Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

• the invention relates to the field of immunology, namely the field of interaction between an antigen and an antibody. More particularly, the invention relates to the use of a synthetic antigen (TCSP), derived from a protein of the unicellular parasite Trypanosoma Cruzi, which is the cause of Chagas' disease, for the detection of antibodies unrelated to Chagas disease, which can diagnose certain autoimmune diseases in a human patient.
• TCSP synthetic antigen
• infectious agents can escape the immune system of a host by interfering with the normal maturation of an effective humoral immune response, and by directing antibodies induced against autoantigens.
• certain infectious agents are capable of triggering an autoimmune response in hosts that exhibit a genetic predisposition.
• Chagas disease is endemic in Latin America and South America, and is a major cause of morbidity and mortality in the countries where it occurs. It is practically absent on other continents. About 16 to 18 million people are infected, and about 50,000 patients die each year. It is the infection with the unicellular parasite Trypanosoma Cruzi (T. cruzi), a member of the order Kinetoplastida and the family Trypanosomatidae, which induces Chagas disease in humans; this protozoan parasite is transmitted by several species of hematophagous insects, and in particular by the triatominae (haematophagous bugs).
• T. cruzi is known to infect cardiac and skeletal muscle cells, glial cells and mononuclear phagocyte cells. After passive penetration in the host cell, the trimastogite form of the parasite differentiates into amastigote form; it divides actively, then the trypomastigote forms are released, thus causing a new cell invasion. About 30% of patients with this disease develop severe cardiac clinical symptoms such as cardiomyopathies (see the article by K. Karratolios et al., "Inflammatory Cardiomyopathy", published in the journal Hellenic Journal of Cardiology, 2006, volume 47 , P. 54-65, and the article by J.
• Comparable cardiac impairment may be observed in patients infected with human immunodeficiency virus (HIV) or other infectious agents outside of any Chagas disease context. Comparable heart attacks may also occur with less frequency in apparently healthy subjects; We know (see the article by F. Kierszenbaum, “Views on the autoimmunity hypothesis for Chagas disease pathogenesis”, published in 2003 in the journal “FEMS Immunology and Medical Microbiology", vol 1545, pp. 1-11, and article by R. Jahns et al., "Pathological autoantibodies in cardiomyopathy", published in Autoimmunity, vol 41 (6), p 454-461, September 2008, showing that these disorders result from autoimmune mechanisms of undetermined origin.
• HCV human immunodeficiency virus
• T. cruzi antigen TCSP
• Antibodies have been described against (adrenergic or cholinergic) receptors without defining their specificity with respect to the TCSP peptide and in no way the involvement of a T. cruzi parasite antigen outside its natural context of Chagas disease. It is known that each stage of the T. cruzi parasite life cycle expresses specific antigenic proteins, as described, for example, in the article by Hoft et al.
• T. cruzi Expresses Various Repetitive Protein Antigens
• TCR70 which is identical to TCR 69
• TCR 70 has been highly conserved, with only occasional substitutions.
• Their frequent occurrence in all isolated fractions and the diversity of these repetitive units suggests that they are involved in the contortion of the destructive role of the immune system. Since these repetitive units are effective modulators of the immune system, it may be thought that other infectious agents use similar strategies, or even the same repetitive units.
• T. cruzi antibodies which are used in blood banks (particularly in Brazil where screening is mandatory), include indirect immunofluorescence (IFA), indirect haemagglutination (IHA), and assay by immunoenzymatic techniques (ELISA).
• IFA indirect immunofluorescence
• IHA indirect haemagglutination
• ELISA assay by immunoenzymatic techniques
• a second subject of the invention is an antigen which specifically binds to the antibody according to the first subject, with the exception of antigens which also bind to antibodies induced by T. cruzi and which are genetically encoded by parasite T. cruzi, that is to say with the exception of antigens comprising the sequence SEQ ID No. 1.
• a third object is the use of a peptide or protein having the sequence
• said peptide or said protein may be a TCSP peptide or a variant of the peptide
• TCSP provided that said variant has a specific activity vis-à-vis the antibodies according to the first subject of the invention.
