WO1997011373A1

WO1997011373A1 - Method for the early detection of an autoimmune disease, diagnostic use thereof and assay kit therefor

Info

Publication number: WO1997011373A1
Application number: PCT/FR1996/001441
Authority: WO
Inventors: Jean Amiral; Anne-Marie Vissac
Original assignee: Societe Diagnostica Stago
Priority date: 1995-09-20
Filing date: 1996-09-16
Publication date: 1997-03-27
Also published as: FR2738916A1; FR2738916B1

Abstract

A method for the early detection of an autoimmune disease using an antigen-antibody-type immune reaction to detect the immune material causing said disease is disclosed. The method comprises (1) contacting a cytokine (Z) with a biological sample thought to contain said immune material, i.e. an autoantibody (anti(Z)) to said cytokine (Z), in order to trigger a reaction anti(Z) + Z ---> anti(Z) - Z; then (2) revealing the resulting reaction product, i.e. the anti(Z) - Z complex, to determine whether or not said antibody anti(Z) is present in the sample. The use of said method for detecting autoantibodies, and an assay kit for carrying out the method, are also disclosed.

Description

METHOD FOR EARLY IDENTIFICATION OF AN AUTOIMMUNE DISEASE, USE IN THE FIELD OF DIAGNOSIS AND DOSAGE NECESSARY FOR IMPLEMENTING THE SAME.

Field of the invention

The present invention relates to the early detection of autoimmune diseases or disorders. More specifically, it relates to a method for identifying these immune diseases or disorders,

The use of this method to detect the presence or absence of the antibodies responsible, and a kit, kit or assay kit for carrying out this method. Prior art

It is known that the communication between the various cells of the immune system (T lymphocytes, B lymphocytes, macrophages, neutrophils, hematopoietic progenitors, etc.) is ensured at least partially by a complex cascade of interactions using, as mediators, particular proteins, namely cytokines.

Among the cytokines, a family of products is known, the 0 "chemokines" (other nomenclatures: "intercrines" or "cytokines of the PF4 superfamily"), which are involved in particular in inflammatory and especially proinflammatory mechanisms during the defense of the the organism vis-à-vis a foreign host, is in monomeric form (single-stranded structure having a molecular weight of approximately 8-10 kD) and especially in polymeric form (mainly the dimeric and tetrameric form), have four Cys groups in their single-stranded structure and are divided into two subfamilies according to the first two Cys encountered going from the N-terminal end to the C-terminal end depending on whether the two said first Cys are separated or not with another amino acid. 0 A distinction is thus made, in particular according to the articles by R. HORUK,

Immunol. Today, 1994, ϋ (No. 4), pages 169-174, from E. BRANDT et al. , Platelets, 1992, 3_, pages 295-305, and of A.E. KOCH et al., J. Invest. Med. , 1995, 42, pages 28-38,

(i) the subfamily of chemokines of the CXC type (where C is Cys and X is an amino acid different from Cys), which comprises in particular IL-8, PF4, GRO, IP-10 and NAP-2 [the definitions of the abbreviations are given below], which is also referred to as the α-intercrine subfamily, and (ii) the subfamily of CC type chemokines (where C is Cys as indicated above), which includes in particular the RANTES and MCP-1, and which is also called the sub-family of β-intercrines.

In short, chemokines act mainly as chemoattractants (i.e. "chemoattractants") and activators of particular leukocytes. Within the same subfamily, each chemokine has activities which are unique, on the one hand, and which overlap or overlap with those of other chemokines, on the other hand. Thus, chemokines which present on the side of the N-terminal an ELR sequence (ie Glu-Leu-Arg) immediately before the first Cys (that is to say an ELRC structure), such as for example GRO, IL -8, PBP, CTAP-III, βTG, NAP-2 and ENA-78 are chemoattractants and activators for neutrophils; CXC chemokines lacking said ELR sequence generally have weak or even zero activities with regard to the attraction of neutrophils, as is notably the case for PF4 and IP-10; and, CC chemokines are chemoattractants and activators for monocytes and T cells, as is notably the case for RANTES, MIP-1 and MCP-1. According to the article by A.E. KOCH et al. above (see in particular page

