FR2707011A1 - Immunological process for the detection and assay of antibodies directed against poly(ADP-ribose) polymerase, application to diagnosis, kit for its implementation - Google Patents

Abstract

The subject of the invention is an immunological process for the detection and assay of antibodies directed against poly(ADP-ribose) polymerase, characterised in that the antigen used is the peptide constituting the zinc finger F1 or F2 of the said polymerase or a homologous peptide or one of their fragments, which have an essentially identical immunological reactivity. This process is applicable to the diagnosis of an autoimmune disease such as disseminated lupus erythematosus and Gougerot-Sjögren's Syndrome, as well as to the diagnosis of precancerous or cancerous states. Another subject of the invention is a kit for the detection of antibodies directed against poly(ADP-ribose) polymerase comprising, as antigen, the peptide F1 or F2, a fragment thereof or a homologous peptide.

Description

The present invention relates to a method of immunodiagnosis of diseases

  autoimmune systemic and cancer states based on research in the

  serum of patients with antibodies to poly (ADP-

ribose) polymerase.

  We know that this enzyme catalyzes ADP-

  ribosylation of cell nucleus proteins, reaction associated with a number of regulatory functions such as DNA repair, gene expression

  and cell differentiation. Its amino sequence

  cides has been described in Biochem. Biophys. Res. Common.

  148 617-622 (1987); the formation of poly (ADP-ribose) is increased in particular in the event of DNA damage, thermal shock and inflammation and the presence of antibodies directed against poly (ADPribose) in the serum of patients suffering from lupus erythematosus disseminated was described in 1977 and later in some patients with Gougerot-Sjôgren syndrome, rheumatoid arthritis, scleroderma or in other patients suffering from an infectious disease such as tuberculosis, leprosy or else being infected with

E. Coli or Klebsiella.

  More recently, the presence of autoantibodies directed against poly (ADPribose) polymerase has been sought in the serum of various persons suffering from autoimmune diseases, but the results mentioned by Yamanaka et al. in J. Clin. Invest. 80 900-904 (1987)

  and 83 180-186 (1989) then by Negri et al. in Autoim-

  munity 6 203-209 (1990) are contradictory, the former finding antibodies in only a small proportion of rheumatic diseases, while the latter finds them in several types of autoimmune diseases, but

  probably because of an experimental artifact.

  We have now found that it was possible to diagnose systemic lupus erythematosus or Gougerot-Sj6gren syndrome by looking in the serum of patients for the presence of antibodies directed against a particular region of the polymerized poly (ADP-ribose) enzyme. - shave. However, it is known that the serum concentration of these antibodies varies with the phases of these diseases, and other means will be necessary in certain cases to diagnose them; the search for antibodies directed against the polymer itself, poly (ADP-ribose), is one of the means already used, but it is not very

  specific and also reveals other autoimmune diseases.

  The present invention relates to an immunological method for detecting and assaying antibodies directed against poly (ADP-ribose) polymerase in a biological sample, characterized in that the antigen used is the peptide forming the zinc fingers Fl or F2 of said poly (ADP-ribose) polymerase,

  fragments thereof or homologous peptides.

  The peptides are shown in FIG. appended, F1 being SEQ ID N 1 and F2 being SEQ ID No 2,

  and respectively include the amino-

  acids located between positions 21 to 56 and 125 to 162

of the enzyme.

  By homologous peptides is meant peptides in which certain amino acids have been replaced by equivalent amino acids, and which exhibit an immunological reactivity substantially identical to that of the peptides constituting the fingers F1

or F2 or fragments thereof.

  As equivalent amino acids, there may be mentioned lysine and arginine, glycine and alanine, aspartic acid

  and glutamic acid, asparagine and glutamine.

  These peptides can be obtained by cloning or by chemical synthesis, for example by the method in

solid phase of Merriefield.

  Autoantibodies against poly (ADP-

  ribose) polymerase are usually found in the serum of patients with systemic autoimmune diseases such as systemic lupus erythematosus

  or the primary or secondary Gougerot-Sjôgren syndromes-

  but they are also thought to be found in the states

  precancerous and cancerous since there is a correlation

  tion between the inhibition of the enzyme and the malignant transformation of the cells, as described by T.

  Boulikas in Anticancer Research 12: 885-898 (1992).

  The present invention therefore also relates to the methods of immunodiagnosis of systemic lupus erythematosus, of Gougerot-Sjôgren syndrome or of certain

  precancerous or cancerous states that consist of performing

  kill the search for antibodies in the patient's serum using a peptide chosen from fingers F1 or F2 of poly (ADP-ribose) polymerase, their fragments and

  their counterparts using an immunoassay.

  The term “immunoassay or immunological detection method” means all the methods well known to those skilled in the art, which consist in bringing together a sample of the biological fluid, in general serum, in which one wishes to search for the antibodies with a certain quantity of the characteristic antigen chosen, to separate the unreacted antigen and to

  reveal the immunological complex possibly formed.