• the antibodies can bind to antigens of an infectious agent, an allergen and / or an autoantigen; said infectious agent may be a pathogen, and the presence of said antibodies is then linked to an infectious or autoimmune disease.
• Said pathogen can also be selected from the group consisting of prions, viruses, prokaryotes, unicellular or multicellular eukaryotes.
• Said infectious or autoimmune disease can be selected from the group consisting of cardiomyopathy, myocarditis, systemic lupus erythematosus.
• Another object of the invention is a method for the detection of antibodies according to the first object in a liquid sample, this method comprising the following steps:
• a biological sample preferably a sample of body fluid and / or supernatant fluid of a cell culture ("liquid sample")
• a biological sample preferably a sample of body fluid and / or supernatant fluid of a cell culture ("liquid sample")
• a biological sample preferably a sample of body fluid and / or supernatant fluid of a cell culture ("liquid sample")
• a peptide or a protein comprising the sequence SEQ ID No. 1, SEQ ID NO: 2, SEQ ID NO: 3, or with a TCSP peptide or a variant of the TCSP peptide, as long as said peptide or said protein exhibits an antibody-specific activity according to claim 1
• the marker may be selected from the group consisting of chemoluminescence markers, agglutination markers
• kits or device for carrying out the method according to the preceding object, comprising: (a) a determined quantity of one or more peptides and / or proteins comprising the sequence SEQ ID No. 1, SEQ ID NO: 2, SEQ ID NO: 3, and / or a TCSP peptide or a variant of the TCSP peptide, as long as said peptides or proteins exhibit antibody-specific activity. according to the first object of the invention; (b) one or more appropriate markers capable of determining the complex formed between said peptide or protein and the antibody according to the first subject of the invention; (c) optionally, a solid support, preferably impregnated with said peptide or said protein.
• the marker may be selected from the group consisting of chemoluminescence markers, agglutination markers, membrane markers, immunoenzymatic markers, radioactive markers.
• an immunodiagnostic method comprising a step of qualitative or quantitative detection of the antibodies according to the first subject by the use of a peptide or a protein comprising the sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or with a TCSP peptide or a variant of the TCSP peptide, as long as said peptide or said protein exhibits an antibody-specific activity according to the first object.
• Yet another object is a method for reducing the level of antibodies according to the first object in a biological fluid sample, such as a blood sample, or in a flow of biological fluid, such as a blood fluid, comprising a step precipitating said antibodies with a peptide or a protein comprising the sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or with a TCSP peptide or a variant of the TCSP peptide, provided that said peptide or said protein has an antibody-specific activity according to the first object.
• the TCSP peptide or variants thereof can also be used to obtain antibodies or antibody fragments having NCRA-equivalent binding activity (Non-Cruzi-Related Antibody, ie, an antibody not induced by T. cruzi); this is yet another object of the invention.
• the peptides or proteins comprising one of the peptide sequences can be used to identify or even isolate the immunogens that induce the NCRA antibodies.
• Computer methods for searching sequence homologies using elaborate algorithms can serve as tools for identifying immunogenic candidates.
• Said immunogenic candidates can be synthesized and then tested for reactivity with biological samples suspected of containing NCRAs.
• TCSP peptide or a variant of the TCSP peptide forms another object of the present invention.
• This interaction results from a homology between the pathogen and the TCSP or the variant of the latter.
• Yet another subject of the invention is represented by the use of a peptide or a protein comprising the sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or a TCSP peptide. or a variant of a TCSP peptide, provided that said peptide or said protein exhibits an activity specific for antibodies according to the first subject of the invention, for obtaining antibodies or fragments of antibodies having equivalent binding activity to NCRA.
• a final subject of the invention is a pharmaceutical preparation comprising antibodies or antibody fragments having NCRA equivalent binding activity, a pharmaceutically acceptable injectable solution and optionally one or more excipients (such as polysorbate and / or sucrose). ) or additives, said antibodies or antibody fragments having been obtained either by use according to the third object in a living organism, leading to the immunization of said organism against the TCSP peptide, or by screening using the TCSP on a library of bacteriophages that express specific antibodies.