34, paragraph "Conclusions"), it is stated that chemokines play an important role in rheumatic inflammations and, since a large number of them are detectable in the serum of rheumatic patients, they can serve as markers for identification or the diagnosis of said inflammations. However, if a person skilled in the art is able to detect a chemokine in a biological medium such as serum by reaction with his specific anti-antibody (chemokine) then revelation of the complex formed, he cannot rely on the identification of a chemokine nor on a possible difference in concentration of a given chemokine compared to a normal value of the healthy subject for diagnosing an autoimmune disease or a risk of such a disease. On the one hand, this way of proceeding would be contrary to the therapy envisaged by other authors which would consist in administering to the subject suffering from an autoimmune disease the chemokine which is lacking. On the other hand, it would not allow easily distinguish free chemokine from chemokine logically linked to (or to) receptor (s) of its target.

We also know, in particular from J. AMIRAL et al. , Thromb.

Haemost., 1992, 68, pages 95-96, J. AMIRAL et al. , Tromb. Haemost. , 1995, 73, pages 21-28, and EP-A-0 495 971, that heparin-induced thrombocytopenia (HIT) type II are caused by an anti autoantibody (Hep-PF4) generated by the body against the heparin-PF4 complex.

It is recalled that HIT is the major complication of heparin therapies. We distinguish type I, where the thrombocytopenia is moderate, reversible, not associated with clinical disorders, and type II much less frequent (at most 5% of patients hospitalized and subjected to heparin therapy) which is characterized by a crisis. severe occurring on average 7 to 15 days after the start of heparin therapy and which can lead to thrombosis. Finally, we know that many cytokines and anti-cytokine antibodies are now marketed for analytical purposes; to this end, see the 1994 and 1995 catalogs of the company R & D SYSTEMS relating to cytokines.

The aforementioned prior art neither describes nor suggests that (i) autoimmune diseases result from the action of anti autoantibody (cytokine), and that (ii) these autoimmune diseases can be detected early by intermediate of the reaction: cytokine + anti (cytokine) -> cytokine-anti (cytokine) Aims of the invention We have just surprisingly discovered that the materials responsible for certain autoimmune diseases, in particular those which are liable to lead to thrombosis, are anti autoantibodies (cytokine) and in particular anti autoantibodies (chemokine).

These autoimmune diseases which include thrombocytopenia, neutropenia, proinflammatory pathologies or those likely to induce thrombosis, can cause (generally very quickly) severe crises ranging from thrombocytopenia and neutropenia to ischemia, thrombosis and amputation.

There is therefore a need for a new technical solution for identifying or detecting said autoimmune diseases at an early stage. According to a first aspect of the invention, it is proposed to provide a new method for early identification of an autoimmune disease, which implements an antigen-antibody type reaction.

According to a second aspect of the invention, it is proposed to use this method to detect the autoantibody responsible for said autoimmune disease.

According to a third aspect of the invention, it is proposed to provide a kit for implementing said method. OBJECT OF THE INVENTION The technical solution which is recommended to satisfy the above-mentioned need consists in seeking the presence or absence of a responsible autoantibody in a liquid biological medium originating from the patient.

According to the invention, a method for the early identification of an autoimmune disease is recommended, said method which implements an immunological reaction of the antigen-antibody type with a view to detecting the immune material responsible for said disease, being characterized in what he understands:

(1) bringing a cytokine (Z) into contact with a liquid biological sample capable of containing said responsible immune material, which is an autoantibody (anti (Z)) directed against said cytokine (Z), in order to trigger the reaction: anti (Z) + Z -> anti (Z) -Z then,

(2) revealing the reaction product thus obtained, the anti (Z) -Z complex, to evaluate the presence or absence of said anti (Z) antibody in said sample.

According to the invention, the use which is recommended is characterized in that a cytokine is used to detect an anti autoantibody (cytokine) capable of being pathogenic by the reaction cytokine -f anti (cytokine) - > anti-cytokine (cytokine) and the revelation of the cytokine-anti complex (cytokine) formed.

