit advantageously includes:
  • kits may also include a suitable solid support.
  • Detection of antibodies against the following antigens PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO 2 -Tyr, NO-Asn, NO-Met, Ig5 (isotype A antibody), Ig12 (isotype A and isotype M antibody), Ig13 (isotype A antibody), Ig16 (isotype A antibody), Ig17 (isotype M antibody) and Ig19 (isotype A antibody), allows in particular to establish the diagnosis of MS.
  • the detection of antibodies directed against the following antigens Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3 (antibody of isotype A and isotype M), Ig5 (antibody of isotype A), Ig12 (isotype antibody A and isotype M), Ig13 (antibody of isotype A and isotype M), Ig16 (antibody of isotype A and isotype M), Ig17 (antibody of isotype A and isotype M), makes it possible to establish the diagnosis of ALS and, according to the antibody titer, to differentiate the 3 classes of this disease. Detection of antibodies to the following antigens:
  • Ig3 isotype A and isotype M antibodies
  • Ig5 isotype A and isotype M antibodies
  • Ig12 isotype A and isotype M antibodies
  • Ig13 isotype M antibodies
  • Ig19 isotype A antibodies
  • PBS salt phosphate buffer
  • NO-Trp NO-tryptophan
  • NO-Asn NO-asparagine
  • NO 2 -Tyr NO 2 -tyrosine
  • SECTION 1 DETERMINATION OF ANTI-NO-CYSTEIN ANTIBODIES
  • MERCK-EUROLAB POLYLABO
  • 10 ⁇ g / ml of NO-Cysteine-G-SAB conjugate solution prepared according to the method described in Boullerne et al., 1995, 1996 and Geffard et al., 1998) are placed per well in carbonate buffer at pH 9, 6 (reaction blanks: SAB-G), overnight at + 4 ° C with stirring, avoiding exposure of the plate to light.
  • PBS buffer containing Tween®, 10% glycerol and 1 g / l of BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS.
  • the plate is rinsed three times with PBS containing Tween®.
  • the wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 ⁇ l of H 2 O 2 (enzyme substrate) and 0.5 ml of 4% OPD are added) and the coloring be done in the dark for 20 ml of citrate phosphate buffer, 10 ⁇ l of H 2 O 2 (enzyme substrate) and 0.5 ml of 4% OPD are added) and the coloring be done in the dark for
  • Antibody titer (DO serum - DO m ⁇ y controls) / DO m ⁇ y controls.
  • SECTION 2 ASSAY OF ANTI-PHOSPHATIDYLINOSITOL ANTIBODIES
  • a Maxisorp® microtiter plate is used. 20 ⁇ g / ml of coating solution are placed per well (Faiderbe et al., 1990; Brochet et al., 1991), prepared from 20 ml of 10 "2 M phosphate buffer + 10 " 3 M CaCl 2 , pH 7, saturated with chloroform, to which 40 ⁇ l of the phosphatidylinositol stock solution (marketed by SIGMA, reference P-5766) have been added with a Hamilton syringe and 100 ⁇ l of a hexane / chloroform mixture (84/16) PI in solution (reaction blanks: SAB).
  • the wells are vortexed until a white precipitate is obtained, then the plate is left overnight at 37 ° C. with stirring, it is dried in an oven for at least 30 min, and rinsed three times at PBS.
  • the serum to be assayed obtained as follows: the day before the assay, prepare a pre-dilution of the serum (1/10 e ) in PBS buffer containing 27 g / l of NaCl and stir at 4 ° C at night. Then centrifuged for 15 min at 10,000 rpm. From the supernatant, diluted to 1/50 th (final dilution to 1/500 th in salty PBS buffer containing 10% glycerol and 1 g / l of SAH-EA).
  • the wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 20 ⁇ l of H 2 O 2 and 1 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 1.0 min. The reaction is stopped with 50 ⁇ l of 4N H 2 SO 4 , and the OD is read at 492 nm.
  • Antibody titer (DO serum - DO m ⁇ y. Controls ) / DO m ⁇ y controls.
  • a Maxisorp microtiter plate is used. 10 ⁇ g / ml of Choline-AG-SAB conjugate solution (prepared according to the method described in Souan et al., 1986) are placed per well in carbonate buffer at pH 9.6 (reaction blanks: SAB), for one overnight at + 4 ° C with stirring. Then poured into the wells of PBS buffer containing Tween®, 10% glycerol and 1 g / l of SAB-AG, in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS.
  • the wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 ⁇ l of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 ⁇ l of 4N H 2 SO 4 , and the OD is read at 492 nm.
  • Antibody titer (DO serum - DO m ⁇ y , controls) / DO m ⁇ y_ controls.
  • Antibody titre (DO serum - DO m ⁇ y controls) / DOmoy. witnesses.
  • a Maxisorp® microtiter plate is used. 20 ⁇ g / ml of FarCys-SAB conjugate solution are placed per well (prepared by activation of FarCys with ethyl chloroformate then coupling to SAB according to the technique indicated for fatty acids in Section 7 below) in buffer carbonate at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C with stirring. It is rinsed three times with PBS. PBS buffer then containing 27 g / l of NaCl and 2 g / l of defatted BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C.
  • the wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 20 ⁇ l of H 2 O 2 and 1 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 ⁇ l of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed in relation to a population of controls as follows:
  • Antibody titer (DO serum - DOmoy. Controls) / DO m ⁇ y , controls.
  • a Maxisorp® microtiter plate is used. 80 ⁇ g / ml of MDA-SAB conjugate solution (prepared according to the method described in Amara et al., 1995) are placed per well in carbonate buffer at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C with stirring. PBS buffer containing Tween®, 10% glycerol and 5 g / l of BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS. Then added serum to be assayed, diluted 1/1000 in PBS buffer containing Tween and 5 g / l BSA and incubated for 2 hours at 37 ° C. It is rinsed three times with PBS.
  • Antibody titre (DO serum - DO m ⁇ y , controls) / DO m ⁇ y_ witnesses.
  • SECTION 8 DETERMINATION OF BACTERIAL ANTI-ANTIGEN ANTIBODIES
  • a Maxisorp® microtiter plate is used. 34 ⁇ g / ml of bacterial antigen lysate, obtained by sonication of a culture of the corresponding bacterium (see Table 1), are placed per well in buffer. carbonate at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C with stirring. PBS buffer then containing 5 g / l of BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 hour at 37 ° C. Rinsed three times with PBS containing Tween®.
  • the wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 ⁇ l of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 ⁇ l of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed in relation to a population of controls as follows:
  • Antibody titer (DO serum - DO m ⁇ y. Controls ) / DO m ⁇ y controls.
  • EXAMPLE 1 QUANTIFICATION OF ANTIBODIES CIRCULATING IN MS Table 1 below collates the results of the immunoenzymatic tests carried out on a large number of subjects according to the method described in the preceding sections. Table 1
  • the specificity of the anti-NO-Cys response vis-à-vis MS has been compared with that of the control neurodegenerative disorders in which radical processes have been found involved: Amyotrophic Lateral Sclerosis, epilepsies and disease Parkinson's. The results showed a significant difference in this response between the various pathologies in favor of MS.
  • Table 2 shows the relationship between the different forms of MS (P: progressive; R: relapsing; RP: relapsing progressive; TEM: controls) from 16 significant conjugates.
  • Figure 1 shows the distribution of data for each form of MS and controls.
  • Anti-NO-Trp antibodies are good indicators for discriminating between progressive and remitting forms (Mann-Whitney p ⁇ 0.0001). Anti-Ach antibodies are relevant for discriminating the remitting form from the other two forms (Mann-Whitney p ⁇ 0.0001).
  • Anti-Ig16 antibodies of isotype A are relevant for discriminating progressive and remitting forms (Mann-Whitney p ⁇ 0.01). These circulating antibodies make it possible to discriminate the forms of
  • class 3 made up of 27% of the sera tested, is characterized by high values especially for the following markers: Ole, Aze, MDA, P), NOCys, Ig3, Ig16, Ig12, Ig13, Ig17 (for all these Ig , the antibodies detected are isotype M) and to a lesser extent Ig5 (for this Ig, the antibody detected is isotype A) - cf.
  • Figure 4 is characterized by high values especially for the following markers: Ole, Aze, MDA, P), NOCys, Ig3, Ig16, Ig12, Ig13, Ig17 (for all these Ig , the antibodies detected are isotype M) and to a lesser extent Ig5 (for this Ig, the antibody detected is isotype A) - cf.
  • the significant antibodies are the antibodies directed against fatty acids, azelaic acid, farnesyl-cysteine, malondialdehyde, phosphatidylinositol, NO-cysteine, NO-phenylalanine and immunoglobulins Ig3 (isotype A and M antibodies), Ig5 (antibodies of isotype A and M), Ig12 (antibody of isotype A and M), Ig13 (antibody of isotype M), Ig17 (antibody of isotype M) and Ig19 (antibody of isotype A), as shown in Table 3 below:

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Abstract

The invention relates to a method for the detection and following of neurodegenerative diseases, comprising the detection of the presence of antibodies of isotype A and/or M against the antigens associated with said diseases. The invention further relates to a kit for carrying out said method.

Description

METHODE ET TROUSSE POUR LE SUIVI DES MALADIES NEURODEGENERATIVESMETHOD AND KIT FOR MONITORING NEURODEGENERATIVE DISEASES
La présente invention a pour objet une méthode et une trousse pour le suivi des maladies neurodégénératives. L'invention trouve notamment application dans le domaine médical et dans le domaine immunologique.The present invention relates to a method and a kit for monitoring neurodegenerative diseases. The invention finds particular application in the medical field and in the immunological field.
Malgré les immenses progrès médicaux de ces cinquante dernières années, un certain nombre de maladies, anciennement connues ou d'apparition récente, restent à proprement parler incurables, malgré un coût important pour la Santé Publique. Citons en particulier les affections neuro-dégénératives, comme la maladie de Par inson, la Sclérose en Plaques (SEP), la Sclérose Latérale Amyotrophique (SLA), la maladie d'Alzheimer, les maladies autoimmunes, les hépatites, les rhumatismes dégénératifs et inflammatoires dont la polyarthrite rhumatoïde (PR).Despite the immense medical progress of the past fifty years, a certain number of diseases, formerly known or of recent appearance, remain strictly speaking incurable, despite a significant cost for Public Health. We can cite in particular the neurodegenerative diseases, such as Parson disease, Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (Alzheimer's disease), Alzheimer's disease, autoimmune diseases, hepatitis, degenerative and inflammatory rheumatism including rheumatoid arthritis (RA).