• excipients such as polysorbate and / or sucrose
• Figure 1 shows the signal intensity distribution related to the presence of antibodies that specifically bind to the TCSP peptide, and which are not necessarily related to T. cruzi infection (these antibodies being called NCRA, Non-Cruzi-Related Antibodies). These data come from an epidemiological test involving an N number of patients belonging to four groups:
• DS Healthy donors (ie, supervised and selected blood donors who are in better health than the general population)
• HIV Africa African Patients Responding Positively to Virus Screening
• HIV Western HIV: Western patients who respond positively to HIV testing.
• Chagas Patients infected with T. cruzi.
• Figure 2 shows the results of an epidemiological study of 79 patients, that is, the rate of NRCA as a function of time (in years) since the inclusion of patients in the study. Description
• Chagas disease refers to a pathological condition caused by infection with the parasite Trypanosoma Cruzi, parenterally or otherwise.
• non-Chagas disease refers to any condition not caused by the Trypanosoma Cruzi parasite, which results in increased reactivity of the antibodies against a novel antigen, referred to herein as TCSP, defined below.
• amino acid we mean natural amino acids and unnatural amino acids.
• Natural amino acids include the L-form of amino acids that can be found in naturally occurring proteins, that is: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).
• the "unnatural” amino acids include the D-form of natural amino acids, the homo forms of certain naturally occurring amino acids (such as: arginine, lysine, phenylalanine, and serine), and the nor forms of leucine and valine. They also include other synthetic amino acids.
• Protein compounds include especially peptides and polypeptides, including derivatives obtained for example by glycosylation, acetylation, phosphorylation, reaction with fatty acids. This term also includes proteins and peptides of natural origin.
• antibodies includes polyclonal and monoclonal antibodies.
• monoclonal antibody refers to an antibody composition having a homogeneous population, regardless of the species, origin and method of obtaining the antibody.
• antibodies includes human antibodies in which at least a portion of the immunoglobulin domains are present, such as antibody fragments and so-called VHVL domains (Variable Heavy and Variable Light Chains), and mini-antibodies.
• NCRA non-Cruzi-Related Antibodies
• TCSP Troposoma Cruzi Synthetic Peptide
• TCSP antigen TCSP peptide
• TCSP protein all mean a new peptide. as defined by the invention, which comprises an amino acid sequence which is also found in the TCR70 and TCR69 proteins, namely SEQ ID No. 1, defined below, which can be isolated in T. cruzi , and which acts as an antigen with NCRA, as well as all variants and functional equivalents, such as its linear or non-linear epitopes recognized by NCRA as defined above.
• SEQ ID N 0 2 defined below.
• autoantigen refers to an epitope present in an endogenous host molecule, which may be recognized by the immune system of said host, to eventually elicit an immune response. This mechanism can lead to an "autoimmune disease”. defined here as a pathological condition caused by an undesired immune response of a host against an autoantigen (also called auto-epitope).
• infectious agent refers here to any agent, living or not, capable of triggering an immune response. More particularly, it refers to pathogens, allergens and haptens.
• the agents . pathogens include prions, viruses, prokaryotes, eukaryotes, isolated or not.
• the allergens include any substances or molecules capable of spontaneously provoking an immune response in a host when said host is exposed to said substances or molecules.
• biological fluid refers to the body liguide of a living organism, such as a human patient, that is to say any liquid taken from a patient, such as serum, plasma, whole blood, urine, cerebrospinal fluid, saliva, or the supernatant fluid of a cell culture.
• the problem is solved by the use of a new antibody that reacts with a new antigen (TCSP), derived from a protein of a unicellular parasite that causes Chagas' disease, the Trypanosome Cruzi (T cruzi).
• TCSP new antigen
• NCRA antibodies antibodies not related to the T. cruzi parasite
• This phenomenon of heterologous recognition is explained by the structural mimetism of the TCSP antigen with another immunogen, endogenous or exogenous, which has immunized the individual and induces NCRA.
