FR2864624A1 - Serological diagnosis of infectious diseases uses microbial antigen immunodetection with monitoring of false positive or negative reactions - Google Patents

Abstract

The in vitro serological diagnostic method for detecting the presence and preferably the quantity of immunoglobulins of a microbial antigen characteristic of a microbial agent in a sample of serum taken from a Class M patient, is new. The in vitro serological diagnostic method for detecting the presence and preferably the quantity of immunoglobulins of a microbial antigen characteristic of a microbial agent in a sample of serum taken from a Class M patient includes monitoring of a possible rheumatoid factor in the test sample by placing the sample in contact with a solid support, e.g. of latex micro-balls, on which a first antigen corresponding to a non-specific Class G immunoglobulin has been fixed, is new. The contact is made in the presence of a first detection substance to see if the non-specific Class G immunoglobulin reacts with it.

Description

METHOD OF SEROLOGICAL DIAGNOSIS OF DISEASES

  INFECTIOUS BY IMMUNODETECTION OF ANTIGEN

  MICROBIAL COMPRISING REACTION CONTROL

FALSE POSITIVE OR NEGATIVE

  The present invention relates to a method for diagnosing human and animal infectious diseases. More particularly, it relates to the field of diagnosis of human and animal infectious diseases by microbial serology. It is a method of indirect diagnosis which is based on the search in the patient's serum of antibodies specific for the infectious microbial agent, namely a bacterium, a virus, a parasite or a fungus responsible for a particular infection. pathology (hereinafter referred to as "microbe").

  In the present invention, the term "patient" is taken broadly, including human patients and also animals. In particular, the term "animal" means: - birds, more particularly poultry raised for their eggs or their flesh, and mammals, especially dogs, cats, horses, cattle such as calves, cows sheep such as sheep and goats such as goats.

  This method of diagnosis is essential for the diagnosis of microbe infections, sampling, culture and / or difficult identification, in particular strict intracellular viruses and bacteria and facultative intracellular bacteria of the genera Rickettsia, Coxiella, Bartonella, Tropheryma, Ehrlichia , Chlamydia, Mycoplasma, Treponema, Borrelia, and Leptospira, for example, for which antigens consisting of whole microbes or fractions or fragments of microbes comprising one or more antigens are used as microbial antigens. It is indeed possible to mechanically break up the microbe by mechanical agitation or by sonication, for example, or to fragment the microbe by an enzymatic process in order to obtain a fraction that retains the antigens that support the serological reaction, object of the present invention. . The fractions thus obtained are separated or further purified from the other components of the microbe and its culture medium by a suitable method, for example by centrifugation or filtration. This is called an antigenic fraction or purified antigen. The entire microbe or any fraction of the microbe is hereinafter referred to as a "particulate or corpuscular antigen" in that the whole microbe or a portion thereof can not be dissolvable but only suspended in a suitable fluid. The microbes or their fraction remain visible as individualizable particles by microscopic observation using an optical microscope or an electron microscope, for example.

  Technically, the microbial serology consists in detecting in the patient's serum an antigen-antibody reaction in which the antigen is represented by all or fraction of the infectious microbial agent to be detected and the antibody is represented by the human or animal immunoglobulins specific for said infectious microbial agent present in the patient's serum. It can be quantified by successively testing a series of increasing dilutions of reason 2 or patient's serum ratio from a first 1:16 or 1:50 dilution of the patient's serum.

  Conventionally, a method of serological diagnosis more particularly comprises the deposition of the antigen on a solid support such as latex microbeads, in particular in the agglutination detection technique in which latex microbeads are covered by the microbial antigen. to be tested, and are agglutinated to each other by the patient's serum exhibiting specific antibodies, this agglutination being visible to the naked eye.

  However, the detection technique by agglutination test is both impractical to implement and insensitive. It also involves the use of large amounts of reagents and the patient's serum, and finally the reading of the results is not automatable. This test of detection by agglutination can not therefore be implemented in the case of tests to be performed in series on a large number of sera.

  According to the method of the invention, therefore, the antigen is deposited, if appropriate, on a solid support of the glass slide type, or titration microplate to implement detection techniques by immunodetection, in particular immunofluorescence, or by technique. enzymatic, in particular of the ELISA type "Enzyme Linked Immunosorbent Assay" or a sheet of nitrocellulose or nylon for detection techniques of the Western-blot type, in which the microbial antigens separated by electrophoresis and then transferred onto the solid support (nitrocellulose sheet or of nylon) react separately from one another with the patient's serum. These detection techniques are well known to those skilled in the art, and include the successive steps of: 1. decomplementation of the serum by heating at 56 ° C. for 30 minutes, 2. contacting the antigen corresponding to the infectious microbial agent fixed on a solid support, with the serum of the patient then incubation under conditions of time, temperature, hygrometry, mechanical agitation and ionic strength of the medium allowing the reaction antigen antibody, 3. cautious and extensive washing to eliminate the serum of the patient not fixed to the solid support, in excess, 4. application of a secondary detection antibody which is an animal immunoglobulin directed against immunoglobulins of the species of the patient in question, for example in the case of a human patient a goat anti-human immunoglobulin immunoglobulin conjugated to a fluorochrome substance, generally fluorescein iso-thiocyanate or an enzyme, usually a peroxidase and incubation under conditions of time, temperature, hygrometry, mechanical agitation and ionic strength of the medium allowing the reaction antigen antibody, 5. cautious and extensive washing to eliminate the labeled immunoglobulin, no fixed, in excess, 6. detection of a reaction by reading, using an appropriate apparatus depending on the marker such as a fluorescence microscope or a microarray reader for the technique of indirect immunofluorescence, or an optical density reader for the enzymatic detection technique of the ELISA type. The Western-Blot reaction is read at the naked eye or after digital acquisition of the gel and analyzed by densitometry software or any appropriate software for image processing.

  The present invention relates more particularly to detection and assay methods in which the presence is detected and, preferably, the amount of class M (IgM) and G (IgG) immunoglobulins specific for a microbial antigen characteristic of a microbe. These determinations provide more accurate and complete information necessary to establish the etiology and follow the evolution of certain infectious diseases. Thus, in general IgM appear earlier. As for IgG, they make it possible to establish the serological status of the patient with respect to a given microbe, in order to establish the serological diagnosis of the infection or to establish the degree of protection of the organism against this microbe. .