To implement the method of the invention, a kit, kit or assay kit is recommended which comprises: at least one Z cytokine, at least one reagent for revealing the formation of the anti (Z) -Z complex, and

- if necessary, at least one anti-antibody (Z) as a positive control. Abbreviations

In the present description, the following abbreviations and acronyms have been used for convenience, which are in particular illustrated in the aforementioned catalogs of 1994 and 1995 from the company R & D SYSTEMS relating to cytokines.

A492 (or A 492 nm) measurement or value of the absorbance under a light having a wavelength of 492 nm BTG see βTG below

CSF colony stimulating factor (in English: "colony stimulating factor"), three CSFs are known, namely the G-CSF, GM-CSF and M-CSF which stimulate the colonies of granulocytes, granulocytes-macrophages and respectively CTAP-III macrophages type III connective tissue activation peptide (in English:

"connective tissue activating peptide III"), it is a derivative of

PBP which does not have the first 9 amino acids of mature PBP EDTA ethylenediaminotetraacetic acid, the disodium salt of this acid is written here in the form Na2 EDTA EGF epithelial growth factor (in English: "épithelial growth factor"), we know several EGFs in particular TGF-α, and

HB-EGF EIA enzyme immunoassay (in English: "enzyme immunoassay")

ENA-78 peptide activating epithelial neutrophils (in English: "épithe¬ lial neutrophil activating peptide-78") comprising 78 amino acids

FGF fibroblast growth factor (in English: "fibroblast growth factor"), there are currently known seven molecules in the family of FGFs which are numbered FGF-1 to FGF-7 FGFa fibrobast acid growth factor (in English: "acidic fibroblast growth factor"), other nomenclature FGF-1 FGFb basic factor of growth of fibrobiastes (in English: "basic fibroblast growth factor"), other nomenclature FGF-2 FIA immunofluorescent test (in English: "fluorescence immunoassay") GAG glycosaminoglycan

GRO product of a growth gene (in English: "growth-related gene") [other nomenclature: MGSA], several GROs are known: GROα whose mature protein comprises 73 amino acids, GROβ and GROγ, (unless otherwise indicated, GRO will denote here GROα or MGSA) HAT thrombocytopenia associated with heparin (in English: "heparin- associated thrombocytopenia")

HB-EGF factor analogous to the epithelial growth factor binding heparin (in English: "heparin-binding EGF-like factor"), it is a particular EGF Hep heparin, in particular normal unfractionated heparin and fractionated heparin; Hep is part of the GAGs

Hep-PF4 complex formed by heparin and platelet factor 4

HGF hepatocyte growth factor (in English: "hepatocyte growth factor") HIT heparin-induced thrombocytopenia, ie thrombocytopenia characterized by the presence of an anti autoantibody (Hep-PF4) (en English: "heparin-induced thrombocytopenia") IP-10 protein inducible by interferon γ (in English: "interferon-γ- inducible protein"), the mature protein of IP-10 contains 77 amino acids IL-8 interleucine 8 (other nomenclature NAP-1), the protein expressed by the corresponding cDNA contains 99 amino acids including a possible signal peptide, at least 4 variants of human IL 8 are known according to the cleavages of the N-terminal end containing 79, 77, 72 and respectively 69 amino acids, the variant of 72 amino acids (which is the most active) was used here.

MBSS m-maleimido-benzoyl-N-hydroxysulfosuccinimide MCP-1 chemotactic protein of type 1 monocytes (in English: "monocytes chemotactic protein-1"), other nomenclature: MCAF (from "monocyte chemotactic and activating factor") MGSA product having a melanoma growth stimulating activity (in English: "melanoma growth-stimulating activity"), see GRO above MIP-1 macrophage inflammatory protein type 1 (in English: "macrophage inflammatory protein-1 "), two neighboring molecules have recently been distinguished: MlP-lα and MlP-l β MW molecular weight

NAP-2 peptide activating type 2 neutrophils (in English:

"neutrophil-activating peptide-2"), mature human NAP-2 contains 70 amino acids and can in particular be obtained by cleavage of the N-terminal end of βTG OPD orthophenylenediamine

PBP basic platelet protein (in English: "platelet basic protein"), the mature human protein contains 94 amino acids, the proteolytic cleavages of the N-terminal end of PBP give CTAP-III (85 amino acids), βTG (81 amino acids ) and respectively NAP-2 (70 amino acids) PDGF platelet-derived growth factor (in English: "platelet- derived growth factor") PF4 platelet factor 4 (in English: "platelet factor-4")