• the peptide sequence of the TCSP antigen is derived from a known protein and present in the Swissprot, Uniprot and TrEMBL databases (Accession Q7M3W1). This protein is known as TCR69; it is similar to the TCR70 protein. According to the invention, the peptide sequence of the protein used to define the NCRA 1 antibody expressed in three-letter amino acid codes, is:
• the immunoreactive variants of this peptide are also retained within the scope of the present invention, provided that they show the same antigenic properties.
• the variants of a peptide sequence may be obtained for example by substitution of one or more amino acids with other chemical entities, provided that the bioactivity of the original sequence is retained; they can also be obtained by adding chemical compounds such as biotin or natural or synthetic polymers, such as polylysine or polysaccharide. All such variants that retain the bioactivity of the original sequences are covered by the present invention.
• the TCSP can be obtained by purification from T. cruzi parasite protein extract, typically leading to TCR69 or TCR70 proteins (see Hoft et al., Cited above), which have the sequences SEQ ID N ° 1, SEQ ID No. 2 and SEQ ID No. 3.
• the TCSP and its variants can also be derived from a chemical synthesis (for example according to the method of Merrifield, well known to those skilled in the art). They can also be obtained by molecular cloning technology, such as I 1 recombinant DNA involving protein expression in microbial expression systems after insertion of a nucleotide sequence encoding the peptide sequence, followed by culturing extraction and purification of the peptide of interest.
• the TCSP antigens and its variants are not suitable for screening for Chagas disease because they lead to false positive reactions.
• the inventors have discovered that the TCSP interacts specifically with the NCRA. This means that the TCSP has a specific peptide sequence, which is able to bind to the NCRA. This binding can be detected by any known method, such as chemiluminescence, agglutination methods, immunoenzymatic or radioactive methods.
• the biomarker antibody is present in biological fluids, and its presence correlates with clinical symptoms in patients with cardiomyopathy, for example, HIV-infected individuals who have developed cardiac complications.
• the present invention relates to the peptide sequence of the TCSP as well as any structural analog capable of binding to the same biomarker antibody and which are not necessarily induced by the T. cruzi parasite (NCRA).
• the invention also relates to an immunoassay method for detecting and monitoring myocarditis and cardiomyopathies in patients following autoimmune infection or inflammation.
• One embodiment of the invention is a method for qualitatively or quantitatively determining the level of NCRA in a biological sample, comprising the following steps: a) Obtaining a biological sample, such as serum, plasma , whole blood, urine, cerebrospinal fluid, saliva, a biopsy specimen, or the supernatant fluid of a cell culture; b) Determination of the NCRA level in this biological sample.
• a biological sample such as serum, plasma , whole blood, urine, cerebrospinal fluid, saliva, a biopsy specimen, or the supernatant fluid of a cell culture.
• the biological sample may be in liquid, gel or solid form. It can come from a patient or from in vitro cultures.
• the present invention also relates to monitoring the treatment of cardiomyopathies, by determining the rate of NCRA.
• the method described above can also be applied to the estimation of the probability of fetal death caused by cardiac arrest in utero, because of the presence of NCRAs in pregnant women. Indeed, trans-placental passage of these antibodies can damage heart cells during embryogenesis. Their effects are all the more significant as the cardiac tissue is still primitive.
• a sample of a biological fluid of the patient, who is a pregnant woman is obtained and the rate of NCRA is determined in that sample.
• the determination of NCRA can lead to a therapeutic strategy in patients with cardiomyopathies or the development of a vaccine against this disease.
• the nature of the immunogen inducing biomarker antibodies is not known; however, the TCSP sequence disclosed in the present invention may lead to characterization of the etiology of the disease, isolation of an infectious or non-infectious agent inducing said NCRAs in human subjects not in contact with the parasite T. cruzi, and thus contribute to the development of new therapeutic strategies.
• anti-TCSP antibodies or antibody fragments can be designed to prevent, by competition, the binding of NCRAs to their biological target site and thereby inhibit their pathogenic effects. The design of such competing antibodies is made possible by this discovery.