  In these methods, two types of secondary detection antibodies labeled with different markers are used, in general animal immunoglobulins directed against the immunoglobulins of the species of the patient in question, for example in the case of a human patient an immunoglobulin of goat anti-human immunoglobulin anti IgM and anti human IgG that react with class M or G specific immunoglobulins complexed with said microbial antigen.

  The specificity of the infectious antigen / serum antibody reaction conditions the specificity and positive predictive value of the serological test based on this reaction, and any binding of a nonspecific antibody to the microbial antigen limits the specificity and predictive value of the serological test. The presence in the test serum, rheumatoid factors or anti-nuclear antibodies, is a source of non-specific binding of antibodies to the microbial antigen as explained below.

  In humans, rheumatoid factors were initially described by Waller [Waller E. Acta Pathol. Microbiol. Scand. 1940, 17: 172-8] and Rose et al. [Rose H.M., Ragan C., Pearce E., Lipmann M.O. Proc. Soc. Exp. Biol. Med. 1948, 68: 1-11] as serum immunoglobulins of rheumatoid arthritis patient capable of agglutinating sheep erythrocytes coated with rabbit class G immunoglobulin (IgG). It has then been shown that some of these rheumatoid factors are class M immunoglobulins (IgM) recognizing the IgG Fc fragment of different species including human IgG.

  Several modifications of the Waller and Rose reaction have resulted in the commercialization of rheumatoid factor testing using an IgG-coated latex bead agglutination method [Singer J.M. Plotz, C.M. Am. J. Med. 1956; 21: 888-92]. The prevalence of rheumatoid factors detected by agglutination is 3.3% (for a titre> 1:20) [Mikkelsen W.M. et al. J. Chron. Dis. 1967, 20: 351-69]. The presence of rheumatoid factors in the serum of a patient - more important in elderly patients - is responsible for false positive results when detecting microbial antigen-specific IgM in serological tests for the indirect diagnosis of diseases infectious [Fuccillo DA et al. Manual of Clinical Laboratory Fourth Edition Immunology, Rose N.R., Macaria E. Conway, Fahey J.L., Friedman H., Penn G.M.

  editors, American Society for Microbiology, Washington D.C., 1992, 545-53]. In fact, these rheumatoid factors bind to the IgG specific for the microbial antigen contained in the serum of the patient, and therefore appear false-positive when detecting specific IgMs in the serological reaction using the microbial antigen in detection tests. agglutination. [Relay C.B. et al. Ann. N. Y. Acad. Sci. 1975, 254: 77-93].

  It is possible to eliminate rheumatoid factors from serum by adsorption by non-specific human IgG which is introduced in excess into the sample. These introduced IgGs are modified by heating or treatment with glutaraldehyde so that they do not interfere with the detection of specific IgGs. This addition of IgG causes precipitation after complexation with the anti IgG substances contained in the sample. But, in this case, the specific IgG and IgM contained in the sample can also be denatured by the modification treatment of IgG introduced into the sample.

  It is also possible to remove total IgM from serum with Staphylococcus aureus protein A or with heterologous antibodies (sheep, for example) directed against human IgM. In this case, however, the formation of heterologous antibody-human IgG complexes results in both adsorption of rheumatoid factors and specific IgM of the microbial antigen.

  Although systematic adsorption of rheumatoid factors is recommended before the serological detection of microbe-specific IgM, there is no systematic control of the efficacy of this adsorption until the implementation of serological reaction.

  Likewise, the presence in the patient's serum of anti-nuclear antibodies limits the specificity of the microbial antigen-serum antibody reaction. Anti-nuclear antibodies are indeed IgG antibodies directed nonspecifically against the set formed by DNA and nuclear proteins of the chromosome of eukaryotic cells and microbes, called histones. These anti-nuclear antibodies therefore bind nonspecifically on any eukaryotic cell including fungi and parasites and on any microbe, bacterium, DNA virus, parasite or fungus. This phenomenon leads to a non-specific positive reaction during serological tests using the detection of IgG specific to a bacterium, a DNA virus, a fungus or a whole parasite or comprising DNA / histone complexes as a microbial antigen. using anti-IgG detection antibodies in a serum containing anti-nuclear antibodies.

  The detection of anti-nuclear antibodies in the serum to be tested, carried out using an immunofluorescence immunodetection technique, has been described [Fritzler MJ In: Manual of Clinical Laboratory Immunology, Fourth Edition, Rose NR, Conway of Macario E., Fahey JL, Friedman H., Penn, GM, (eds.). American Society for Microbiology, Washington, D.C. 1992, pp. 724-729] using mammalian cells including human cells such as Hep2 cells as antigen. Under these conditions, the prevalence of antinuclear antibodies detected on pure serum is 2% in the general population, 20% among parents of patients with rheumatoid arthritis, and 75% among the elderly without apparent clinical pathology [Mc Carty GA, et al. Antinuclear antibodies: contemporary techniques and clinical applications to connective tissue diseases. Oxford University Press, New York, 1984]. In this method, the cells consist of a stack of confluent cells deposited and read manually. This deposit of confluent cells is too viscous to be robotisable by deposition with a syringe-type robot, and the reading is not automatable because only manual deposition allows to deposit a large quantity of cells so as to ensure sufficient detection of the reaction Cell-anti-nuclear antibodies. In these publications, there is no mention of the need for systematic monitoring of the presence of anti-nuclear antibodies in immunodetection tests of microbial antigens. In addition, detection with confluent cells manually deposited on solid support is not applicable for routine laboratory tests.

  There is therefore no method published to date, for the control or elimination of anti-nuclear antibodies before performing a serological test for the diagnosis of infectious diseases so as to ensure the absence of the result a false positive reaction applicable in routine laboratory. Therefore, no serology protocol published in the reference manuals nor any commercially available serological test systematically includes the screening of antinuclear antibodies or rheumatoid factor as a prerequisite for the realization or interpretation of the serology.

  In practice, to date, in laboratory routine immunodetection tests, it is only possible to detect false positives due to the presence of rheumatoid factor (or antinuclear antibodies) by performing tests on a plurality of cells. microbial antigens whose concomitant presence is not likely, but this type of verification, it is understood, significantly increases the cost in terms of material and labor and the duration of tests.