  The antigen can be immobilized on a solid phase, such as the wells of a titration microplate or of polymer microbeads, by adsorption or by chemical coupling; the attached antibody can then be

  revealed by the formation of a complex with an anti

  antibody, which has been previously labeled in a conventional manner in this field by an enzyme, the

  presence on the solid phase will result in conver-

  sion of its substrate, when it is introduced into the medium, into a colored, fluorescent or luminescent product;

  the anti-antibody can also be radiolabelled.

  Other more or less complex methods are known and described in particular in the work

  "Laboratory Technics in Biochemistry and Molecular Biolo-

  gy ", vol. 19, edited by R.H. Burdon and P.H. van Knippen-

  berg - Elsevier (1988), to whom a person skilled in the art can

refer.

  The present invention also relates to a kit for detecting antibodies directed against poly (ADP-ribose) polymerase, characterized in that it comprises an antigenic peptide chosen from the fingers F1 and F2 of said polymerase, a homologous peptide or one of their fragments having a substantially identical immunological reactivity, optionally immobilized on a solid support, and optionally a

  labeled antibody capable of binding to the antibody- complex

  antigen formed, and means for demonstrating the

  attachment of said antibody to said complex.

  - In what follows, we describe the results of the study of sera from 235 known subjects with various autoimmune diseases and from 42 healthy volunteers,

  by the process of the invention with an immunoen-

  zymatic on solid phase called ELISA.

  97 patients had systemic or active systemic lupus erythematosus (SLE), 67 had primary Gougerot-Sjôgren syndrome (pSS) and 16 had secondary lupus associated syndrome (sSS) while 38 had juvenile polyarthritis (JCA) and 17 a

mixed connectivity (MCTD).

  The F2 peptide (Fig.) Synthesized by the Merriefield method or for comparison the recombinant human poly (ADP-ribose) polymerase enzyme described in Gene 114 279-283 (1992) was immobilized in the wells of a microtiter plate. ) as well as the calf enzyme of similar structure, by adsorption in a conventional manner at 370 ° C. from their solutions in a carbonate buffer (0.05 M, pH 9.6), at a rate of 100 ng / ml for the enzymes and 1 pM for the peptide. Poly (ADP-ribose) of 90 + 10 units with a branch density of 2 + 0.5%, prepared as described in Biochem. Cell. Biol. 65,668-673 (1987) was also immobilized from a solution in Tween phosphate buffered saline of 50 ng / ml, by the method

  cited in Clin. Exp. Immunol. 86 124-133 (1991).

  The sera tested were diluted to 1/1000 and the revelation was carried out by adding human anti-IgG conjugated to black radish peroxidase, using as substrate tetramethylbenzidine in

presence of H202.

  The sera were declared positive when the optical density measured was greater than a value defined using normal sera, it being understood that the optical density of less than 2.5% of these normal sera

  could be greater than this value.

  The results obtained are shown in the table below. The whole recombinant enzyme does not give a detectable reaction under the conventional conditions chosen; on the other hand poly (ADP-ribose) gives interesting results, as underlined

other authors.

  The F2 peptide gives an unexpected result, when we refer to that obtained with the whole enzyme, human or natural recombinant calf, especially since very few sera have been found

  positive with the enzyme and negative with the peptide.

BOARD

  Positive subjects with with Disease Nb subjects Poly (ADP-ribose) Whole enzyme Peptide F2 LED n = - 97 41 (42.3%) 5 (5.2%) 34 (35.1%) pSS n = 67 14 (20.9%) 6 (9.0%) 28 (41.8%) sSS n = 16 7 (43.8%) 2 (12.5%) 9 (56.3%) JCA n = 38 1 (2.6%) I (2.6%) 0 (0%) MCTD n = 17 2 (11.8%) 1 (5.9%) 1 (5.9%) normal n = 42 1 (2 , 4%) 0 (0%) I (2.4%)

Claims (6)

  1. Immunological method for detecting and assaying antibodies directed against poly (ADP-ribose) polymerase in a biological sample, characterized   in that the antigen used is the peptide consisting   killing the finger F1 or the finger F2 of the zinc of said polymerase or a homologous peptide or one of their fragments, exhibiting an immunological reactivity substantially identical.   2. Method according to claim 1, character-   laughed at in that the antigen is the F2 finger of zinc from said polymerase.   3. Method according to one of claims 1   and 2, characterized in that the biological sample to be studied is brought into contact with the immobilized antigen and   that the immunological complex is revealed with an anti body marked by an enzyme.   4. Method for diagnosing an autoimmune disease chosen from systemic lupus erythematosus and Gougerot-Sjôgren syndrome, characterized in that the search for serum antibodies directed against poly (ADP-ribose) polymerase is carried out by a process   according to one of claims 1 to 3.   5. Method for diagnosing a precanc-   red or cancerous, characterized in that the   search for serum antibodies against poly-   (ADP-ribose) polymerase by a process according to one of claims 1 & 3.   6. Kit for detecting antibodies directed against poly (ADP-ribose) polymerase, characterized in that it comprises an antigenic peptide chosen from fingers F1 and F2 of said polymerase or a homologous peptide or one of their fragments , presenting a   substantially identical immunological reactivity,   you immobilized on a solid support, and possibly   a labeled antibody capable of binding to the antibody-antigen complex formed, and means for   evidence of attachment of said antibody to said complex.