• the measurement of NCRA longitudinally may indicate a change in cardiomyopathy and the effectiveness of its possible treatment.
• the subtraction of these same NCRAs from the bloodstream e.g. by immunoadsorption techniques, can reduce or even completely eliminate their pathogenic effects.
• the invention is based on the surprising discovery of the antigenic properties of a protein isolated from the parasite T. cruzi in subjects who have never been in contact with this parasite. Indeed, the TCSP antigen exhibits exceptional "cross-reactive" activity with NCRAs that are prevalent in living human subjects outside the endemic areas of the parasite. This is clearly a mechanism of peptide mimicry; these antibodies induce false-positive reactions when tested for Chagas disease.
• an embodiment of the present invention is to employ the TCSP polypeptide or its immunoreactive variants for the detection of NCRA.
• These NCRA antibodies can also bind to self antigens, or antigens of infectious organisms with more or less affinity and specificity than those of TCSP binding.
• the present invention provides a polypeptide as described above for the identification or isolation of an infectious or noninfectious agent inducing antibodies which in turn may cause an autoimmune disease.
• the present invention also provides a polypeptide as described above, wherein said infectious agent is a relatively common virus or microbe among subjects seemingly healthy and widespread, especially in HIV-infected individuals, such as mycoplasmas or other opportunistic infections.
• the present invention also makes it possible to employ a polypeptide as described above, wherein said autoimmune disease is selected from those described in cardiac pathologies such as cardiomyopathies and myocarditis.
• the present invention provides a method for detecting antibodies against the TCSP polypeptide, by contacting the TCSP, or a variant, with the NCRAs present in a biological fluid sample, detecting the binding of the NCRAs in the said biological sample by methods known to those skilled in the art
• the present invention provides an assay method for the detection of antibodies against the TCSP polypeptide, comprising reagents or tools for detecting antigen-antibody binding using methods known to those skilled in the art. immunoassay.
• the TCSP polypeptide or variants thereof are employed, provided that the latter have a specific activity against NCRAs 1 to improve the specificity of serological screening assays for the disease. of Chagas. It is clear from the results shown in Figure 1 that NCRAs, detected in screening tests, may be from an origin other than that of T. cruzi infection. Therefore, inhibition of these antibodies by the TCSP antigen may improve the specificity of Chagas disease diagnostic tests.
• the immunoreactivity tests in the present invention were carried out by an Enzyme-Linked Immunosorbent Assay (ELISA) technique, known to those skilled in the art; however any other technique for detecting or measuring the antigen-antibody binding can also be applied to the present invention, in particular to implement the new immunodiagnostic method which forms one of the objects of the invention.
• ELISA Enzyme-Linked Immunosorbent Assay
• Another aspect of the invention is the therapeutic use of the TCSP peptide or variants thereof for obtaining antibodies or antibody fragments having NCRA equivalent binding activity.
• antibodies or antibody fragments are first prepared by injecting a living organism (eg, animals) against the TCSP peptide.
• a pharmaceutical preparation comprising these antibodies or antibody fragments, a pharmaceutically acceptable injectable solution and optionally adjuvants (such as polysorbate, sucrose) is prepared.
• this pharmaceutical preparation is administered (eg injected) to a patient to compete with the pathogenic NCRAs.
• the rate of pathogenic NCRA is decreased, and the clinical symptoms of the patient improve.
• Yet another aspect of the invention is a method for reducing the level of antibodies according to the invention in a sample of biological fluid, and in particular a blood sample or in a blood stream, comprising a step of retaining said antibodies to using a peptide or a protein comprising the sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or with a TCSP peptide or a variant of the TCSP peptide, provided that said peptide or said protein has a specific activity against the antibodies according to the invention.
• This method can be implemented for diagnostic purposes, for the purpose of treating a quantity of biological fluid or for therapeutic purposes by plasmapheresis. It can be implemented statically or in a flow of said biological fluid.
• the biological fluid comes into contact with a solid support on which is fixed said peptide or said protein or protein comprising the sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or said TCSP peptide or a variant of said TCSP peptide.
• the antibodies if present, then precipitate on this solid support and are removed from the biological fluid.