  There are known methods and diagnostic kits involving the use of a solid support on which soluble proteins are covalently attached, but these covalent chemical couplings are complex and expensive to perform. The non-covalent attachment of soluble protein or particulate antigen to a solid support has been proposed by physical or physico-chemical adsorption on the support, as part of the solid-base immunodetection test protocols, but the stability of the fixation is insufficient. One difficulty lies in the fact that it is necessary to thoroughly wash the solid support beforehand to eliminate residues of marking elements that may interfere with the reading of the results, whereas these washes make the physical adsorption of the substances on the solid support too unstable. . Another difficulty is that it is not possible to deposit with robots of corpuscular antigens whether cells or whole or partial bacteria as mentioned above.

  A first object of the present invention is to provide a reliable immunodetection test for microbial antigens, in particular particulate antigens, by a method and tools that are simple to implement and to perform for routine laboratory application within the context of the present invention. serial tests, in the case where the test serum may contain rheumatoid factors or antinuclear antibodies.

  Another object of the present invention is to provide a solid support preparation method allowing robotic deposition and non-covalent attachment by physical adsorption on the support of control antigens of the presence of rheumatoid factor and anti-nuclear antibody and microbial antigen, consisting of particulate proteins or antigens as explained above, as well as automated reading of the control of absence of rheumatoid factor and anti-nuclear antibodies and the result of the immunological reaction in a patient serum test involving a microbial serology reaction of IgM or specific IgG by adsorption with the microbial antigen particulate or corpuscular deposited on a solid support.

  Likewise, no commercially available serological test systematically includes the control of the introduction and the reactive value (or immunological reactivity) of the anti-IgM or anti-IgG detection secondary antibodies used, and another original purpose of the present invention. is to provide a test including this reactivity control.

  To this end, the present invention provides a method for in vitro serological diagnosis of microbial agent by immunodetection in which the presence is detected and, preferably, the amount of patient immunoglobulins, in particular human, is measured for a patient of the patient. human species, of class M specific for a microbial antigen characteristic of said microbial agent, in a patient serum sample to be tested, in particular human, by detection and, preferably, quantification of an immunological reaction complex between said antigen a microbial to be detected and a said class M specific immunoglobulin, using a first detection substance comprising a first labeling element, preferably a first labeling antibody, reacting only with a said class M immunoglobulin, of the species of the patient, in particular human, characterized in that the presence of rheumatoid factor in said sample to be tested by contacting said test sample with a solid support on which a first antigen corresponding to a non-specific class G immunoglobulin, preferably an immunoglobulin of the patient's particular human species, has been fixed, the setting contact being made in the presence of said first detection substance, and it is verified whether said non-specific class G immunoglobulin fixed on the solid support, reacted with said first detection substance.

  If the sample comprises rheumatoid factors (anti-IgG IgM), these can react with the immunoglobulins (IgG1) fixed on the solid support, in the form of the following complex with said first detection substance (Ac, anti IgM * '): S-IgG, IgM anti-IgG-Act IgM * ').

  When the absence of rheumatoid factor is established, the detection of a reaction between said first detection substance and said microbial antigen (Agmic) is indeed evidence of the presence in the test serum of class M-specific immunoglobulins. microbial antigen (IgM anti Agmic) by formation of the complex (S-Agmic-IgM anti Ag-IgM anti IgM * 1), and not the presence of IgG specific for said microbial antigen (anti Agmic IgG), which could in effect, in the presence of rheumatoid factor, forming a false positive complex (S-Agmic-IgG anti-Agmic-IgM anti-IgG-Act anti IgM * 1).

  Furthermore, as explained below, when said first antigen consists of an IgG of the particular human patient species, this makes it possible to check the presence and reactivity of a second detection substance specifically recognizing serum IgGs. of the patient's species.

  Advantageously, in a diagnostic method according to the invention, the amount of immunoglobulin of the patient, in particular a class G human patient specific for a said microbial antigen, is detected by detection and, preferably, quantifying an immunological reaction complex between said microbial antigen and a class G specific immunoglobulin with a second detection substance comprising a second labeling element, preferably a second labeling antibody comprising a second marking element different from said first marking element, said second detection substance only reacting with a said class G human immunoglobulin, especially of the patient's species, characterized in that the possible presence is checked; anti-nuclear antibody in said test sample by contacting said test sample with a solid support on which has been fixed a second antigen comprising DNA / histone complexes, preferably comprising nuclei of nucleated cells of the species of the patient, in particular human, more preferably, all or part of cells of origin of the species of the patient, in particular human, in continuous line, and it is checked whether said second detection substance has reacted with said second antigen fixed on the solid support.

  In the case of a human patient, it is possible, in particular, to use non-confluent human fibroblast cells in suspension, in particular HL60 cells.

  If the sample to be tested comprises antinuclear antibodies (which are IgG, in particular human), they can react with said second antigen (Ag2) and be detected by said second labeling substance (anti IgG * 2 Ac2) since that It is a substance reacting with IgG of the patient's species, in particular human, and forming the anti-IgG * 2 anti-Ag2-Ac2 complex (S-Ag2-IgG).

  If, in addition, a complex is detected on the solid support to which said first antigen (IgG1,) is attached, a complex between said first antigen and said second marking substance, namely necessarily a complex (S-IgG1-Ac2 anti IgG * 2 ), this confirms the absence of rheumatoid factor.

  The detection of a reaction between said first detection substance and said second solid-supported antigen (Ag2) necessarily signifies that a complex is formed with antinuclear antibodies (Ab anti-nucl) according to (S-Ag2- IgG anti-IgM IgG anti IgM * IgM * 1), and therefore that the sample includes both rheumatoid factor and antinuclear antibodies.

  Once the absence of anti-nuclear antibodies has been established, the detection of a reaction of said second labeling substance with said microbial antigen is evidence of the presence of IgG specific for said microbial antigen and for the formation of a anti-IgG-anti IgG * 2 anti-IgM-IgG complex and not a false positive complex resulting from the reaction of the anti-nuclear antibodies with the microbial antigen according to the complex (S-Agmic-Ac anti-nucl. -Ac2 anti IgG * 2).

  Preferably, when the presence of immunoglobulins of the patient, in particular human, class G specific for a said microbial antigen is detected using a second said detection substance comprising a second marking element. A check is made of the presence and reactivity of said second detection substance by contacting said solid support, on which said first antigen corresponding to a said immunoglobulin of the patient's species, in particular human, non-specific, has been fixed. class G, with a solution containing a said second anti-IgG labeling antibody and not containing rheumatoid factor, and verifying whether said second detection substance has reacted with said first antigen fixed on the solid support.

  More preferably, the reactivity of said second detection substance introduced into said serum sample to be tested is verified after verifying the absence of rheumatoid factor and anti-nuclear antibodies in said sample.