Dans ces maladies, le diagnostic nosologique est posé, certains mécanismes et étapes du processus lésionnel sont connus, des traitements symptomatiques sont opposés, souvent très chers, mais les résultats ne sont pas à la hauteur des espérances. La Sclérose en Plaques est une pathologie démyélinisante multiloculaire du système nerveux central affectant la substance blanche du cerveau et de la moelle épinière, très lourdement invalidante, d'évolution insidieuse et imprévisible.In these diseases, the nosological diagnosis is made, certain mechanisms and stages of the lesion process are known, symptomatic treatments are opposed, often very expensive, but the results are not up to expectations. Multiple Sclerosis is a multilocular demyelinating pathology of the central nervous system affecting the white matter of the brain and spinal cord, very disabling, with an insidious and unpredictable course.
En France, la SEP touche 1 individu sur 1000 en pleine maturité (40 à 60 cas pour 100 000 habitants), soit 50 000 français, et il faut compter 2000 nouveaux cas chaque année. Dans le monde, on recense 2 millions de cas.In France, MS affects 1 in 1000 individuals in full maturity (40 to 60 cases per 100,000 inhabitants), or 50,000 French, and there are 2,000 new cases each year. Worldwide, there are 2 million cases.
La maladie débute chez l'adulte jeune, entre 15 et 50 ans (20 et 40 ans surtout). Son incidence est deux fois plus grande chez la femme que chez l'homme.The disease begins in young adults, between 15 and 50 years old (especially 20 and 40 years old). Its incidence is twice greater in women than in men.
La SEP comprend trois grandes formes cliniques distinctes : rémittente, progressive et rémittente progressive. La Sclérose Latérale Amyotrophique (SLA) ou Maladie de Charcot est une affection neurologique d'évolution rapide, d'étiologie inconnue. Elle est caractérisée par une atteinte dégénérative des motoneurones du cerveau, du tronc cérébral et de la corne antérieure de la moelle épinière, tandis que les neurones sensitifs ne sont pas touchés et que les fonctions intellectuelles restent intactes, laissant les patients pleinement conscients de la progression de leur maladie et de la détérioration de leurs capacités fonctionnelles.MS includes three main distinct clinical forms: relapsing, progressive and relapsing progressive. Amyotrophic Lateral Sclerosis (ALS) or Charcot's disease is a rapidly evolving neurological disorder of unknown etiology. It is characterized by degenerative damage to the motor neurons of the brain, brainstem and anterior horn of the spinal cord, while the sensory neurons are not affected and the intellectual functions remain intact, leaving patients fully aware of the progression of their illness and the deterioration of their functional capacities.
La prévalence de la SLA est égale à celle de la SEP. Elle est 5 fois supérieure à celle de la maladie de Huntington. Chaque année, meurent plus de malades atteints de SLA que de SEP ou de maladie de Huntington.The prevalence of ALS is equal to that of MS. It is 5 times higher than that of Huntington's disease. Each year, more people with ALS die than MS or Huntington's disease.
La SLA survient le plus souvent entre 45 et 70 ans, atteignant 1,5 à 2 fois plus les hommes que les femmes. Elle concerne des sujets de plus en plus jeunes : il existe des cas à début précoce avant 40 ans.ALS most often occurs between the ages of 45 and 70, reaching 1.5 to 2 times more men than women. It concerns increasingly young people: there are cases with early onset before 40 years of age.
Son incidence, c'est-à-dire le nombre de nouveaux cas chaque année, a tendance à augmenter dans le monde pour une raison inconnue. En France, il y aurait 5 à 10 000 personnes atteintes, et il apparaît environ 1100 nouveaux cas chaque année. L'évolution de cette maladie est dramatique, toujours mortelle.Its incidence, that is, the number of new cases each year, tends to increase worldwide for an unknown reason. In France, there are 5 to 10,000 people affected, and there are about 1100 new cases each year. The evolution of this disease is dramatic, always fatal.
Elle se fait vers une aggravation progressive, avec installation d'un état grabataire et d'une insuffisance respiratoire par atteinte de la musculature intercostale et diaphragmatique, menant rapidement au décès (en 3 à 5 ans). La SLA comprend une hétérogénéité clinique évolutive qui laisse à penser à une hétérogénéité biologique.It is done towards a progressive aggravation, with installation of a bedridden state and a respiratory insufficiency by attack of the intercostal and diaphragmatic musculature, leading quickly to death (in 3 to 5 years). ALS includes an evolving clinical heterogeneity that suggests biological heterogeneity.
La polyarthrite rhumatoïde (PR), anciennement appelée polyarthrite chronique évolutive, est une maladie inflammatoire polyarticulaire chronique responsable de dégâts ostéocartilagineux entraînant une impotence fonctionnelle douloureuse par déformation et ankylose.Rheumatoid arthritis (RA), formerly known as active chronic arthritis, is a chronic polyarticular inflammatory disease responsible for osteocartilaginous damage causing painful functional impotence by deformation and ankylosis.
C'est donc une maladie potentiellement sévère qui cause un handicap important chez plus de la moitié des patients, 10 ans après le début des symptômes, et réduit de plusieurs années leur espérance de vie.It is therefore a potentially severe disease which causes significant disability in more than half of the patients, 10 years after the onset of symptoms, and reduces their life expectancy by several years.
Sa prévalence en France serait d'environ 1 % : c'est le rhumatisme inflammatoire chronique le plus fréquent. Il touche 7000 nouveaux individus par an, atteignant plus souvent la femme encore jeune.Its prevalence in France is around 1%: it is the most frequent chronic inflammatory rheumatism. It affects 7000 new individuals per year, more often reaching young women.
La Spondylarthrite (SPR) est une affection rhumatismale chronique se manifestant par une atteinte au niveau lombo-sacré entraînant une ankylose progressive. Cette affection immunologique et inflammatoire est liée à une surexpression de l'antigène d'histocompatibilité B27 (retrouvé dans 90% des cas).Spondyloarthritis (SPR) is a chronic rheumatic disease manifested by an attack on the lumbosacral level resulting in progressive ankylosis. This immunological and inflammatory condition is linked to an overexpression of the B27 histocompatibility antigen (found in 90% of cases).
L'importance de ces maladies tant au niveau du nombre de personnes touchées qu'au niveau de coût pour la santé publique, n'est donc pas négligeable.The importance of these diseases both in terms of the number of people affected and in terms of cost to public health, is therefore not negligible.
A ce jour, il n'existe pas de test biologique pour évaluer le stade et/ou prévoir l'évolution de ces maladies. Seul un examen clinique, éventuellement complété par des examens complémentaires, permet de poser le diagnostic de ces maladies.To date, there is no biological test to assess the stage and / or predict the evolution of these diseases. Only a clinical examination, possibly supplemented by additional examinations, enables the diagnosis of these diseases to be made.
Il existe donc un réel besoin de pouvoir disposer d'un test diagnostique évolutif spécifique, susceptible de pallier aux insuffisances de la clinique.There is therefore a real need to be able to have a specific evolving diagnostic test, capable of overcoming the inadequacies of the clinic.
Le Demandeur a mis en évidence que l'occurrence des maladies neurodégénératives était associée à des modifications antigéniques, intégrées par les cellules immunocompétentes, se traduisant par la production d'anticorps circulants d'isotypie A et/ou M. Ces anticorps sont dirigés contre les antigènes indiqués dans le Tableau 1 ci-après, montrant les modifications antigéniques et les facteurs bactériens participant à la chronicité de ces maladies : Tableau 1The Applicant has demonstrated that the occurrence of neurodegenerative diseases is associated with antigenic modifications, integrated by immunocompetent cells, resulting in the production of circulating antibodies of isotype A and / or M. These antibodies are directed against antigens indicated in Table 1 below, showing the antigenic modifications and the bacterial factors participating in the chronicity of these diseases: Table 1
La détection de ces anticorps peut donc rentrer dans le cadre d'un diagnostic général des maladies neurodégénératives, et dans le suivi de ces maladies. The detection of these antibodies can therefore be part of a general diagnosis of neurodegenerative diseases, and in the monitoring of these diseases.
Un tel procédé de détection de ces anticorps dans un fluide biologique peut comprendre les étapes suivantes :Such a method for detecting these antibodies in a biological fluid can comprise the following steps:
- mise en contact dudit fluide avec chacun des antigènes suivants :- bringing said fluid into contact with each of the following antigens:
Pal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO2-Tyr, NO-Phe, NO-Arg, NO-Asn, NO-Met, NO-Cr, NO-SAB, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17, Ig19 etPal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO 2 -Tyr, NO-Phe, NO-Arg, NO-Asn, NO -Met, NO-Cr, NO-SAB, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17, Ig19 and
- mise en évidence des complexes éventuellement formés entre lesdits antigènes et les anticorps correspondants.- highlighting of the complexes possibly formed between said antigens and the corresponding antibodies.
Comme cela est indiqué dans le Tableau 1, lorsqu'on détecte la présence d'un anticorps dirigé contre un antigène bactérien "Ig", cet anticorps peut être d'isotypie A et/ou d'isotypie M.As indicated in Table 1, when the presence of an antibody directed against a bacterial "Ig" antigen is detected, this antibody can be isotype A and / or isotype M.
Les antigènes non-bactériens autre que la sérum albumine bovine éventuellement nitrosylée sont avantageusement conjugués.The non-bacterial antigens other than possibly nitrosylated bovine serum albumin are advantageously conjugated.
Par " antigène conjugué " on entend au sens de la présente invention un antigène (tel que Pal, FarCys, NO-Cys, etc.) couplé à une molécule porteuse, de préférence la sérum albumine bovine, soit directement soit par l'intermédiaire de glutaraldéhyde ou d'anhydride glutarique. Un tel couplage est réalisé de manière bien connue de l'homme du métier, par exemple comme décrit dans Boulleme et al., 1995, 1996 ; Geff ard et al., 1998. Les antigènes bactériens sont obtenus par sonication de cultures des bactéries indiquées dans le Tableau 1.By "conjugated antigen" is meant within the meaning of the present invention an antigen (such as Pal, FarCys, NO-Cys, etc.) coupled to a carrier molecule, preferably bovine serum albumin, either directly or via glutaraldehyde or glutaric anhydride. Such coupling is carried out in a manner well known to those skilled in the art, for example as described in Boulleme et al., 1995, 1996; Geff ard et al., 1998. The bacterial antigens are obtained by sonication of cultures of the bacteria indicated in Table 1.