• Another aspect of the invention is a pharmaceutical preparation comprising antibodies or antibody fragments having NCRA equivalent binding activity, a pharmaceutically acceptable injectable solution and optionally one or more adjuvants (such as polysorbate and / or sucrose) .
• These antibodies or antibody fragments can be obtained in different ways. In one embodiment, they can be obtained by screening using the TCSP on a bacteriophage library that express specific antibodies. In another embodiment, they can be obtained in a living organism, by using a peptide or protein comprising the sequence SEQ ID N 0 1, SEQ ID No. 2 or SEQ ID No. 3 for the detection of the antibodies that bind in a specific manner to a peptide having the sequence SEQ ID N C 1 and which are not induced in a host following its T. cruzi infection, said use leading to the immunization of said organism against the TCSP peptide.
• Yet another aspect of the invention is the use of the TCSP, and in particular the TCSP comprising the sequence SEQ ID No. 1, SEQ ID No. 2 or SEQ ID No. 3, in a vaccine composition, for immunizing a human at least partially against a disease other than Chagas disease, and in particular against autoimmune cardiomyopathy, myocarditis and systemic lupus erythematosus.
• This vaccination can be done by injecting a single dose or a plurality of doses.
• the TCSP can be used as such, as a peptide, or in biotinylated form and / or bound to an avidin derivative, and in particular to an avidin derivative obtained by known chemical and enzymatic modification. under the name NeutraLite Avidin.
• Said composition may comprise solvents, additives, buffers and pharmacologically acceptable carriers, as well as, if appropriate, other active ingredients.
• a peptide or a protein comprising the sequence SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, or a TCSP peptide or a variant of the TCSP peptide, is used. as long as said peptide or said protein exhibits an activity specific to the antibodies according to the invention, for identifying or isolating a pathogen which specifically binds to NCRAs or which is specifically recognized by NCRA.
• This new method identifies pathogens that can induce diseases other than Chagas disease that induce NCRA formation.
• the TCSP may be advantageous to label the TCSP, for example using a chromophore or a fluophore, or another easily detectable chemical entity (enzyme, radioactive isotope). , biotin, hapten, etc.).
• a chromophore or a fluophore or another easily detectable chemical entity (enzyme, radioactive isotope). , biotin, hapten, etc.).
• Example 1 These examples correspond to screening tests carried out on an N number of several populations of humans. Tables 1 and 2 and Figure 1 show results from these screening tests. We describe here the ELISA diagnostic technique used for these tests, as well as the successive stages of the tests
• the TCSP peptide represented by a synthetic peptide of sequence SEQ ID No. 1, was adsorbed on polystyrene microplates at a concentration of 1 ⁇ g / ml. The Free binding sites were then saturated with immunologically neutral proteins (in this case albumin). The microplates were then rinsed with a wash buffer and dried to be ready for use. Samples to be tested were incubated in the wells after dilution 1/20 in dilution buffer; antibodies not fixed on the antigenic surface were removed by three wash cycles. The specific TCSP antibodies fixed on the microplates were detected by an antibody against human IgG and labeled with an enzyme tracer. A chromogenic substrate of the enzyme employed served to reveal the binding of the anti-TCSP by measuring the optical density corresponding to the absorption of the chromogen in an adequate range of wavelengths, and in particular to a length of wave of about 450 nm.
• T. cruzi uses a highly immunogenic motif that could be used by other pathogens as well, and / or that could give rise to autoimmune antibodies.
• Reaction of the TCSP peptide with the NCRAs present in patient sera by interaction of the sera samples and solid surfaces on which the TCSP was previously adsorbed. 4. Reaction of the TCSP with the sera of patients infected with the T. cruzi parasite: Since the TCSP antigen is derived from a protein of the T. cruzi parasite, it is difficult, by conventional techniques, to distinguish the antibodies induced by T. cruzi infection of those induced by the mechanism of autoimmunity (NCRA). In an ELISA using the TCSP as an antigen, it was found that both classes of antibodies are confounded; the EIA result is not discriminating.

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