  If said second detection substance is reactive in the absence of rheumatoid factor, the following complex (anti-IgG S-IgG1-Ac2) with said first antigen (IgG) must be detected. In this case, the absence of reaction of said second detection substance with said second antigen is evidence of the absence of anti-nuclear antibodies.

  In an advantageous embodiment, a check is made of the presence and the reactivity of said first marking substance which is an anti-immunoglobulin M antibody of the patient's species, in particular human, by putting into contact a support. solid on which is fixed a third antigen corresponding to an immunoglobulin of the patient's species, in particular human, non-specific class M (IgM1), with a solution containing a said first marking substance and not containing rheumatoid factor, and checking whether said first substance reacts with said third antigen fixed on the solid support.

  If said first detection substance is present and is well reactive, an anti-IgM * 1 S-IgM1-Aci complex must be detected. Thus, the absence of detection of a complex comprising said first marker at said first antigen (IgG1) and, where appropriate, at the level of said second antigen fixed on solid support, is the proof of the absence of rheumatoid factor.

  More particularly, the reactivity of said first detection substance is verified after checking for the absence of rheumatoid factor. Otherwise, in the presence of rheumatoid factor, no reaction of said first detection substance with said third antigen could be detected. formation of a subsequent complex with rheumatoid factor (S-IgM1-IgG anti IgM). However, in the presence of both said first and second detection substances, a reaction between said third antigen and said second detection substance is detected by the detection of the complex (IgM-IgM1 IgG anti IgM Ace anti IgG * 2).

  More particularly, the deposition of IgG of the patient's species, in particular human, y-specific (specific to the gamma chain of immunoglobulins of the patient's species, in particular human) as said first antigen makes it possible to positively control the introduction of conjugated anti-IgG immunoglobulin during the serological reaction as well as its immunological reactivity (qualitative aspect). Indeed, this conjugated antibody will also bind to the IgG deposition which will therefore be recognized during the IgG detection phase specific to the serological reaction. Also, the deposition of γ-specific immunoglobulin class M (IgM) as said third antigen by robotic deposition of an IgM solution of the patient's species, in particular human, y-specific to detect the introduction and the Immunological reactivity (qualitative aspect) of the conjugated anti-IgM immunoglobulin during the serological reaction is an important innovation because this type of positive control is not systematically used currently during serological reactions.

  The present invention thus allows the systematic detection of rheumatoid factors, and the systematic detection of anti-nuclear antibodies, in a serum used for an indirect immunofluorescence serological diagnosis, after the robotic deposition of immunoglobulins of the patient's species, in particular class G (IgG) and nucleated cells of the patient's species, in particular human, on the support of the serological reaction. Also, the robotized deposition of immunoglobulins of the patient's species, in particular human, IgG and IgM, makes it possible to control the presence of the anti-immunoglobulin secondary antibodies M and G respectively of the patient's species, in particular human, used.

  A frequent error in the performance of serological tests, especially for battery serological tests carried out on a large number of test sera, is due to defects in the introduction of test sera, in particular by pipetting. These errors occur in particular in the steps that involve the displacement of the sample to be tested, in particular by pipetting, certain containers, in particular containing the solid support on which the antigen to be detected is deposited, may not be inadvertently filled with the serum. of the species of the patient, in particular human, to be tested. Serum pipetting is known to have a 1% error risk, due to a purely technical problem of no pipette pipetting, or to human error due to inadvertent pipetting.

  These errors impose the introduction of controls in the realization of the reaction. The systematic incorporation during each new manipulation of a negative control serum that is to say not containing antibodies specific for the antigen to be tested makes it possible to interpret the positive reactions. Similarly, the incorporation of a positive control serum, that is to say containing the antibody specific for the antigen tested with a known titre makes it possible to check the quality of the antigen and the conjugated immunoglobulin.

  However, there is currently no control of whether the serum to be tested has been introduced into the serological test. If, inadvertently, the serum to be tested is not introduced into the serological test, the bacterial antigen-serum antibody reaction will certainly not exist, and the test will be interpreted, falsely, as negative (false-negative). In the present invention, it is used that the protein A of Staphylococcus aureus (Staphylococcus aureus) has an affinity for serums of animal origin, in particular the horse, the cattle, the pig, the rabbit, the guinea pig, the mouse ; to a lesser degree hamster, rat and sheep. On the other hand, chick and goat serum do not react with protein A. Protein A is a 42 kDa polypeptide, and is a wall constituent of Staphylococcus aureus strains, similar but different proteins are characterized. on the surface of bacteria of the genus Streptococcus [Langone JJ Adv. Immunol. 1982, 32: 157-251]. This property of Staphylococcus aureus protein A has already been used in a serological test in humans for the serological diagnosis of infectious endocarditis [Rolain JM, Lecam C, Raoult D. Simplified serological diagnosis of endocarditis due to Coxiella burnetii and Bartonella. Clin. Diag. Lab. Immunol. 2003; 10: 1147-8].

  The present invention thus comprises the introduction of an introduction control of the serum to be tested during bacterial serology reactions.

  For this purpose, an antigen containing protein A is introduced and, in particular, introducing a Staphylococcus aureus bacterium as a control antigen that the test sample contains a serum of the patient's species, in particular of human origin, and to detect an antigen-immunoglobulin reaction of the patient's species, in particular human, with a specific substance.

  Since protein A reacts with animal and human immunoglobulins nonspecifically, even in the case of significant infectious pathology, it is possible to use this protein A as a positive control of the introduction of a serum of the species of the patient, in particular human, in the sample to be tested.

  More specifically, the present invention provides a method for in-vitro serological diagnosis in which the presence of antibodies specific for an infectious microbial agent is detected in a test sample, characterized in that it is controlled that said sample to be tested. contains a serum of the patient's species, in particular human, by detecting whether immunoglobulins of the patient's species, in particular human, react with an antigen containing the protein A of a bacterium Staphylococcus aureus.