RANTES chemokine CC whose name is the acronym for "Regulated upon activation, normal T cell Expressed and Secreted", the mature human protein contains 68 amino acids RIA radioimmunoassay (in English: "radio immunoassay") RT room temperature (15-25 °) βTG β-thromboglobulin (other nomenclature: BTG), derived from PBP and

CTAP-III, in particular by loss of 3 amino acids at the N-terminal end of CTAP-III TGF-α transforming growth factor of type α (in English: "transforming growth factor-α"), substance belonging to the set TGF-β transforming growth factor β-type EGFs (in English: "transforming growth factor-β"), five molecules TGF-β 1 (or TGF-β) and TGF-β2 to TGF are currently known -β5 which have 30 to 80% similarity to TGF-β 1 VEGF vascular endothelial growth factor (in French: "vascular endothelial growth factor") Brief description of the drawings

The accompanying drawings are provided to illustrate the present invention with respect to the detection of the responsible autoantibodies. In these drawings:

- Figures la, lb, le, ld and respectively represent it graphically the detection of anti autoantibodies (chemokine) in the plasma of patients 1, 2, 3, 4 and respectively 5 with various chemokines and a blank control (C) , in the presence (I) or in the absence (II) of Hep, and

- Figure 2 shows the kinetic evolution of the number of wafers (PC) over time expressed in days (d).

Detailed description of the invention

The autoantibodies responsible or characteristic of autoimmune diseases and disorders are directed against cytokines and among these mainly (from a statistical point of view) against chemokines.

The mechanism leading to the formation of said anti autoantibodies (cytokine) has not yet been elucidated or determined. Nevertheless, the overall result which is observed is that autoimmune diseases, such as thrombocytopenia, neutropenia and proinflammatory pathologies, are characterized by the presence in the organism of patients (mainly in plasma and serum) of anti autoantibody (cytokine).

It is assumed, but this is a theory which does not bind the Applicant, that (A) a given cytokine Z binds to a body Y, which is (i) a target cell (Y \) of Z, (ii) a portion of the wall of a foreign cell (Y2) comprising at least one of the receptors for Z (for this purpose see the aforementioned article by R. HORUK which reports the presence of the erythrocyte receptor CK, which fixes IL -8, on the malaria parasite in humans (Plasmodium vivax) or (iii) a molecule capable of fixing said cytokine, to form a ZY complex such as Z-Yj,

Z-Y2 or Z-Y3;

(B) the body Y of said complex binds to another cell (X) of the organism

(the adhesion of neutrophils to platelets is described in particular by GB NASH in Thrombos. Res., 1994, 74, Suppl. 1, pages S3-S 1 1), to form a ZYX complex; as a variant, said ZYX complex can be obtained by reaction of Z with YX; and, (C) the organism which does not recognize ZYX generates antibodies and in particular the anti-cytokine autoantibodies characteristic according to the invention, which it is proposed to detect quickly before the onset or the arrival of the crisis severe, which leads to thrombosis amputation (of the fingers, toes or limbs) or death.

This mechanism bears similarities to that which leads to

HITs of type II, it differs from them however by the nature of the responsible antibodies, namely the anti (cytokine), whereas the said HITs are heparino-dependent and characterized by the presence of anti (Hep-

PF4).

In addition, according to said mechanism, the presence of a GAG amplifies the manifestations and the severity of the pathologies linked to the existence of anti-antibodies (cytokine), since the GAGs bind to a large number of cytokines and practically to all chemokines. It turns out that in particular Hep [high molecular weight heparin (MW = 30,000-6,000 approximately), "standard" heparin (MW = approximately 12,000), low molecular weight heparin (MW = 6,000-3,000 approximately)] and chondroitin-sulfuric acids (in particular dermatan sulfate) have a great affinity for chemokines. Furthermore, since GAGs such as Hep fix cytokines, they have the possibility of conveying them to other cells (for example erythrocytes, endothelium, etc. for Hep) which are devoid of receptors for said cytokines; new targets are then obtained for autoantibodies.