Le fluide biologique peut être, en particulier : un sérum, un plasma, du sang total, de l'urine, un liquide cérébrospinal.The biological fluid can be, in particular: a serum, a plasma, whole blood, urine, a cerebrospinal fluid.
Le procédé mis en oeuvre peut être avantageusement du type " sandwich ". Les complexes éventuellement formés peuvent être mis en évidence à l'aide d'un anticorps anti-immunoglobuline A ou M humaine, couplé à un marqueur, par exemple un marqueur fluorescent, le système biotine/streptavidine, un élément (radio)isotopique ou une enzyme.The process used can advantageously be of the "sandwich" type. The complexes possibly formed can be demonstrated using an anti-human immunoglobulin A or M antibody, coupled to a marker, for example a fluorescent marker, the biotin / streptavidin system, a (radio) isotopic element or a enzyme.
Le procédé conforme à l'invention est avantageusement mis en oeuvre à l'aide d'un support solide convenable.The process according to the invention is advantageously carried out using a suitable solid support.
Comme support solide, on peut utiliser tout dispositif adapté à la manipulation de suspensions cellulaires et préférentiellement des tubes, des supports magnétiques particulaires ou des plaques rigides ou souples de microtitration en polyéthylène, polystyrène, chlorure de polyvinyle ou nitrocellulose comportant des micropuits.As solid support, any device suitable for handling cell suspensions can be used, preferably tubes, particulate magnetic supports or rigid or flexible microtiter plates made of polyethylene, polystyrene, polyvinyl chloride or nitrocellulose comprising microwells.
Lorsqu'on utilise une plaque de microtitration préactivée ayant des terminaisons NH2, il n'est pas nécessaire de "coupler" les antigènes à une molécule porteuse.When using a pre-activated microtiter plate with NH 2 endings, there is no need to "couple" the antigens to a carrier molecule.
L'expression " un anticorps couplé à un élément (radio)isotopique " signifie que l'anticorps porte, soit sur un élément propre de sa structure, par exemple les résidus de tyrosine constitutifs, soit sur un radical approprié qui lui a été fixé, un isotope radioactif permettant de le doser par comptage de la radioactivité qui lui est associée.The expression "an antibody coupled to an (radio) isotopic element" means that the antibody carries, either on a specific element of its structure, for example the tyrosine residues constituting, or on an appropriate radical which has been attached to it, a radioactive isotope allowing it to be determined by counting the radioactivity associated with it.
Lorsque l'anticorps est couplé à une enzyme celle-ci, associée à l'emploi de réactifs appropriés, permet une mesure quantitative de cet anticorps. Le substrat et les réactifs sont choisis de sorte que le produit final de la réaction ou de la séquence des réactions provoquées par l'enzyme et mettant en oeuvre ces substances soit :When the antibody is coupled to an enzyme, this, associated with the use of appropriate reagents, allows a quantitative measurement of this antibody. The substrate and the reagents are chosen so that the final product of the reaction or of the sequence of reactions caused by the enzyme and using these substances is:
- ou bien une substance colorée ou fluorescente qui diffuse dans le milieu liquide environnant les cellules et qui fait l'objet de la mesure finale spectrophotométrique ou fluorimétrique, respectivement,- or a colored or fluorescent substance which diffuses into the liquid medium surrounding the cells and which is the subject of the spectrophotometric or fluorimetric final measurement, respectively,
- ou bien une substance colorée insoluble qui se dépose sur les cellules et les parois sur lesquelles elles sont fixées et qui peut faire l'objet, soit d'une mesure photométrique par réflexion, soit d'une évaluation à l'œil éventuellement par rapport à une gamme de teintes étalonnées.- or an insoluble colored substance which is deposited on the cells and the walls on which they are fixed and which can be the subject, either of a photometric measurement by reflection, or of an evaluation with the eye possibly compared to a range of calibrated shades.
Lorsque l'on utilise un élément radio-isotopique, comme par exemple l'iode 125, la radioactivité associée à l'échantillon est comptée dans un compteur gamma selon toute modalité appropriée.When a radioisotopic element, such as iodine 125, is used, the radioactivity associated with the sample is counted in a gamma counter according to any appropriate method.
Lorsque l'on utilise une enzyme, l'apparition d'un produit coloré ou fluorescent est obtenue en ajoutant au support solide une solution contenant le substrat de l'enzyme et un ou plusieurs réactifs auxiliaires permettant d'obtenir finalement comme produit de réaction, soit un produit coloré soluble dans le milieu, soit un produit coloré insoluble, soit un produit fluorescent soluble, comme cela a été expliqué précédemment. On mesure ensuite le signal lumineux provenant des échantillons ainsi traités, à l'aide de l'appareillage adapté à chaque cas : photomètre en transmission ou en réflexion ou fluorimètre respectivement. Lorsque le support solide est une plaque de microtitration, la lecture du signal lumineux peut être effectuée séquentiellement dans tous les puits d'une même plaque par l'emploi de lecteurs automatisés d'usage courant dans les laboratoires de biologie. On peut utiliser comme enzyme la phosphatase alcaline, dont les substrats préférentiels sont le paranitrophénylphosphate pour une lecture finale spectrophotométrique ou le méthyl-4- umbelliférylphosphate pour une lecture fluorométrique ou le bromo- 5-chloro-4-indolyl-3-phosphate pour obtenir un produit de réaction coloré insoluble. On peut de même utiliser comme enzyme la β- galactosidase dont le substrat préférentiel est l'orthonitrophényl-β-D- galactopyranoside.When an enzyme is used, the appearance of a colored or fluorescent product is obtained by adding to the solid support a solution containing the substrate of the enzyme and one or more auxiliary reagents making it possible finally to obtain as reaction product, either a colored product soluble in the medium, or an insoluble colored product, or a soluble fluorescent product, as has been explained previously. The light signal from the samples thus treated is then measured, using the apparatus adapted to each case: transmission or reflection photometer or fluorimeter respectively. When the solid support is a microtiter plate, the reading of the light signal can be carried out sequentially in all the wells of the same plate by the use of automated readers commonly used in biology laboratories. As an enzyme, alkaline phosphatase can be used, the preferred substrates of which are paranitrophenylphosphate for a spectrophotometric final reading or methyl-4-umbelliferylphosphate for fluorometric reading or bromo-5-chloro-4-indolyl-3-phosphate for obtaining a insoluble colored reaction product. It is likewise possible to use the enzyme β- galactosidase whose preferential substrate is orthonitrophenyl-β-D- galactopyranoside.
Préférentiellement, on peut coupler, de manière conventionnelle, les anticorps anti-lg à la peroxydase. Les réactifs utilisés pour révéler la peroxydase conjuguée aux anticorps anti-lg contiennent de l'eau oxygénée, substrat de l'enzyme, et un chromogène approprié par exemple de l'orthophénylènediamine, la diamino-3,3' benzidine ou la TMB (3,3',5,5'-tétraméthylbenzidine) pour obtenir un produit final de réaction insoluble, ou bien l'acide parahydroxyphénylpropionique pour obtenir un produit de réaction fluorescent soluble dans le milieu. La réaction colorimétrique est arrêtée avec de l'acide sulfurique.Preferably, anti-Ig antibodies can be coupled in a conventional manner to peroxidase. Reagents used to reveal peroxidase conjugated to anti-Ig antibodies contain hydrogen peroxide, enzyme substrate, and a suitable chromogen such as orthophenylenediamine, 3,3-diamino benzidine or TMB (3 , 3 ', 5,5'-tetramethylbenzidine) to obtain a final insoluble reaction product, or else parahydroxyphenylpropionic acid to obtain a fluorescent reaction product soluble in the medium. The colorimetric reaction is stopped with sulfuric acid.
Un autre mode préférentiel de réalisation de l'invention est l'utilisation d'anticorps anti-immunoglobuline couplés à l'acétylcholinestérase.Another preferred embodiment of the invention is the use of anti-immunoglobulin antibodies coupled to acetylcholinesterase.
L'acétylcholinestérase est couplée à l'anticorps en utilisant préférentiellement un procédé dérivé de celui décrit dans le brevet français N° 2 550 799 ou un procédé qui comporte schématiquement la préparation de fragments de l'anticorps par une technique connue, la modification de l'enzyme par réaction avec un agent hétérobifonctionnel approprié et enfin le couplage des produits ainsi obtenus.The acetylcholinesterase is coupled to the antibody, preferably using a process derived from that described in French Patent No. 2,550,799 or a process which schematically comprises the preparation of fragments of the antibody by a known technique, the modification of the enzyme by reaction with an appropriate heterobifunctional agent and finally the coupling of the products thus obtained.
La révélation de l'activité enzymatique est réalisée de préférence selon la technique bien connue qui emploie l'acétylthiocholine comme substrat de l'enzyme et le réactif d'Ellman, ou acide dithio-5,5' nitro-2 benzoïque comme chromogène, selon toute variante adaptée au cas examiné, par exemple celle décrite dans Anal. Chem. 57 (1985) 1170-The revelation of the enzymatic activity is preferably carried out according to the well-known technique which employs acetylthiocholine as substrate of the enzyme and the reagent of Ellman, or dithio-5,5 '2-nitro-benzoic acid as chromogen, according to any variant adapted to the case under consideration, for example that described in Anal. Chem. 57 (1985) 1170-
1173.1173.
Les chromogènes cités sont utilisés tels quels ou sous forme de sels solubles dans l'eau. L'invention a également pour objet une trousse pour la mise en oeuvre du procédé décrit ci-dessus. Cette trousse comprend avantageusement :The chromogens mentioned are used as such or in the form of water-soluble salts. The invention also relates to a kit for implementing the method described above. This kit advantageously includes:
- chacun des antigènes suivants : Pal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO2-Tyr, NO-Phe, NO-- each of the following antigens: Pal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO 2 -Tyr, NO-Phe, NO-
Arg, NO-Asn, NO-Met, NO-Cr, NO-SAB, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17 et Ig19 ;Arg, NO-Asn, NO-Met, NO-Cr, NO-SAB, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17 and Ig19;
- éventuellement au moins un anticorps anti-immunoglobuline humaine, d'isotypie A et/ou M tel que défini précédemment. Cette trousse peut également comprendre un support solide convenable.- optionally at least one human anti-immunoglobulin antibody, isotype A and / or M as defined above. This kit may also include a suitable solid support.
Ce test permet notamment :This test allows in particular:
- d'établir un diagnostic précoce de la maladie, en particulier chez les personnes à risque, et donc de ne pas prendre de retard dans le traitement de la maladie ;- to establish an early diagnosis of the disease, in particular in people at risk, and therefore not to fall behind in the treatment of the disease;
- de suivre l'évolution de la maladie, et donc de pouvoir adapter le traitement en conséquence.- to follow the evolution of the disease, and therefore to be able to adapt the treatment accordingly.