  More particularly, according to the present invention, it is previously checked that said tested sample does indeed contain a serum of the patient's species, in particular human, by detecting whether immunoglobulins of the patient's species, in particular human, react with a fourth antigen containing the protein A of a bacterium Staphylococcus aureus, preferably by contacting said sample with a solid support to which is attached a said fourth antigen, in the presence of a third detection substance which is a substance reacting with a immunoglobulin of the patient's species, especially human, and not with said fourth antigen, preferably an anti-immunoglobulin antibody of the patient's species, in particular human, and not reacting with the protein A. In an advantageous embodiment said fourth antigen is a whole bacterium Staphylococcus aureus comprising protein A. More particularly use the Staphylococcus aureus bacteria deposited in public collections such as bacteria deposited at AC.C.C. under N 29213 and at C.N.C.M. of the Institut Pasteur (France) under the number 65.8T as described in the publication mentioned above [Rolain JM, Lecam C, Raoult D. Simplified serological diagnosis of endocarditis due to Coxiella burnetii and Bartonella. Clin. Diag. Lab. Immunol. 2003; 10: 1147-8].

  Furthermore, apart from the standard strains, any bacterial strain identified as Staphylococcus aureus can be used as AgI antigen.

  Advantageously, the presence of a said product of the reaction is detected with an anti-immunoglobulin immunoglobulin of the patient's species which is an immunoglobulin of animal origin, preferably in the case of a human patient, an immunoglobulin of goat or chick.

  Preferably, said detection substance, preferably, if appropriate, said third detection substance, is an animal immunoglobulin, more preferably in the case of a human patient, a goat or chick immunoglobulin.

  Also preferably, said third detection substance corresponds to said first or second detection substance and, preferably, said first and second detection substances are an immunoglobulin of animal origin in the case of a human patient, respectively anti-IgM. or IgG anti marked respectively by two different marking elements.

  Advantageously, said detection substance is an antibody conjugated to a marking element which is a fluorescent substance. And, in particular, it is verified whether a reaction product complex between said fourth antigen and said third detection substance is detected by fluorescence.

  Advantageously, in a diagnostic method according to the invention, the dose of said immunoglobulin of the species of the test patient of class M or, where appropriate, of class G, specific to the test, is detected and, where appropriate, quantified. said microbial antigen in the sample to be tested by automated reading using a reading device suitable for said marking elements.

  As marking element of said detection substances, it is therefore advantageous to use enzymatic labeling, radioactive or fluorescent, the latter type of fluorescent labeling being preferred.

  The term fluorescent labeling means that the antibody has been made fluorescent by coupling or complexing with a suitable fluorescent agent such as fluorescein iso (thio) cyanate or any other substance which emits detectable radiation after its illumination, each substance being characterized by the wavelength at which it is to be illuminated (excitation wavelength) and the wavelength of the radiation it emits (emission wavelength).

  The expression "radioactive labeling" means that the antibody carries a radioactive isotope making it possible to measure it by counting the radioactivity associated with it, the isotope being able to be carried either on one element of the structure of the antibody, for example the residues constitutive tyrosine, or on a suitable radical which has been fixed to it.

  Enzyme tagging means that the specific antibody is coupled or complexed to an enzyme that, when associated with the use of appropriate reagents, allows quantitative measurement of that specific antibody.

  The substrate and the reagents are chosen so that the final product of the reaction or of the reaction sequence caused by the enzyme and employing these substances is: either a colored or fluorescent substance which diffuses in the liquid medium surrounding the tested sample and which is the subject of either the final spectrophotometric or fluorimetric measurement, respectively, or an evaluation by the eye; optionally with respect to a range of calibrated colors, or an insoluble colored substance which is deposited on the tested sample and which may be the subject of either a reflection-based photometric measurement or an evaluation at the eye, possibly with respect to a range of calibrated shades.

  When a fluorescent detection substance is used, the fluorescence associated with the test sample is read directly on a suitable apparatus capable of detecting the radiation at the emission wavelength and quantifying it.

  When using a radioactive probe, such as for example iodine 125, the radioactivity associated with the sample tested is completed in a gamma counter in any appropriate manner and for example after solubilization of the cells with an alkaline solution (for example a solution soda) and recovery of the solution containing the radioactivity using an absorbent pad.

  When an enzyme is used as a labeling element on the detection antibody, the appearance of a colored or fluorescent product is obtained by adding a solution containing the substrate of the enzyme and one or more auxiliary reagents for finally obtaining as a reaction product, either a colored product soluble in the medium, an insoluble color product, or a soluble fluorescent product, as explained above. The light signal coming from the samples thus treated is then measured using the apparatus adapted to each case: photometer in transmission, or in reflection or fluorimeter respectively. Alternatively, the coloration obtained can also be evaluated by eye, possibly with the aid of a range of calibrated colored solutions.

  By using alkaline phosphatase as the enzyme, the coupling of this enzyme with the detection antibody is carried out according to the method proposed by Boehringer Mannheim-Biochemica. The preferred substrates for this enzyme are paranitrophenyl phosphate for a fluorometric reading or 5-bromo-4-chlorobenzyl phosphate for a fluorometric reading or 5-bromo-4-chloroindolyl-6-phosphate to obtain an insoluble color reaction product. It is also possible to use as the enzyme [3-galactosidase whose preferred substrates are orthonitrophenyl [1-D-galactropyranoside or 4-methylumbelliferyl] D-galactopyranoside.

  Detection antibodies can also be coupled to peroxidase. In that case; the coupling method is derived from that described by M. B. Wilson and P. K. Nakane in Immunofluorescence and related staining techniques, W. Knapp, K. Kolubar, G. Wicks ed. Elsevier / North Holland. Amsterdam 1978, p. 215-224.

  The reagents used to reveal the peroxidase conjugated to the detection antibodies contain oxygenated water, substrate of the enzyme, and a suitable chromogen, for example orthophenylenediamine or 2-azino-2'-bis (ethyl-3) acid. thiazolin-6-sulfonic acid) or ABTS to obtain a final reaction product colored and soluble in the medium or 3,3-diamino-benzidine or 3-amino-9-ethyl carbazole or 4-chloro-anaphthol to obtain a product final insoluble reaction, or parahydroxyphenyl propionic acid to obtain a fluorescent reaction product soluble in the medium.

  Another embodiment of the invention is the use of detection immunoglobulin coupled to acetylcholinesterase.

  The acetylcholinesterase is coupled to the antibody, preferably using a method 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 a suitable heterobifunctional agent and finally the coupling of the products thus obtained. Other known methods of constructing immunoenzymatic conjugates can also be used in this case.

  The revelation of the enzymatic activity specifically related to the antigen recognized by the acethylcholinesterase conjugate is preferably carried out according to the well-known technique which employs acethylthiocholine as substrate for the enzyme and Ellman's reagent, or acid 5-dithio-5'-nitro-2-benzoic as chromogen, according to any variant adapted to the case examined, for example that described by Pradelles et al., in Anal. Chem. 1985, 57: 1170-1173.