Among the cytokines, from which the organism can generate autoantibodies responsible (or characteristic) for thrombo-penetrant, neutropenic, pro-inflammatory pathologies or liable to induce thrombosis, mention may be made of CSFs (in particular G-CSF, GM-CSF and MCSF), EGFs (in particular TGF-α), FGFs (in particular FGFa and GFGb),

HGF, PDGF, TGF-β and VEGF.

Given the frequency of these pathologies in humans, cytokine Z will advantageously be chosen from the group consisting of chemokines. Among said chemokines from which the organism generates autoantibodies responsible (or characteristic) for thrombo-penetrant, neutropenic, pro-inflammatory pathologies or liable to induce thrombosis, there may be mentioned - substances of the CXC type, where C denotes Cys and X an amino acid residue different from Cys, and

- CC type substances, where C denotes Cys, namely: among the CXC chemokines comprising the ELR fragment as indicated above: GROs (in particular GROα, GROβ and GROγ), IL 8, PBP, PBP derivatives ( CTAP-III, βTG and NAP-2) and ENA-78, among CXC chemokines not comprising said ELR fragment: PF4 and IP-10, and among CC chemokines: RANTES, MCP-1 and MIP-2.

To implement the method of the invention, use is made of a biological sample capable of containing one of the responsible or characteristic autoantibodies. This biological sample will be an aqueous liquid and more precisely a body liquid. If one can effectively look for and detect an anti (cytokine) antibody in urine, saliva, synovial fluid or pus, it is rather recommended that said body fluid is plasma or serum which, compared to others body fluids, higher antibody concentrations.

In practical terms, the liquid biological sample (advantageously plasma or serum) will be sought for the presence or absence of an anti-autoantibody (Z) directed against a cytokine belonging to the subfamily of chemokines of the type CXC and chosen from the group consisting of PBP, CTAP-III and β-TG, and better still the presence or absence of an anti (Z) autoantibody directed against a cytokine belonging to the subfamily of chemokines of the type CXC and chosen from the group consisting of IL-8, NAP-2, GRO, IP-10 and ENA-78. If necessary, one can search in said sample for the presence or absence of an anti-autoantibody (Z) directed against a cytokine belonging to the subfamily of chemokines of the CC type and chosen from the group consisting of RANTES, MIP-1 and MCP- 1.

The revelation of the anti (Z) -Z complex, which was formed in stage (1), is carried out in stage (2) according to a method known per se, in particular an EIA, RIA, FIA method. In the tests reported below, an EIA sandwich technique, according to which one reacts

- the anti (Z) antibody, which may be contained in the plasma or the serum, with the corresponding antigen Z immobilized, - after rinsing, the anti (Z) -Z complex with a labeled anti (antibody) [such as anti (IgG), anti (IgA), anti (IgM) or anti (IgG, A, M) coupled with pero¬ xydase], then

- after rinsing, the resulting complex with the OPD / H2O2 system for revealing and measuring A492. The cytokines that are used according to the invention are human cytokines (because the cytokines of animal origin are not generally 100% identical to the corresponding human cytokines). These human cytokines are prepared by genetic engineering from the corresponding cDNAs; for the preparation of PF4, it is also possible to use the extraction and purification process described in the aforementioned document EP-A-0 495 971.

The purpose of using the method according to the invention is to detect an anti (chemokine) antibody directed against a chemokine of the CXC or CC type, in particular for the purpose of early diagnosis of immune diseases which in particular result in thrombocytopenia, neutropenia and syndromes. inflammatory and thrombosis.

In particular, said use relates to the detection of an anti (Il-8), anti (NAP-2), anti (GRO), anti (ENA-78) or respectively anti (IP-10) antibody, by reaction with a chemokine chosen from IL-8, NAP-2, GRO, ENA-78 and respectively IP-10, for the early diagnosis of immune diseases, in particular chosen from thrombocytopenic, neutropenic, proinflammatory pathologies and capable of inducing thrombosis .