La détection des anticorps dirigés contre les antigènes suivants : PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO2-Tyr, NO-Asn, NO-Met, Ig5 (anticorps d'isotypie A), Ig12 (anticorps d'isotypie A et d'isotypie M), Ig13 (anticorps d'isotypie A), Ig16 (anticorps d'isotypie A), Ig17 (anticorps d'isotypie M) et Ig19 (anticorps d'isotypie A), permet en particulier d'établir le diagnostic de la SEP. La quantification des anticorps dirigés contre les antigènes suivants : NO2-Tyr, NO-Tyr, Ach et Ig16 (anticorps d'isotypie A), permet de discriminer les formes particulières de la SEP (progressive, rémittente, rémittente progressive).Detection of antibodies against the following antigens: PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO 2 -Tyr, NO-Asn, NO-Met, Ig5 (isotype A antibody), Ig12 (isotype A and isotype M antibody), Ig13 (isotype A antibody), Ig16 (isotype A antibody), Ig17 (isotype M antibody) and Ig19 (isotype A antibody), allows in particular to establish the diagnosis of MS. The quantification of the antibodies directed against the following antigens: NO 2 -Tyr, NO-Tyr, Ach and Ig16 (antibody of isotype A), makes it possible to discriminate the particular forms of MS (progressive, relapsing, relapsing progressive).
De même, la détection des anticorps dirigés contre les antigènes suivants : Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3 (anticorps d'isotypie A et d'isotypie M), Ig5 (anticorps d'isotypie A), Ig12 (anticorps d'isotypie A et d'isotypie M), Ig13 (anticorps d'isotypie A et d'isotypie M), Ig16 (anticorps d'isotypie A et d'isotypie M), Ig17 (anticorps d'isotypie A et d'isotypie M), permet d'établir le diagnostic de la SLA et, en fonction du titre en anticorps, de différencier les 3 classes de cette maladie. La détection des anticorps dirigés contre les antigènes suivants :Likewise, the detection of antibodies directed against the following antigens: Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3 (antibody of isotype A and isotype M), Ig5 (antibody of isotype A), Ig12 (isotype antibody A and isotype M), Ig13 (antibody of isotype A and isotype M), Ig16 (antibody of isotype A and isotype M), Ig17 (antibody of isotype A and isotype M), makes it possible to establish the diagnosis of ALS and, according to the antibody titer, to differentiate the 3 classes of this disease. Detection of antibodies to the following antigens:
Pal, Ole, Myr, PI, MAD, FarCys, Aze, NO-Cys, NO-Phe, Ig3 (anticorps d'isotypie A et d'isotypie M), Ig5 (anticorps d'isotypie A et d'isotypie M), Ig12 (anticorps d'isotypie A et d'isotypie M), Ig13 (anticorps d'isotypie M) et Ig19 (anticorps d'isotypie A), permet d'établir le diagnostic de la PR.Pal, Ole, Myr, PI, MAD, FarCys, Aze, NO-Cys, NO-Phe, Ig3 (isotype A and isotype M antibodies), Ig5 (isotype A and isotype M antibodies), Ig12 (isotype A and isotype M antibodies), Ig13 (isotype M antibodies) and Ig19 (isotype A antibodies) are used to diagnose RA.
Dans la description et les revendications, on utilise les abréviations suivantes :In the description and the claims, the following abbreviations are used:
SAB : sérum albumine bovineSAB: bovine serum albumin
G : résidu de glutaraldéhydeG: glutaraldehyde residue
AG : résidu d'anhydride glutariqueGA: glutaric anhydride residue
PBS : tampon phosphate saléPBS: salt phosphate buffer
OPD : ortho-phénylènediamineOPD: ortho-phenylenediamine
H2O2 : eau oxygénéeH 2 O 2 : hydrogen peroxide
H2SO4 : acide sulfuriqueH 2 SO 4 : sulfuric acid
PI : phosphatidylinositolPI: phosphatidylinositol
CaCI2 : chlorure de calciumCaCI 2 : calcium chloride
NaCI : chlorure de sodiumNaCI: sodium chloride
DO : densité optiqueDO: optical density
SAH : sérum albumine humaineSAH: human albumin serum
SAH-EA : sérum albumine humaine sur laquelle est accrochée de l'éthylènediamine ig = immunoglobulineSAH-EA: human albumin serum on which ethylenediamine is attached ig = immunoglobulin
Ach : acétyl cholineAch: acetyl choline
Ole : acide oléiqueOle: oleic acid
Pal : acide palmitique Myr : acide myristique FarCys : Farnésyl cystéinePal: palmitic acid Myr: myristic acid FarCys: Faresyl cysteine
MDA : malondialdéhydeMDA: malondialdehyde
(néo-épitope résultant de la lipoperoxydation)(neo-epitope resulting from lipoperoxidation)
(avec R = protéine)(with R = protein)
NO-Cys : NO-cystéineNO-Cys: NO-cysteine
(néoépitope dû à la nitrosylation de la cystéine)(neoepitope due to nitrosylation of cysteine)
OH S CH2 — CH NH — (CH2)5 -NH2 OH S CH 2 - CH NH - (CH 2 ) 5 -NH 2
COOHCOOH
NO-Trp : NO-tryptophaneNO-Trp: NO-tryptophan
(néoépitope dû à la nitrosylation du tryptophane)(neoepitope due to nitrosylation of tryptophan)
NO-Asn : NO-asparagineNO-Asn: NO-asparagine
(néoépitope dû à la nitrosylation de l'asparagine)(neoepitope due to nitrosylation of asparagine)
ON — NH-C I l — CH 2 C I H-NH2 i-ON - NH-C I l - CH 2 CI H-NH 2 i-
COOH NO-Tyr : NO-tyrosineCOOH NO-Tyr: NO-tyrosine
(néoépitope dû à la nitrosylation de la tyrosine)(neoepitope due to nitrosylation of tyrosine)
NO2-Tyr : NO2-tyrosineNO 2 -Tyr: NO 2 -tyrosine
(néoépitope dû à la formation de peroxynitrite : NO+O2 ")(neoepitope due to peroxynitrite formation: NO + O 2 " )
-CH2-CH-NH-(CH2) — NH2 COOH-CH 2 -CH-NH- (CH 2 ) - NH 2 COOH
NO-His : NO-histidineNO-His: NO-histidine
(néoépitope dû à la nitrosylation de l'histidine)(neoepitope due to nitrosylation of histidine)
NO-Phe : NO-phénylalanineNO-Phe: NO-phenylalanine
(néoépitope dû à la nitrosylation de la phénylalanîne)(neoepitope due to the nitrosylation of phenylalanine)
NO-Met : NO-méthionineNO-Met: NO-methionine
(néoépitope dû à la nitrosylation de la méthionine)(neoepitope due to nitrosylation of methionine)
NO-Arg : NO-arginine NO-Arg: NO-arginine
(néoépitope dû à la nitrosylation de l'arginine)(neoepitope due to nitrosylation of arginine)
ON — NH-Ç — N — CH2-CH2-CH CH-COOHON - NH-Ç - N - CH 2 -CH 2 -CH CH-COOH
IIII
NH NHNH NH
NO-Cr : NO-créatineNO-Cr: NO-creatine
(néoépitope dû à la nitrosylation de la créatine)(neoepitope due to nitrosylation of creatine)
ON — NH-C — N — CH^COOHON - NH-C - N - CH ^ COOH
NH CH,NH CH,
L'invention sera mieux comprise à l'aide des sections et exemples ci-après, donnés à titre purement illustratif.The invention will be better understood with the aid of the sections and examples below, given purely by way of illustration.
SECTION 1 : DOSAGE DES ANTICORPS ANTI-NO-CYSTEINE On utilise une plaque de microtitration Maxisorp® commercialisée par la Société MERCK-EUROLAB (POLYLABO), comportant 96 puits. On place par puits 10 μg/ml de solution de conjugué NO-Cystéine-G-SAB (préparé selon la méthode décrite dans Boullerne et al., 1995, 1996 et Geffard et al., 1998) dans du tampon carbonate à pH 9,6 (blancs de réaction : SAB-G), pendant une nuit à + 4°C sous agitation, en évitant l'exposition de la plaque à la lumière. On verse ensuite dans les puits du tampon PBS contenant du Tween®, 10% glycérol et 1 g/l de SAB, afin de saturer la surface des puits en protéine, ce qui obtenu après 1 h à 37°C. On rince trois fois au PBS.SECTION 1: DETERMINATION OF ANTI-NO-CYSTEIN ANTIBODIES A Maxisorp® microtiter plate sold by the company MERCK-EUROLAB (POLYLABO) is used, comprising 96 wells. 10 μg / ml of NO-Cysteine-G-SAB conjugate solution (prepared according to the method described in Boullerne et al., 1995, 1996 and Geffard et al., 1998) are placed per well in carbonate buffer at pH 9, 6 (reaction blanks: SAB-G), overnight at + 4 ° C with stirring, avoiding exposure of the plate to light. PBS buffer containing Tween®, 10% glycerol and 1 g / l of BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS.
On ajoute ensuite le sérum à doser, dilué au 1/10006 dans du tampon PBS contenant du Tween®, 10 % glycérol et 1 g/l de SAB-G , et on incube pendant 2 heures à 37°C. On ajoute enfin dans les puits une solution au 1/5000e, dans du tampon PBS contenant du Tween® et 1 g/l de SAB, d'anticorps anti- IgM humaines (commercialisé par Sanofi Pasteur sous la référence 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C.Then add the serum to be assayed, diluted 1/1000 6 in PBS buffer containing Tween®, 10% glycerol and 1 g / l of SAB-G, and incubate for 2 hours at 37 ° C. Finally added to the wells a solution e to 1/5000 in PBS buffer containing Tween and 1 g / l BSA, human IgM antibody (commercially available from Sanofi Pasteur under reference 75061) labeled with peroxidase, and incubated for 1 hour at 37 ° C.
On rince la plaque trois fois au PBS contenant du Tween®.The plate is rinsed three times with PBS containing Tween®.
On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 10 μl de H2O2 (substrat de l'enzyme) et 0,5 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendantThe wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 μl of H 2 O 2 (enzyme substrate) and 0.5 ml of 4% OPD are added) and the coloring be done in the dark for
10 mn. On arrête la réaction avec 50 μl d'H2SO4 4N, et on lit la DO à10 mins. The reaction is stopped with 50 μl of H 2 SO 4 4N, and the OD is read.