  Advantageously, in the diagnostic method according to the invention, the presence is detected and, where appropriate, the amount of class M or, if appropriate, class G immunoglobulin specific for said microbial antigen is measured in said sample. test serum, by contacting a said microbial antigen fixed on a solid support, with said serum sample containing a said first or respectively second detection substance reacting specifically with the class M or, respectively, the immunoglobulin class G of the species of the test patient specific for said microbial antigen.

  Advantageously, said microbial antigen is a particulate or corpuscular antigen as described above, consisting of inactivated whole microbe or microbe fraction.

  In a particular embodiment, said microbial antigen is selected from microorganisms comprising a bacterium, a virus, a parasite or a fungus.

  More particularly, said microbial antigen is an intracellular bacterium, and in particular, said microbial antigen is selected from bacteria of the genus Rickettsia, Coxiella, Bartonella, Tropheryma, Ehrlichia, Chlamydia, Mycoplasma, Treponema, Borrelia, and Leptospira, this list not being not exhaustive.

  More particularly, said microbial antigen is a bacterium responsible for endocarditis.

  In another embodiment, said antigen is a microbial antigen is a viral antigen, in particular a virus selected from H.I.V. viruses, C.M.V. or Epstein-Barr, this list is not exhaustive.

  In a preferred embodiment of the diagnostic method according to the invention, a plurality of antigens chosen from said first, second, third and fourth antigens and at least one said microbial antigen, preferably a plurality of antigens, are used. microbial antigens, fixed on the same solid support.

  More preferably, at least one said microbial antigen and said first, second and third and, preferably, fourth antigen are fixed on the same solid support and only two detection substances are used with different labeling elements, preferably the first ones. and second detection substances being animal immunoglobulins, more preferably, in the case of a human patient, a goat immunoglobulin.

  In this latter embodiment, a protocol of succession of controls is as follows: 1) It is verified that said fourth antigen containing protein A reacts with one of the detection substances. Otherwise, we stop the test, that is to say we do not take into account this sample.

  2) If said first antigen (IgG1) reacts with said first detection substance (anti IgM * Ac1), the serum sample comprises rheumatoid factors, so again the detection tests are not taken into account. and quantifying specific Ig Ms.

  3) If said first antigen does not react with the second detection substance (anti IgG * 2 Ac2), said second detection substance is not present or not reactive. The result of the test concerning the detection of IgG specific for the microbial antigen is not taken into account.

  4) If said first antigen reacts with the second detection substance, there is no rheumatoid factor and said second substance is reactive: it is possible to continue the test, that is to say, to take into account the results provided subsequent checks on reactions with the second, third and fourth antigens.

  5) If said second antigen containing a DNA / histone complex reacts with said second detection substance, antinuclear antibodies are present and the specific IgG detection and quantification tests are not taken into account.

  6) If said second antigen reacts with said first detection substance, rheumatoid factors and antinuclear antibodies are present and the test is stopped.

  7) If said second antigen does not react, and said first and second detection substances are present and reactive, there is no anti-nuclear antibody, and can be continued subject to the following verification.

  8) It is verified that said third antigen reacts with said first detection substance. If said third antigen (IgM) lo does not react, said first substance is not present or does not react, and the test is stopped.

  In summary, the result of the reaction with said microbial antigen is only taken into account if the following cumulative conditions are met: said fourth antigen reacts, said first antigen reacts with said second detection substance, said second antigen does not react with said second antigen does not react, and said third antigen reacts with said first detection substance.

  The present invention also provides a diagnostic kit useful for the implementation of a method according to the invention, characterized in that it comprises a said solid support on which is fixed at least one said first antigen, and a said first detection substance, and, if appropriate, a second said detection substance and a said solid support on which is fixed at least one said second antigen, and where appropriate a said solid support on which is fixed at least one said third antigen and, where appropriate, a solid support to which at least one said fourth antigen is attached.

  Preferably, in a kit according to the invention, the microbial antigen and the various so-called first, second, third and, preferably, fourth antigens are fixed on the same solid support, and in that it comprises as detection substance said first and second detection substance only.

  Advantageously, in the methods and diagnostic kits according to the invention, a solid glass or plastic slide, a titration tube or a well of a plastic microtiter plate is used as the solid support, and more particularly as a support. solid, any device suitable for handling cell and bacterial suspensions and in particular tubes, glass or polymer slides, jewelry tubes or rigid microtiter plates made of polyethylene, polystyrene or chloride may be used.

  The subject of the present invention is also a method for preparing a solid support on which at least one antigen selected from a said microbial antigen, a said first and, where appropriate, a second, third or fourth antigen is detection by automated reading using a said first and, where appropriate, second detection substance, useful in a method according to the invention or a kit according to the invention, characterized in that one carries out a washing prior to said solid support with a solution of an ethanol / acetone mixture, preferably at 50/50, then robotically deposited said antigens with a deposition robot preferably comprising a syringe and stabilized the fixing of said antigens by adsorption on said solid support by treatment with alcohol, preferably methanol or ethanol, which alcohol is then removed and preferably the fixing of said antigens by staining, in particular with the red Ponceau dye.

  A binder, such as, for example, bovine serum albumin, may be used at a final concentration of 1-5%, or egg yolk at a final concentration of 1-10% to stabilize the binding of said antigens to said solid support.

  The prewash solution makes it possible to clean the support of any trace of detection substance or other residual and, in particular, to eliminate any fluorescence while retaining the physical adsorption faculty of the support with respect to the said antigens subsequently deposited.

  The alcohol stabilization treatment stabilizes the physical adsorption binding of both proteins, such as IgG and IgM, as well as particulate antigens.

  Advantageously, a microbial antigen is deposited in a robotic manner and, if appropriate, a second antigen and, where appropriate, a fourth antigen, in the form of a suspension of corpuscles of non-confluent cells or whole microbes, in particular bacteria or fractions. cells or microbes, especially bacteria.

  The determination of the appropriate preliminary washing of the solid support, in particular of the glass slide has given rise to numerous tests. This treatment aims to clean this support perfectly to eliminate fluorescent artifacts while preserving the subsequent fixation of the antigens in a manner compatible with their mode of preservation but also, preserving otherwise the integrity at least the immunological reactivity of the antigens. It has been shown, after much unsuccessful research, that the rinsing and cleaning of the slides in the aqueous phase would not allow a subsequent fixation of the antigens to be deposited; the same was true for rinsing with surfactant molecules such as Tween 20. Cleaning with alcohols was not enough to remove most of the fluorescent artifacts. It is therefore at the end of these multiple tests that has been optimized a solid support cleaning protocol, including the glass slide, with a mixture of ethanol 50% - acetone 50% and air drying.