Other advantages and characteristics of the invention will be better understood on reading the description which will follow of exemplary embodiments and test results. Of course, all of these elements are given by way of illustration and are not limiting. Example 1

Plasma trials were initiated in 5 hospitalized patients undergoing heparin therapy with unfractionated Hep (CALCIPARINE®, standard heparin of MW = approximately 12,000 in the form of calcium salt) or fractionated Hep (FRAXIPARINE®, heparin of MW = approximately 4,500) and presented the symptoms of a type II HIT but without the characteristic presence of anti antibodies (Hep-PF4). These patients actually had Type II HAT. The presence or absence of anti (PF4) / anti (Hep-PF4) autoantibodies [ie principally anti (PF4) when Hep was not present in the coating containing PF4, or anti (Hep-PF4) when Hep was present in said coating], anti (IL-8), anti (NAP-2), anti (βTG), anti (CTAP-III) and respectively anti (PBP) according to a method EIA sandwich.

In the microcuvettes of analysis plates (COVALINK®) are introduced 200 μl of a solution of MBSS at 125 mg / 1, incubated for 4 h at RT (preactivation of the walls) and washed with 1 M phosphate buffer at pH 7.5. 200 μl of a solution of 20 μg / ml of mature human protein (rhPF4, rhIL-8, rhNAP-2, rhβTG, rhCTAP-III or respectively rhPBP) are introduced in combination with increasing standard Hep concentrations (0 to 2 IU / ml) and let stand overnight at RT. Rinsed to remove each unbound protein and then saturate with i the coating buffer containing 10% v / v goat serum. In the microcuvettes thus coated, 200 μl of plasma are incubated (prediluted at 1: 100 using a diluent consisting of 0.05 M phosphate buffer of 0.15 M NaCl and 10% goat serum) for lh at RT. The microcuvettes are rinsed with a solution of 0.025 M phosphate buffer, 0.15 M NaCl, 0.1% of TWEEN® 20, at pH 7.5. Add the conjugate [ie anti (IgG, A, M) overall coupled to peroxidase, at a concentration of 3 μg / ml in the plasma diluent] in the microcuvettes. After rinsing, the development of the coloring is carried out with the OPD / H2O2 system for 3 minutes at RT and then the reaction is stopped with 3M of sulforic acid. A492 is then measured in 0.5 h. The negative control test designated C (absence of antibody) was carried out by replacing the patient's plasma, which was diluted, with the dilution medium. If necessary, positive control tests can be undertaken with said dilution medium containing a given quantity of an antibody identical to that which it is desired to seek.

For the determination of the number of platelets, the blood of 5 patients was collected on Na2 EDTA. For all other tests, blood has was collected on trisodium citrate, conventional anticoagulant (at the rate of 9 volumes of blood for 1 volume of trisodium citrate), and centrifuged for 20 minutes at 3000 g, the citrate plasma constituting the supernatant being collected, distributed in vials of determined volume and frozen at -80 ° C for storage, said plasma being thawed at 37 ° C for 0.25 h before use.

For the detection of anti (PF4) / anti (Hep-PF4), anti (IL-8), anti (NAP-2), anti (βTG), anti (CTAP-III) and anti (PBP) antibodies, respectively results obtained were recorded in table I and represented graphically in figures la to the for the 5 patients (numbered 1 to 5). If the value measured for the absorbance is such that (α) A492 <0.30, it is considered having regard to control C that the plasma tested is devoid of the antibody sought, (β) 0.30 <A492 ≤ 0, 50, it is considered that there is a doubt as to the presence of said antibody, and

(γ) 0.50 <A492, it is considered that the plasma tested contains the antibody sought.

Furthermore in FIG. 1, it was indicated that the anti (Z) -Z complex was obtained in the presence (I) or in the absence (II) of Hep. Table II contains the clinical data of said patients.

Figure 2 represents, in the number of platelets (PC) / days system, the numbering of the platelets of each patient, each arrow (in bold in said figure 2) indicating the day on which he stopped administering Hep for each patients. All of these results show that patients 1-3

(see figures la-lc) presented in their plasma the anti-IL-8 antibody responsible for HAT thrombocytopenia type II, and that patients 4-5 (see figures ld-le) presented in their plasma the antibody anti (NAP-2) responsible for HAT type II thrombocytopenia. In addition, antibodies directed against "precursors" of NAP-2 (essentially CTAP-III and

PBP), were detected in the patient's plasma 4.