492 nm.492 nm.
Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante :The results are then expressed in relation to a population of controls as follows:
Titre en anticorps = (DO sérum - DOmθy témoins) / DOmθy témoins.Antibody titer = (DO serum - DO mθ y controls) / DO mθ y controls.
SECTION 2 : DOSAGE DES ANTICORPS ANTI-PHOSPHATIDYLINOSITOL On utilise une plaque de microtitration Maxisorp®. On place par puits 20 μg/ml de solution de coating (Faiderbe et al., 1990 ; Brochet et al., 1991), préparée à partir de 20 ml de tampon phosphate 10"2M + CaCI2 10"3M, pH 7, saturée en chloroforme, auquel on a ajouté 40 μl de la solution mère de phosphatidylinositol (commercialisé par SIGMA, référence P-5766) avec une seringue Hamilton et 100 μl d'un mélange hexane/chloroforme (84/16) pour faire passer le PI en solution (blancs de réaction : SAB). On passe les puits au vortex jusqu'à l'obtention d'un précipité blanc, puis on laisse la plaque une nuit à 37 °C sous agitation, on la sèche à l'étuve pendant au moins 30 mn, et on rince trois fois au PBS. On ajoute ensuite le sérum à doser, obtenu de la manière suivante : la veille du dosage, on prépare une pré-dilution du sérum (1/10e) dans du tampon PBS contenant 27 g/l de NaCI et on agite à 4°C pendant la nuit. On centrifuge ensuite 15 mn à 10 000 tr/mn. A partir du surnageant, on dilue au 1/50e (dilution finale au 1/500e dans du tampon PBS salé contenant 10 % de glycérol et 1 g/l de SAH-EA). On ajoute enfin dans les puits une solution au 1/5000e, dans du tampon PBS contenant du Tween® et 1 g/l de SAB, d'anticorps anti- IgM humaines (SANOFI-PASTEUR, référence 75061) marqués à la peroxydase, et on incube pendant 2 heures à 37°C. On rince la plaque trois fois au PBS contenant du Tween®.SECTION 2: ASSAY OF ANTI-PHOSPHATIDYLINOSITOL ANTIBODIES A Maxisorp® microtiter plate is used. 20 μg / ml of coating solution are placed per well (Faiderbe et al., 1990; Brochet et al., 1991), prepared from 20 ml of 10 "2 M phosphate buffer + 10 " 3 M CaCl 2 , pH 7, saturated with chloroform, to which 40 μl of the phosphatidylinositol stock solution (marketed by SIGMA, reference P-5766) have been added with a Hamilton syringe and 100 μl of a hexane / chloroform mixture (84/16) PI in solution (reaction blanks: SAB). The wells are vortexed until a white precipitate is obtained, then the plate is left overnight at 37 ° C. with stirring, it is dried in an oven for at least 30 min, and rinsed three times at PBS. Then add the serum to be assayed, obtained as follows: the day before the assay, prepare a pre-dilution of the serum (1/10 e ) in PBS buffer containing 27 g / l of NaCl and stir at 4 ° C at night. Then centrifuged for 15 min at 10,000 rpm. From the supernatant, diluted to 1/50 th (final dilution to 1/500 th in salty PBS buffer containing 10% glycerol and 1 g / l of SAH-EA). Finally added to the wells a solution e to 1/5000 in PBS buffer containing Tween and 1 g / l BSA, human IgM antibodies (Sanofi-Pasteur, reference 75061) labeled with peroxidase, and incubated for 2 hours at 37 ° C. The plate is rinsed three times with PBS containing Tween®.
On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 20 μl de H2O2 et 1 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 1.0 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm.The wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 20 μl of H 2 O 2 and 1 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 1.0 min. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm.
Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante :The results are then expressed in relation to a population of controls as follows:
Titre en anticorps = (DO sérum - DOmθy. témoins) / DOmθy témoins.Antibody titer = (DO serum - DO mθ y. Controls ) / DO mθ y controls.
SECTION 3 : DOSAGE DES ANTICORPS ANTI-AŒTYLCHOLlNESECTION 3: ASSAY OF ANTI-AETYLCHOLINE ANTIBODIES
On utilise une plaque de microtitration Maxisorp. On place par puits 10 μg/ml de solution de conjugué Choline-AG-SAB (préparé selon la méthode décrite dans Souan et al., 1986) dans du tampon carbonate à pH 9,6 (blancs de réaction : SAB), pendant une nuit à + 4°C sous agitation. On verse ensuite dans les puits du tampon PBS contenant du Tween®, 10% glycérol et 1 g/l de SAB-AG, afin de saturer la surface des puits en protéine, ce qui obtenu après 1 h à 37°C. On rince trois fois au PBS. On ajoute ensuite le sérum à doser, dilué au 1/1000e dans du tampon PBS contenant du Tween® et 1 g/l de SAB-AG , et on incube pendant 2 heures à 37°C. On rince la plaque trois fois au PBS contenant du Tween®.A Maxisorp microtiter plate is used. 10 μg / ml of Choline-AG-SAB conjugate solution (prepared according to the method described in Souan et al., 1986) are placed per well in carbonate buffer at pH 9.6 (reaction blanks: SAB), for one overnight at + 4 ° C with stirring. Then poured into the wells of PBS buffer containing Tween®, 10% glycerol and 1 g / l of SAB-AG, in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS. Then added serum to be assayed, diluted 1/1000 in PBS buffer containing Tween and 1 g / l BSA-AG, and incubated for 2 hours at 37 ° C. The plate is rinsed three times with PBS containing Tween®.
On ajoute enfin dans les puits une solution au 1/10 000e, dans du tampon PBS contenant du Tween® et 1 g/l de SAB, d'anticorps anti- IgM humaines (SANOFI-PASTEUR, référence 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C. On rince la plaque trois fois au PBS contenant du Tween®.Finally added to the wells a solution to 1/10 000th in PBS buffer containing Tween and 1 g / l BSA, human IgM antibodies (Sanofi-Pasteur, reference 75061) labeled with peroxidase, and incubated for 1 hour at 37 ° C. The plate is rinsed three times with PBS containing Tween®.
On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 10 μl de H2O2 et 0,5 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 10 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm.The wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 μl of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm.
Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante :The results are then expressed in relation to a population of controls as follows:
Titre en anticorps = (DO sérum - DOmθy, témoins) / DOmθy_ témoins.Antibody titer = (DO serum - DO mθ y , controls) / DO mθ y_ controls.
SECTION 4 : DOSAGE DES ANTICORPS ANTI-ACIDE AZELAIQUESECTION 4: ASSAY OF AZELAIC ACID ANTIBODIES
On utilise une plaque de microtitration Maxisorp. On place par puitsA Maxisorp microtiter plate is used. We place by well
10 μg/ml de solution de conjugué Aze-SAB (préparé selon la méthode décrite dans Daverat et al., 1989) dans du tampon carbonate à pH 9,6 (blancs de réaction : SAB), pendant une nuit à + 4°C sous agitation. On verse ensuite dans les puits du tampon PBS contenant du Tween®, 10% glycérol et 1 g/l de SAH-EA, afin de saturer la surface des puits en protéine, ce qui obtenu après 1 h à 37°C. On rince trois fois au PBS. On ajoute ensuite le sérum à doser, dilué au 1/5006 dans du tampon PBS contenant du Tween®, 10 % glycérol et 1 g/l de SAH-EA , et on incube pendant 2 heures à 37°C. On rince la plaque trois fois au PBS contenant du Tween®. On ajoute enfin dans les puits une solution au 1/10 000e, dans du tampon PBS contenant du Tween® et 1 g/l de SAB, d'anticorps anti- IgM humaines (SANOFI-PASTEUR, référence 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C. On rince la plaque trois fois au PBS contenant du Tween®. On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 10 μl de H2O2 et 0,5 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 10 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm. Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante :10 μg / ml of Aze-SAB conjugate solution (prepared according to the method described in Daverat et al., 1989) in carbonate buffer at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C under agitation. Then poured into the wells of PBS buffer containing Tween®, 10% glycerol and 1 g / l of SAH-EA, in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS. Then add the serum to be assayed, diluted 1/500 6 in PBS buffer containing Tween®, 10% glycerol and 1 g / l of SAH-EA, and incubate for 2 hours at 37 ° C. The plate is rinsed three times with PBS containing Tween®. Finally added to the wells a solution to 1/10 000th in PBS buffer containing Tween and 1 g / l BSA, human IgM antibodies (Sanofi-Pasteur, reference 75061) labeled with peroxidase , and incubate for 1 hour at 37 ° C. The plate is rinsed three times with PBS containing Tween®. The wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate buffer phosphate, 10 μl of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 minutes. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed with respect to a population of controls as follows:
Titre en anticorps = (DO sérum - DOmθy témoins) / DOmoy. témoins.Antibody titre = (DO serum - DO mθ y controls) / DOmoy. witnesses.
SECTION 5 : DOSAGE DES ANTICORPS ANTI-FARNESOL-CYSTEINESECTION 5: ASSAY OF ANTI-FARNESOL-CYSTEINE ANTIBODIES
On utilise une plaque de microtitration Maxisorp®. On place par puits 20 μg/ml de solution de conjugué FarCys-SAB (préparé par activation de FarCys au chloroformiate d'éthyle puis couplage à la SAB selon la technique indiquée pour les acides gras dans la Section 7 ci-après) dans du tampon carbonate à pH 9,6 (blancs de réaction : SAB), pendant une nuit à + 4°C sous agitation. On rince trois fois au PBS. On verse ensuite dans les puits du tampon PBS contenant 27 g/l de NaCI et 2 g/l de SAB délipidée, afin de saturer la surface des puits en protéine, ce qui obtenu après 1 h à 37°C. On rince trois fois au PBS. On ajoute ensuite le sérum à doser, dilué au 1/2506 dans du tampon PBS contenant 27 g/l de NaCI et 2 g/l de SAB délipidée, et on incube pendant 2 heures à 37°C. On rince trois fois au PBS.A Maxisorp® microtiter plate is used. 20 μg / ml of FarCys-SAB conjugate solution are placed per well (prepared by activation of FarCys with ethyl chloroformate then coupling to SAB according to the technique indicated for fatty acids in Section 7 below) in buffer carbonate at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C with stirring. It is rinsed three times with PBS. PBS buffer then containing 27 g / l of NaCl and 2 g / l of defatted BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS. Then add the serum to be assayed, diluted to 1/250 6 in PBS buffer containing 27 g / l of NaCl and 2 g / l of defatted BSA, and incubate for 2 hours at 37 ° C. It is rinsed three times with PBS.