  The present invention also relates to the deposition of particulate or corpuscular microbial antigens by a deposition robot. According to the present invention, the inventors have developed, after numerous unsuccessful attempts, robotized deposition conditions of corpuscular microbial antigens (inactivated whole microbe or inactivated whole microbe fraction) suspended in a deposition medium. Indeed, only homogeneous solutions of one or more molecules are currently deposited on solid support by the deposition robots. To do this, the concentration of these corpuscular antigens is calibrated before deposition by counting inactivated microbial particles by fluorescence activated cell sorting (FACS-scanned) and then deposited robotically on a solid support. The empirical development of these calibrated and robotic deposits has involved several steps. On the one hand, several tests have been carried out concerning the most appropriate mode of conservation of the deposited antigens. After several unsuccessful attempts, it has been developed a preservation in a PBS-0.1% sodium acid buffer at -80 ° C. for certain antigens or a preservation at + 4 ° C. in 100% ethanol for others. antigens (cells such as HL60 cells for example). Moreover, several tests were carried out to determine the optimal concentration for each of the antigens tested, the sub-optimal concentrations giving undetectable deposits, the supra-optimal concentrations causing sedimentation of the corpuscular antigens during deposition and therefore a significant variation of the amount of antigen deposited. Finally, deposits of corpuscular antigens containing microbial DNA (bacteria, viruses, parasites or microscopic fungi) are calibrated by application of a fluorescent dye (intercalent molecule) whose excitation and emission wavelengths. are advantageously chosen according to those used by the fluorochrome labeling the conjugated immunoglobulins. This fluorescent, nonspecific labeling of the antigens can be carried out before they are deposited by the deposition robot or after it. The fluorescent marker is advantageously chosen for its stability in the light of day.

  Advantageously, the cells are calibrated at 108 cells per milliliter.

  Other features and advantages of the present invention will become apparent in the light of the following examples which refer to FIGS. 1 to 4.

  FIG. 1 represents the detection of robotic deposits of microbes of the Staphylococcus aureus species calibrated at 109 bacteria / ml stained with the fluorescent dye Hoescht 33342 of Example 1.

  FIG. 2 shows the deposits of Ig M illuminated with a labeled secondary antibody of Example 2, three concentrations of Ig M were deposited from top to bottom: 10 mg / ml, 1 mg / ml and 0.2 mg / ml, six deposits were made by concentration.

  FIG. 3 represents the serological reaction using a serum representing specific IgGs against Coxiella burnetii, Staphylococcus aureus, IgG and Coxiella burnetii spots are "lit", in Example 3.

  FIG. 4 represents a plate stained with a fluorescent dye, making it possible to visualize all the 8 deposited antigens, HL60 cells, Staphylococcus aureus, Coxiella burnetii, Legionella pneumophila, Bartonella henselae, Bartonella quintana, Bartonella vinsonii subsp. berkoffii, and Bartonella elizabethae in Example 3.

  Example 1: Deposition of said second and fourth antigens on solid support.

  HL60 cells (N ATCC CCL 240) are continuous-line human fibroblast cells used for the detection of anti-nuclear antibodies. After culture and production according to the usual protocols, the concentration of HL 60 cells was quantified using a cell counter (BPC / Yeast Microcytes, BioDETECT AS, Olso, Norway) and this concentration was reported to 108 cells. / mL in sterile BPS buffer, pH = 7.4 to obtain a suspension of non-confluent cells that can be deposited by deposition robot.

  Staphylococcus aureus (ATCC Deposit No. 29213) was cultured on 5% sheep blood agar and then harvested in sterile PBS buffer containing 0.1% sodium azide. The inoculum was measured using the same cell counter and calibrated at 09 bacteria / mL which is the optimum concentration given the constraints of absence of sedimentation during deposition and deposition in sufficient quantities of particles staphylococcal and then preserved by freezing at - 80 C.

  These HL60 cells and Staphylococcus aureus bacteria were deposited at a concentration of 109 CFU / mL determined by FACS-scan (Microcytes) of the glass slides (Reference LLR2-45, CML, Nemours, France). Cells and bacteria were deposited on the solid support using a deposition robot (Arrayer 427TM, Affymetrix, MWG Biotech SA, Courtaboeuf, France). The deposits were air-dried for 30 minutes in the "spotter" chamber, then fixed in a 100% methanol bath for 10 minutes, and then dried again in the open air. The efficiency of the robotic deposition on the slides, after the ethanol fixation and after the baths required by the following indirect immunofluorescence reaction, was successfully verified by the fluorescent dye Hoescht 333-42 which is excited. at 350 nanometers and emitting at 460 nanometers (Molecular Probes). Figure 1 illustrates the robotic deposition of 6 columns of 6 deposits each of Staphylococcus aureus.

  EXAMPLE 2 Deposition of the First Antigen (IgG) and Third Antigen (IgM) on a Solid Support of Class G Human Immunoglobulins Yspecific (IgG) (Serotec, Cergy Saint-Christophe, France) Diluted at a Concentration of 5 mg / ml, and class-specific M immunoglobulins (IgM (Serotec) diluted to a concentration of 10 mg / mL to obtain well-homogeneous spots, were robotically deposited using a deposition robot (model 427, Arrayer, Affymetrix, Inc., CA) on a solid support consisting of a glass slide (Reference LLR2-45, CML, Nemours, France).