Furthermore, as Figures la to show that the results are not influenced by the presence of Hep, the A492 values being identical in the presence (I) or in the absence (II) of Hep, it is clear that the autoantibodies responsible are directed against proteins (here IL-8, NAP-2 and, where appropriate, precursors of NAP-2) and not against complexes of Hep with said proteins.

TABLE I

Absorbance at 492 nm when detecting antibodies

TABLE π

Clinical data

Example 2

In a batch of 815 patients with thrombocytopenia or neutropenia, the presence in the plasma of anti (Hep-PF4), anti (PF4), anti (IL-8), anti (NAP-2), anti (βTG), anti (CTAP-III) and anti (PBP) respectively. 17 patients were selected in whom the plasma lacked these antibodies and then, according to the process described in Example 1, replacing the products PF4, IL-8, NAP-2, βTG, CTAP-III and PBP respectively with ENA- 78, GRO, TFG-α, FGFa, FGFb, PDGF, and GM-CSF respectively, the presence of anti (ENA- 78), anti (GRO), anti (TFG-α), anti ( FGFa), anti (FGFb), anti (PDGF), and respectively anti (GM-CSF). The distribution of autoimmune diseases was that shown in Table III

TABLE III

Claims

1. Method for the early identification of an autoimmune disease, said method, which implements an immunological reaction of the antigen-antibody type with a view to detecting the immune material responsible for said disease, being characterized in that it comprises:

(1) bringing a cytokine (Z) into contact with a biological sample capable of containing said responsible immune material, which is an autoantibody (anti (Z)) directed against said cytokine (Z), in order to trigger the reaction

anti (Z) + Z -> anti (Z) -Z then, (2) the revelation of the reaction product thus obtained, the anti (Z) -Z complex, to evaluate the presence or absence of said anti (Z ) in said sample.

2. Method according to claim 1, characterized in that Z is a cytokine chosen from the group consisting of chemokines.

3. Method according to claim 1, characterized in that the cytokine

Z is a chemokine of the CXC type, where C denotes Cys and X an amino acid residue different from Cys.

4. Method according to claim 1, characterized in that the cytokine

Z is a chemokine of the CC type, where C denotes Cys.

5. Method according to claim 1, in which the presence or absence of an anti-autoantibody (Z) directed against a cytokine belonging to the subfamily of chemokines of the CXC type and chosen from the group consisting of IL-8, NAP-2, GRO, IP-10 and ENA-78.

6. Method according to claim 1, in which the presence or absence of an anti-autoantibody (Z) directed against a cytokine belonging to the subfamily of chemokines of the CXC type and chosen from the group consisting of is sought. by PBP, CTAP-III and β-TG.

7. Method according to claim 1, in which the presence or absence of an anti-autoantibody (Z) directed against a cytokine is sought. belonging to the subfamily of CC type chemokines and chosen from the group consisting of RANTES, MIP-1 and MCP-1.

8. Use of the method according to claim 1, said use being characterized in that a cytokine is used to detect an anti antibody (cytokine) capable of being pathogenic by the cytokine + anti reaction (cytokine) -> cytokine -anti (cytokine) and the revelation of the cytokine-anti complex (cytokine) formed.

9. Use according to claim 8, for detecting an anti (chemokine) antibody directed against a chemokine of the CXC or CC type, in particular for the purpose of early diagnosis of immune diseases which in particular result in thrombocytopenia, neutropenia, inflammatory syndromes and thromboses.

10. Use according to claim 8, for detecting an anti (Il-8), anti (NAP-2), anti (GRO), anti (ENA-78) or anti (IP-10) antibody, by reaction with a chemokine chosen from IL-8, NAP-2, GRO, ENA-78 and respectively IP-10, for the early diagnosis of immune diseases, in particular chosen from thrombocytopenic, neutropenic, proinflammatory pathologies and likely to induce thrombosis.

11. Dosing kit for implementing the method according to any one of claims 1 to 7, characterized in that it comprises:

- at least one Z cytokine,

at least one reagent for revealing the formation of the anti (Z) -Z complex, and

- if necessary, at least one anti-antibody (Z) as a positive control.