On ajoute enfin dans les puits une solution au 1/50006, dans du tampon PBS contenant du Tween® et 1 g/l de SAB, d'anticorps anti- IgM humaines (SANOFI-PASTEUR, référence 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C. On rince la plaque trois fois au PBS contenant du Tween®.Finally, a 1/5000 6 solution is added to the wells, in PBS buffer containing Tween® and 1 g / l of SAB, of anti-human IgM antibodies (SANOFI-PASTEUR, reference 75061) labeled with peroxidase, and incubated for 1 hour at 37 ° C. The plate is rinsed three times with PBS containing Tween®.
On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 20 μl de H2O2 et 1 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 10 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm. Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante :The wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 20 μl of H 2 O 2 and 1 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed in relation to a population of controls as follows:
Titre en anticorps = (DO sérum - DOmoy. témoins) / DOmθy, témoins.Antibody titer = (DO serum - DOmoy. Controls) / DO mθ y , controls.
SECTION 6 : DOSAGE DES ANTICORPS ANTI-MDASECTION 6: ASSAY OF ANTI-MDA ANTIBODIES
On utilise une plaque de microtitration Maxisorp®. On place par puits 80 μg/ml de solution de conjugué MDA-SAB (préparé selon la méthode décrite dans Amara et al., 1995) dans du tampon carbonate à pH 9,6 (blancs de réaction : SAB), pendant une nuit à + 4°C sous agitation. On verse ensuite dans les puits du tampon PBS contenant du Tween®, 10% glycérol et 5 g/l de SAB, afin de saturer la surface des puits en protéine, ce qui obtenu après 1 h à 37°C. On rince trois fois au PBS. On ajoute ensuite le sérum à doser, dilué au 1/1000e dans du tampon PBS contenant du Tween® et 5 g/l de SAB, et on incube pendant 2 heures à 37°C. On rince trois fois au PBS.A Maxisorp® microtiter plate is used. 80 μg / ml of MDA-SAB conjugate solution (prepared according to the method described in Amara et al., 1995) are placed per well in carbonate buffer at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C with stirring. PBS buffer containing Tween®, 10% glycerol and 5 g / l of BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 h at 37 ° C. It is rinsed three times with PBS. Then added serum to be assayed, diluted 1/1000 in PBS buffer containing Tween and 5 g / l BSA and incubated for 2 hours at 37 ° C. It is rinsed three times with PBS.
On ajoute enfin dans les puits une solution au 1/5000e, dans du tampon PBS contenant du Tween® et 5 g/l de SAB, d'anticorps anti- IgM humaines (SANOFI-PASTEUR, référence 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C. On rince la plaque trois fois au PBS contenant du Tween®.Finally added to the wells a solution e to 1/5000 in PBS buffer containing Tween and 5 g / l BSA, human IgM antibodies (Sanofi-Pasteur, reference 75061) labeled with peroxidase, and incubated for 1 hour at 37 ° C. The plate is rinsed three times with PBS containing Tween®.
On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 10 μl de H2O2 et 0,5 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 10 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm. Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante : Titre en anticorps = (DO sérum - DOmθy. témoins) / DOmθy témoins. SECTION 7 : DOSAGE DES ANTICORPS ANTI-ACIDES GRAS On utilise une plaque de microtitration Maxisorp®. On place par puits 50 μg/ml de solution de conjugué Ole-BSA, Pal-BSA ou Myr-BSA (préparés selon la méthode décrite dans Maneta et al., 1987 ; Amara et al., 1994 a et b, Constans et al., 1995 ; Boullerne et al., 1996) dans du tampon carbonate contenant du CaCI2 10"3M, pH 9,6 (blancs de réaction : SAB délipidée), pendant une nuit à + 4°C sous agitation. On rince trois fois au PBS contenant du CaCI2 10"3M. On ajoute ensuite le sérum à doser, dilué au 1/1000e dans du tampon PBS contenant du CaCI2 10'3M et 1 g/l de SAB délipidée, et on incube pendant 2 heures à 37°C. On rince trois fois au PBS contenant du CaCI2 10'3M.The wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 μl of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed relative to a population of controls as follows: Antibody titre = (DO serum - DO mθ y. Controls ) / DO mθ y controls. SECTION 7: ASSAY OF ANTI-FATTY ACID ANTIBODIES A Maxisorp® microtiter plate is used. 50 μg / ml of Ole-BSA, Pal-BSA or Myr-BSA conjugate solution are placed per well (prepared according to the method described in Maneta et al., 1987; Amara et al., 1994 a and b, Constans et al ., 1995; Boullerne et al., 1996) in carbonate buffer containing 10 −3 M CaCl 2 , pH 9.6 (reaction blanks: defatted SAB), overnight at + 4 ° C. with stirring. Rinsing three times to PBS containing CaCl 2 10 "3 M. Then add the serum to be assayed, diluted to 1/1000 th in PBS buffer containing CaCl 2 10 ' 3M and 1 g / l of defatted BSA, and incubate for 2 hours at 37 ° C. Rinsed three times with PBS containing CaCl 2 10 '3 M.
On ajoute enfin dans les puits une solution au 1/5000e, dans du tampon PBS contenant contenant du CaCI2 10"3M et 1 g/l de SAB délipidée, d'anticorps anti-lgM humaines (SANOFI-PASTEUR, référence 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C. On rince la plaque trois fois au PBS contenant du Tween®. On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 10 μl de H2O2 et 0,5 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 10 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm. Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante : Titre en anticorps = (DO sérum - DOmθy, témoins) / DOmθy_ témoins.Finally added to the wells a solution e to 1/5000 in PBS containing containing CaCl February 10 "3 M and 1 g / l defatted BSA, anti-human IgM antibodies (SANOFI-PASTEUR, reference 75061 ) labeled with peroxidase, and incubated for 1 hour at 37 ° C. The plate is rinsed three times with PBS containing Tween®. The wells are emptied by inverting the plate. Reactive reagent ( for 20 ml of citrate phosphate buffer, 10 μl of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed relative to a population of controls as follows: Antibody titre = (DO serum - DO mθ y , controls) / DO mθ y_ witnesses.
SECTION 8 : DOSAGE DES ANTICORPS ANTI-ANTIGENES BACTERIENS On utilise une plaque de microtitration Maxisorp®. On place par puits 34 μg/ml de lysat d'antigène bactérien, obtenu par sonication d'une culture de la bactérie correspondante (cf. Tableau 1), dans du tampon carbonate à pH 9,6 (blancs de réaction : SAB), pendant une nuit à + 4°C sous agitation. On verse ensuite dans les puits du tampon PBS contenant 5 g/l de SAB, afin de saturer la surface des puits en protéine, ce qui obtenu après 1h à 37°C On rince trois fois au PBS contenant du Tween®.SECTION 8: DETERMINATION OF BACTERIAL ANTI-ANTIGEN ANTIBODIES A Maxisorp® microtiter plate is used. 34 μg / ml of bacterial antigen lysate, obtained by sonication of a culture of the corresponding bacterium (see Table 1), are placed per well in buffer. carbonate at pH 9.6 (reaction blanks: SAB), overnight at + 4 ° C with stirring. PBS buffer then containing 5 g / l of BSA is then poured into the wells in order to saturate the surface of the wells with protein, which is obtained after 1 hour at 37 ° C. Rinsed three times with PBS containing Tween®.
On ajoute ensuite le sérum à doser, dilué au 1/500e dans du tampon PBS contenant du Tween® et 2,5 g/l de SAB, et on incube pendant 2 heures à 37°C. On rince trois fois au PBS contenant du Tween®. On ajoute enfin dans les puits une solution au 1/50006, dans du tampon PBS contenant du Tween® et 2,5 g/l de SAB, d'anticorps anti- IgA ou anti-lgM humaines (SANOFI-PASTEUR, références 75041 et 75061) marqués à la peroxydase, et on incube pendant 1 heure à 37°C. On rince la plaque trois fois au PBS contenant du Tween®. On vide les puits par retournement de la plaque. On ajoute dans chaque puits du réactif de révélation (pour 20 ml de tampon citrate phosphate, on ajoute 10 μl de H2O2 et 0,5 ml d'OPD à 4 %) et on laisse la coloration se faire à l'obscurité pendant 10 mn. On arrête la réaction avec 50 μl d'H2SO44N, et on lit la DO à 492 nm. Les résultats sont ensuite exprimés par rapport à une population de témoins de la façon suivante :Then add the serum to be assayed, diluted 1/500 e in PBS buffer containing Tween® and 2.5 g / l of BSA, and incubate for 2 hours at 37 ° C. Rinsed three times with PBS containing Tween®. Finally, a 1/5000 6 solution is added to the wells, in PBS buffer containing Tween® and 2.5 g / l of SAB, of anti-human IgA or anti-IgM antibodies (SANOFI-PASTEUR, references 75041 and 75061) labeled with peroxidase, and incubated for 1 hour at 37 ° C. The plate is rinsed three times with PBS containing Tween®. The wells are emptied by inverting the plate. Development reagent is added to each well (for 20 ml of citrate phosphate buffer, 10 μl of H 2 O 2 and 0.5 ml of 4% OPD are added) and the coloring is allowed to take place in the dark for 10 min. The reaction is stopped with 50 μl of 4N H 2 SO 4 , and the OD is read at 492 nm. The results are then expressed in relation to a population of controls as follows:
Titre en anticorps = (DO sérum - DOmθy. témoins) / DOmθy témoins.Antibody titer = (DO serum - DO mθ y. Controls ) / DO mθ y controls.
EXEMPLE 1 : QUANTIFICATION D'ANTICORPS CIRCULANTS DANS LA SEP Le Tableau 1 ci-après rassemble les résultats des tests immunoenzymatiques effectués sur un grand nombre de sujets selon la méthode décrite dans les sections qui précèdent. Tableau 1EXAMPLE 1: QUANTIFICATION OF ANTIBODIES CIRCULATING IN MS Table 1 below collates the results of the immunoenzymatic tests carried out on a large number of subjects according to the method described in the preceding sections. Table 1
* anticorps d'isotypie M ** anticorps d'isotypie A Une réponse anticorps statistiquement significative a été trouvée en faveur de la SEP pour 23 conjugués sur 33.* isotype M antibody ** isotype A antibody A statistically significant antibody response was found in favor of MS for 23 out of 33 conjugates.
A titre d'exemple, la spécificité de la réponse anti-NO-Cys vis-à-vis de la SEP a été confrontée à celle des affections neurodégénératives contrôles dans lesquelles des processus radicalaires ont été trouvés impliqués : Sclérose Latérale Amyotrophique, épilepsies et maladie de Parkinson. Les résultats ont montré une différence significative de cette réponse entre les différentes pathologies en faveur de la SEP.For example, the specificity of the anti-NO-Cys response vis-à-vis MS has been compared with that of the control neurodegenerative disorders in which radical processes have been found involved: Amyotrophic Lateral Sclerosis, epilepsies and disease Parkinson's. The results showed a significant difference in this response between the various pathologies in favor of MS.
Le Tableau 2 ci-dessous montré la relation entre les différentes formes de SEP (P : progressive ; R : rémittente ; RP : rémittente progressive ; TEM : témoins) à partir de 16 conjugués significatifs. Table 2 below shows the relationship between the different forms of MS (P: progressive; R: relapsing; RP: relapsing progressive; TEM: controls) from 16 significant conjugates.
La figure 1 montre la répartition des données pour chaque forme de SEP et les témoins. Figure 1 shows the distribution of data for each form of MS and controls.
antigène NO yr NOTrp Ach igiβ 1NO yr NOTrp antigen Ach igi β 1
P 2 0,0409 0,0006 <0,0001 0,0491 n SEP P 39 38 117 109 n SEP R 59 54 112 107 n SEP RP 40 39 71 68 n Témoin 126 148 229 225P 2 0.0409 0.0006 <0.0001 0.0491 n SEP P 39 38 117 109 n SEP R 59 54 112 107 n SEP RP 40 39 71 68 n Control 126 148 229 225
0 anticorps d'isotypie A ; 2 test de Kruskal-Wallis)0 isotype A antibodies; 2 Kruskal-Wallis test)
Chaque conjugué discrimine, de manière particulière, les formes cliniques de SEP:Each conjugate discriminates, in a particular way, the clinical forms of MS:
Les anticorps anti-N02-Tyr sont particulièrement pertinents pour discriminer les formes progressive et rémittente-progressive (p de Mann-Whitney entre ces deux échantillons = 0,0167).Anti-NO2-Tyr antibodies are particularly relevant for discriminating between progressive and remittent-progressive forms (Mann-Whitney p between these two samples = 0.0167).
On note des valeurs faibles pour les SEP progressives.There are low values for progressive MS.
Les anticorps anti-NO-Trp sont de bons indicateurs pour discriminer les formes progressive et rémittente (p de Mann-Whitney < 0,0001). Les anticorps anti-Ach sont pertinents pour discriminer la forme rémittente des deux autres formes (p de Mann-Whitney < 0,0001).Anti-NO-Trp antibodies are good indicators for discriminating between progressive and remitting forms (Mann-Whitney p <0.0001). Anti-Ach antibodies are relevant for discriminating the remitting form from the other two forms (Mann-Whitney p <0.0001).
Les anticorps anti-lg16 d'isotypie A sont pertinents pour discriminer les formes progressive et rémittente (p de Mann-Whitney < 0,01). Ces anticorps circulants permettent de discriminer les formes deAnti-Ig16 antibodies of isotype A are relevant for discriminating progressive and remitting forms (Mann-Whitney p <0.01). These circulating antibodies make it possible to discriminate the forms of
SEP et surtout le passage d'une forme évolutive à une autre (forme rémittente/forme progressive). Cette évolution n'est identifiable par aucune autre approche clinique ou exploratoire actuellement. EXEMPLE 2 : QUANTIFICATION D'ANTICORPS CIRCULANTS DANS LA SLAMS and especially the passage from one evolutionary form to another (remitting form / progressive form). This development cannot be identified by any other clinical or exploratory approach at present. EXAMPLE 2: QUANTIFICATION OF CIRCULATING ANTIBODIES IN ALS
Les Figures 2 à 4 mettent en évidence les 3 classes de SLA :Figures 2 to 4 highlight the 3 classes of ALS:
• la classe 1, constituée par 39% des sérums testés (118 au total), est caractérisée par des valeurs fortes pour les antigènes suivants :• class 1, made up of 39% of the sera tested (118 in total), is characterized by strong values for the following antigens:
FarCys, Ig3, Ig5, Ig16, Ig12, Ig13 et dans une moindre mesure Ig17 (pour toutes ces Ig, les anticorps détectés sont d'isotypie A) - cf. Figure 2 ;FarCys, Ig3, Ig5, Ig16, Ig12, Ig13 and to a lesser extent Ig17 (for all these Ig, the antibodies detected are isotype A) - cf. Figure 2;
• la classe 2, constituée par 34 % des sérums testés, est caractérisée par des valeurs faibles pour l'ensemble des 21 antigènes• class 2, made up of 34% of the sera tested, is characterized by low values for all 21 antigens
(avec les médianes < 0 pour 19 d'entre eux). Seuls FarCys et PI s'élèvent au même niveau que les témoins - cf. Figure 3 ;(with medians <0 for 19 of them). Only FarCys and PI rise to the same level as the cookies - cf. Figure 3;
• la classe 3, constituée par 27 % des sérums testés, est caractérisée par des valeurs élevées surtout pour les marqueurs suivants : Ole, Aze, MDA, P), NOCys, Ig3, Ig16, Ig12, Ig13, Ig17 (pour toutes ces Ig, les anticorps détectés sont d'isotypie M) et dans une moindre mesure Ig5 (pour cette Ig, l'anticorps détecté est d'isotypie A) - cf. Figure 4.• class 3, made up of 27% of the sera tested, is characterized by high values especially for the following markers: Ole, Aze, MDA, P), NOCys, Ig3, Ig16, Ig12, Ig13, Ig17 (for all these Ig , the antibodies detected are isotype M) and to a lesser extent Ig5 (for this Ig, the antibody detected is isotype A) - cf. Figure 4.
Ces profils biologiques sont à rattacher à des types évolutifs différents de la SLA.These biological profiles are to be linked to different evolutionary types of ALS.
De plus, 80% des patients testés restent dans le même groupe d'origine au cours de l'évolution clinique de leur maladie.In addition, 80% of the patients tested remain in the same origin group during the clinical course of their disease.
EXEMPLE 3 : QUANTIFICATION DES ANTICORPS CIRCULANTS DANS LA PREXAMPLE 3: QUANTIFICATION OF CIRCULATING ANTIBODIES IN RA
Dans la PR (tous stades et évolutions confondus), les anticorps significatifs sont les anticorps dirigés contre les acides gras, l'acide azélaïque, le farnésyl-cystéine, le malondialdéhyde, le phosphatidylinositol, NO-cystéine, NO-phénylalanine et les immunoglobulines Ig3 (anticorps d'isotypie A et M), Ig5 (anticorps d'isotypie A et M), Ig12 (anticorps d'isotypie A et M), Ig13 (anticorps d'isotypie M), Ig17 (anticorps d'isotypie M) et Ig19 (anticorps d'isotypie A), comme le montre le Tableau 3 ci-après : In RA (all stages and trends combined), the significant antibodies are the antibodies directed against fatty acids, azelaic acid, farnesyl-cysteine, malondialdehyde, phosphatidylinositol, NO-cysteine, NO-phenylalanine and immunoglobulins Ig3 (isotype A and M antibodies), Ig5 (antibodies of isotype A and M), Ig12 (antibody of isotype A and M), Ig13 (antibody of isotype M), Ig17 (antibody of isotype M) and Ig19 (antibody of isotype A), as shown in Table 3 below:
Tableau 3Table 3
* anticorps d isotypie M ** anticorps d isotypie A Il apparaît donc que les titres en anticorps circulants d'isotypie M sont une aide au suivi des malades atteints de PR. * isotype M antibody ** isotype A antibody It therefore appears that the circulating antibodies of isotype M are an aid in monitoring patients with RA.
REFERENCES BIBLIOGRAPHIQUESBIBLIOGRAPHICAL REFERENCES
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• AMARA A., CHAUGIER C, RAGNAUD J.-M. and GEFFARD M. Circulating autoantibodies directed against conjugated fatty acids in sera of HIV-1 infected patients. Clinical and Expérimental Immunology, (1994 b) 96, 379-383.• AMARA A., CHAUGIER C, RAGNAUD J.-M. and GEFFARD M. Circulating autoantibodies directed against conjugated fatty acids in sera of HIV-1 infected patients. Clinical and Experimental Immunology, (1994 b) 96, 379-383.
• AMARA A., CONSTANS J., CHAUGIER C, SEBBAN A., DUBOURG L., PEUCHANT E., PELLEGRIN J.L, LENG B., CONRI C. and GEFFARD M. Autoantibodies to malondialdehyde-modified epîtope in connective tissue diseases and vasculitides. Clinical and Exp. Immunol., (1995) 101, 1-6.• AMARA A., CONSTANS J., CHAUGIER C, SEBBAN A., DUBOURG L., PEUCHANT E., PELLEGRIN JL, LENG B., CONRI C. and GEFFARD M. Autoantibodies to malondialdehyde-modified epîtope in connective tissue diseases and vasculitides . Clinical and Exp. Immunol., (1995) 101, 1-6.
• BOULLERNE A., PETRY K.G., MEYNARD M. and GEFFARD M. Indirect évidence for NO involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S- nitrosocysteine. J. of Neuroimmunol., (1995) 60, 117-124.• BOULLERNE A., PETRY K.G., MEYNARD M. and GEFFARD M. Indirect evidence for NO involvement in multiple sclerosis by characterization of circulating antibodies directed against conjugated S-nitrosocysteine. J. of Neuroimmunol., (1995) 60, 117-124.
• BOULLERNE A. L, PETRY K.G. and GEFFARD M. Circulating antibodies directed against conjugated fatty acids in sera of patients with mutiple sclerosis. J. of Neuroimmunol., (1996) 65, 75-81.• BOULLERNE A. L, PETRY K.G. and GEFFARD M. Circulating antibodies directed against conjugated fatty acids in sera of patients with mutiple sclerosis. J. of Neuroimmunol., (1996) 65, 75-81.
• BROCHET B., FAIDERBE S., AUDHUY S., GOSSET L, GEFFARD M. and ORGOGOZO J.M. Antibodies against phosphatidylinositol in multiple sclerosis. Current Concepts in Multiple Sclerosis - Proceedings of the 6th congress of the ECTRIMS. (Wiethôlter M., Dichgans J.), MERTIN J., Eds Excerpta Medica, Amsterdam, (1991) 97-102.• BROCHET B., FAIDERBE S., AUDHUY S., GOSSET L, GEFFARD M. and ORGOGOZO JM Antibodies against phosphatidylinositol in multiple sclerosis. Current Concepts in Multiple Sclerosis - Proceedings of the 6th congress of the ECTRIMS. (Wiethôlter M., Dichgans J.), MERTIN J., Eds Excerpta Medica, Amsterdam, (1991) 97-102.
• CONSTANS J., CONRI C, PELLEGRIN J.L, SERGEANT C, SIMONOFF M., PEUCHANT E., DUBOURG L., THOMAS M.J., PELLEGRIN I., BROSSARD G., BARBEAU P., AMARA A., GEFFARD M., CLERC M., FLEURY H. et LENG B. Stress oxydatif et infection à VIH : un concept à préciser et une voie thérapeutique à explorer. Annales de Médecine Interne, (1995) 146, 514-520.• CONSTANS J., CONRI C, PELLEGRIN JL, SERGEANT C, SIMONOFF M., PEUCHANT E., DUBOURG L., THOMAS MJ, PELLEGRIN I., BROSSARD G., BARBEAU P., AMARA A., GEFFARD M., CLERC M., FLEURY H. and LENG B. Oxidative stress and HIV infection: a concept to be clarified and a therapeutic path to explore. Annals of Internal Medicine, (1995) 146, 514-520.
• DAVERAT P., GEFFARD M. and ORGOGOZO J.M. Identification and characterization of anti-conjugated azelaic acid antibodies in multiple sclerosis. J. of Neuroimmunology, (1989), 22, 129-134.• DAVERAT P., GEFFARD M. and ORGOGOZO J.M. Identification and characterization of anti-conjugated azelaic acid antibodies in multiple sclerosis. J. of Neuroimmunology, (1989), 22, 129-134.
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• GEFFARD M., BODET D., CLAUDEPIERRE P., METZGER J.M. et SIBILIA J. Anticorps sériques circulants dirigés contre des antigènes modifiés par le NO dans les affections neurologiques et rhumatismales. Immunoanal. Biol. Spéc, (1998) 13, 209-217.• GEFFARD M., BODET D., CLAUDEPIERRE P., METZGER J.M. and SIBILIA J. Circulating serum antibodies directed against antigens modified by NO in neurological and rheumatic diseases. Immunoanal. Biol. Specs, (1998) 13, 209-217.
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• SOUAN M.L, GEFFARD M., LEBRUN-GRANDIE P. and ORGOGOZO J.M. Détection of anti-acetyleholine antibodies in myasthénie patients. Neurosc. Letters, (1986) 64, 23-28. • SOUAN M.L, GEFFARD M., LEBRUN-GRANDIE P. and ORGOGOZO J.M. Detection of anti-acetyleholine antibodies in myasthenia gravis patients. Neurosc. Letters, (1986) 64, 23-28.

Claims

REVENDICATIONS
1. Procédé de détection des anticorps associés à l'occurrence ou l'évolution d'une maladie neurodégénérative dans un fluide biologique, qui comprend les étapes suivantes :1. Method for detecting antibodies associated with the occurrence or progression of a neurodegenerative disease in a biological fluid, which comprises the following steps:
- mise en contact dudit fluide avec chacun des antigènes suivants :- bringing said fluid into contact with each of the following antigens:
Pal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO2-Tyr, NO-Phe, NO-Arg, NO-Asn, NO-Met, NO-Cr, NO-SAB, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17, Ig19, etPal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO 2 -Tyr, NO-Phe, NO-Arg, NO-Asn, NO -Met, NO-Cr, NO-SAB, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17, Ig19, and
- mise en évidence des complexes éventuellement formés entre lesdits antigènes et les anticorps correspondants.- highlighting of the complexes possibly formed between said antigens and the corresponding antibodies.
2. Procédé de détection des anticorps associés à l'occurrence ou l'évolution d'une maladie neurodégénérative dans un fluide biologique, qui comprend les étapes suivantes :2. Method for detecting antibodies associated with the occurrence or progression of a neurodegenerative disease in a biological fluid, which comprises the following steps:
- mise en contact dudit fluide avec chacun des antigènes suivants :- bringing said fluid into contact with each of the following antigens:
PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO2-Tyr, NO-Asn, NO-Met, Ig5, Ig12, Ig13, Ig16, Ig17 et Ig19, etPI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO 2 -Tyr, NO-Asn, NO-Met, Ig5, Ig12, Ig13, Ig16, Ig17 and Ig19, and
- mise en évidence des complexes éventuellement formés entre lesdits antigènes et les anticorps correspondants.- highlighting of the complexes possibly formed between said antigens and the corresponding antibodies.
3. Procédé de détection des anticorps associés à l'occurrence ou l'évolution d'une maladie neurodégénérative dans un fluide biologique, qui comprend les étapes suivantes :3. Method for detecting antibodies associated with the occurrence or progression of a neurodegenerative disease in a biological fluid, which comprises the following steps:
- mise en contact dudit fluide avec chacun des antigènes suivants :- bringing said fluid into contact with each of the following antigens:
Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3, Ig5, Ig12, Ig13, Ig16 et Ig17, et - mise en évidence des complexes éventuellement formés entre lesdits antigènes et les anticorps correspondants.Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3, Ig5, Ig12, Ig13, Ig16 and Ig17, and - highlighting of the complexes possibly formed between said antigens and the corresponding antibodies.
4. Procédé de détection des anticorps associés à l'occurrence ou l'évolution d'une maladie neurodégénérative dans un fluide biologique, qui comprend les étapes suivantes :4. Method for detecting antibodies associated with the occurrence or progression of a neurodegenerative disease in a biological fluid, which comprises the following steps:
- mise en contact dudit fluide avec chacun des antigènes suivants :- bringing said fluid into contact with each of the following antigens:
Pal, Ole, Myr, PI, MAD, FarCys, Aze, NO-Cys, NO-Phe, Ig3, Ig5, Ig12, Ig13, Ig17 et lg19, etPal, Ole, Myr, PI, MAD, FarCys, Aze, NO-Cys, NO-Phe, Ig3, Ig5, Ig12, Ig13, Ig17 and lg19, and
- mise en évidence des complexes éventuellement formés entre lesdits antigènes et les anticorps correspondants.- highlighting of the complexes possibly formed between said antigens and the corresponding antibodies.
5. Procédé selon l'une des revendications 1 à 4, dans lequel les antigènes non-bactériens autres que NO-SAB sont couplés à une molécule porteuse, de préférence la sérum albumine bovine.5. Method according to one of claims 1 to 4, wherein the non-bacterial antigens other than NO-SAB are coupled to a carrier molecule, preferably bovine serum albumin.
6. Procédé selon l'une des revendications 1 à 5, dans lequel le complexe éventuellement formé est mis en évidence à l'aide d'un anticorps anti-immunoglobuline humaine d'isotypie M ou, le cas échéant, d'isotypie A, ledit anticorps étant marqué de préférence par une enzyme.6. Method according to one of claims 1 to 5, in which the complex optionally formed is demonstrated using an anti-human immunoglobulin antibody of isotype M or, if appropriate, of isotype A, said antibody preferably being labeled with an enzyme.
7. Procédé selon la revendication 6, dans lequel l'enzyme est la peroxydase.7. The method of claim 6, wherein the enzyme is peroxidase.
8. Procédé selon l'une des revendications 1 à 7, qui est mis en oeuvre à l'aide d'un support solide, de préférence une plaque de microtitration. 8. Method according to one of claims 1 to 7, which is implemented using a solid support, preferably a microtiter plate.
9. Procédé selon la revendication 1, dans lequel la maladie dégénérative est la sclérose en plaques, la sclérose latérale amyotrophique, la maladie de Parkinson, la polyarthrite rhumatoïde ou la spondylarthrite.9. The method of claim 1, wherein the degenerative disease is multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease, rheumatoid arthritis or spondyloarthritis.
10. Trousse pour la mise en oeuvre du procédé selon la revendication 1, qui comprend chacun des antigènes suivants : Pal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO2-Tyr, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17 et Ig19.10. Kit for implementing the method according to claim 1, which each comprises the following antigens: Pal, Ole, Myr, PI, MAD, Ach, FarCys, Aze, NO-Cys, NO-Tyr, NO-Trp, NO-His, NO 2 -Tyr, Ig3, Ig5, Ig12, Ig13, Ig16, Ig17 and Ig19.
11. Trousse pour la mise en oeuvre du procédé selon la revendication 2, qui comprend chacun des antigènes suivants : PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO2-Tyr, NO-Asn, NO-Met, Ig5, Ig12, Ig13, Ig16, Ig17 et lg19.11. Kit for implementing the method according to claim 2, which each comprises the following antigens: PI, Ach, Aze, NO-Cys, NO-Tyr, NO-Trp, NO 2 -Tyr, NO-Asn, NO -Met, Ig5, Ig12, Ig13, Ig16, Ig17 and lg19.
12. Trousse pour la mise en oeuvre du procédé selon la revendication 3, qui comprend chacun des antigènes suivants : Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3, Ig5, Ig12, Ig13, Ig16 et Ig17.12. Kit for implementing the method according to claim 3, which each comprises the following antigens: Ole, PI, MAD, Aze, NO-Cys, FarCys, Ig3, Ig5, Ig12, Ig13, Ig16 and Ig17.
13. Trousse pour la mise en oeuvre du procédé selon la revendication 4, qui comprend chacun des antigènes suivants : Pal, Ole, Myr, PI, MAD, FarCys, Aze, NO-Cys, NO-Phe, Ig3, Ig5, lg12, Ig13, Ig17 et Ig19.13. Kit for implementing the method according to claim 4, which each comprises the following antigens: Pal, Ole, Myr, PI, MAD, FarCys, Aze, NO-Cys, NO-Phe, Ig3, Ig5, Ig12, Ig13, Ig17 and Ig19.
14. Trousse selon l'une des revendications 10 à 13, dans laquelle les antigènes non-bactériens autres que NO-SAB sont couplés à une molécule porteuse, de préférence la sérum albumine bovine.14. Kit according to one of claims 10 to 13, wherein the non-bacterial antigens other than NO-SAB are coupled to a carrier molecule, preferably bovine serum albumin.
15. Trousse selon l'une des revendications 10 à 14, qui comprend également au moins un anticorps anti-immunoglobuline humaine d'isotypie M et/ou au moins un anticorps anti- immunoglobuline humaine d'isotypie A. 15. Kit according to one of claims 10 to 14, which also comprises at least one anti-immunoglobulin antibody human isotype M and / or at least one anti-human immunoglobulin antibody isotype A.
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