  The deposits were made by transfer of the antigen suspension from a well of microtiter plate containing 25 μL of suspension, in a volume of 1 mL deposited, at 25 ° C. and 55% humidity in the robot enclosure. deposit. These conditions were controlled by a thermohygrometer. The deposits of a size of 200 .mu.m were air-dried for 30 minutes at 37.degree. C. in the enclosure of the deposition robot, then fixed in a 100% ethanol bath for 10 minutes and then dried again. outdoors. The efficiency of the robotic deposition and then the ethanol fixation after the baths required by the indirect immunofluorescence reaction was verified by the indirect immunofluorescence technique.

  human sera containing rheumatoid factors and 10 s human sera not containing rheumatoid factors (negative controls) for immunodetection of rheumatoid factors, [qualitative detection by agglutination test of sensitized latex beads (Rhumalatex Fumouze, Fumouze Laboratories, Levallois-Perret France)] and were each deposited on an IgG deposit in three dilutions each, 1:32, 1:64 and 1: 128. After a first wash, an indirect immunofluorescence reaction was performed using a goat immunoglobulin (reference A-11013, Molecular Probes, Eugene, USA) as a secondary anti-human IgM antibody, coupled to Alexa 488 which is a fluorescent molecule excited at 488 nanometers and emitting at 540 nanometers and using the same immunoglobulin coupled to Axa 594 (A-21216, Molecular Probes, Eugene, USA) which is a fluorescent molecule excited at 594 nanometers and emitting at 640 nanometers in a second experiment. The reaction was read by fluorescence microscopy and showed fluorescent detection in all sera containing rheumatoid factors in the form of a bright spot for each of the 3 dilutions tested and no fluorescence in the sera not containing these. factors. This example illustrates that it is possible to perform robotic deposition of IgG and human IgM on a solid support under conditions compatible with performing an indirect immunofluorescence reaction for the detection of rheumatoid factors. Also, this example illustrates that it is possible to perform the detection of rheumatoid factors by an indirect immunofluorescence technique.

  Figure 2 illustrates the Ig deposits illuminated with a Alexa-594-labeled secondary antibody, three concentrations of Ig M were deposited from top to bottom: 10 mg / ml, 1 mg / ml ml and 0.2 mg / ml, six deposits were made by concentration. The concentration of 10 mg / ml makes it possible to obtain the most homogeneous spots.

  Example 3: A serological diagnosis of Coxiella burnetii infection was carried out according to the method described in the present invention. For the realization of the various serological tests, 8 antigens were used: HL 60 cells (as said second antigen), Staphylococcus aureus (as said fourth antigen), human IgG (as said first antigen) and Coxiella burnetii Nine Mile, phase II (N ATCC VR 616), Legionella pneumophila, Bartonella henselae, Bartonella quintana, Bartonella vinsonii subsp. berkoffii, and Bartonella elizabethae (as said microbial antigens).

  After culture and production according to the usual protocols, the concentration of HL 60 cells was quantified using a cell counter (BPC / Yeast Microcytes, BioDETECT AS, Olso, Norway) and this concentration was reported at 10 ° C. cells / mL in sterile BPS buffer, pH = 7.4 to obtain a suspension of non-confluent cells deposited by deposition robot. These cells were stored in 100% ethanol at + 4 ° C. before their robotic deposition.

  Staphylococcus aureus was cultured on 5% sheep blood agar then harvested in sterile PBS buffer containing 0.1% sodium acid. The inoculum was measured using the same cell counter and calibrated at 100 bacteria / ml and then stored by freezing at -80 ° C.

  Class G human immunoglobulins (IgG) (Serotec, Cergy Saint-Christophe, France) were diluted extemporaneously at a concentration of 5 mg / ml.

  Finally, the bacterium Coxiella burnetii Nine Mile phase II was cultured on cell support and purified according to the usual protocols and quantified by the Microcyte counter in order to standardize its concentration at 109 bacteria / mL which is the optimum concentration given the constraints of absence. sedimentation during deposition and deposition in sufficient quantity of the bacterial particles. This antigen was stored at -80 ° C in PBS buffer, 0.1% sodium acid.

  These 4 antigens were deposited on the solid support (Reference LLR2-45, CML, Nemours, France) using a deposition robot (Arrayer 427TM, Affymetrix, MWG Biotech SA, Courtaboeuf, France).

  The solid support consisted of a glass slide previously cleaned with 50% ethanol 50% acetone and then dried in ambient air. The deposits were air-dried for 30 minutes in the enclosure of the deposition robot, then fixed in a 100% methanol bath for 10 minutes, and then dried again in the open air. The deposits were made by transfer of the antigen suspension from a well of microtiter plate containing 25 μL of suspension, in a volume of 1 mL deposited, at 25 ° C. and 55% humidity in the robot enclosure. deposit. These conditions were controlled by a thermohygrometer. The deposits of a dimension of 200 .mu.m were air-dried for 30 minutes at 37.degree. C. in the enclosure of the deposition robot, then fixed in a 100% methanol bath for 10 minutes, and then dried again at the open air.

  To carry out the serological test, 5 human sera containing rheumatoid factors, 5 human sera containing antinuclear antibodies, 10 human sera with IgG anti-Coxiella burnetii phase II antibodies at a titre of 1: 200 and antibodies of the type IgM anti-Coxiella burnetti phase II at a titre of 1:50 and 10 negative control human sera containing none of these 3 types of antibody were used and were each deposited on an IgG deposit in three dilutions each, 1: 32, 1:64 and 1: 128.

  After a first wash, an indirect immunofluorescence reaction was performed: using a first secondary antibody consisting of a goat anti-human IgM immunoglobulin, coupled to Alexa 488 (A11013, Molecular Probes, Eugene, USA) which is a fluorescent molecule excited at 488 nanometers and emitting at 540 nanometers - - then a second secondary antibody consisting of a goat immunoglobulin anti human IgG coupled to the Alexa 594 labeling element (A-21216, Molecular Probes, Eugene, USA ) which is a fluorescent molecule excited at 594 nanometers and emitting at 640 nanometers in a second experiment.

  These reactions were performed at room temperature, in a humid chamber, with 15 minutes of incubation with the serum to be tested, followed by washing with sterile PBS buffer and then incubation for 5 minutes with the conjugated antibodies, before second rinse. The reactions were read by fluorescence microscopy and gave rise to digitized recordings of the spots. During this test, the 30 sera or human sera "turned on" the S. aureus deposit, that is to say reacted with said fourth antigen, only sera containing no rheumatoid factors "turned on" the IgG deposit when reading the Alexa 488 marker, only the sera containing anti-nuclear antibodies "turned on" the HL60 cell deposition, and only the sera containing antibodies against C. burnetii "lit" the deposition of C. burnetii. All reactions using the anti-IgG conjugated secondary antibodies "lit" the IgG deposition. Observed titres of anti-C antibodies. burnetii were concordant with those found by the referenced method (indirect microimmunofluorescence) [Dupont HT, Thirion X, Raoult D.Q io fever serology: cutoff determination for microimmunofluorescence. Clin Diagn Lab Immunol. 1994; 1: 189-96].

  FIG. 3 represents the serological reaction using a serum exhibiting specific IgG against Coxiella burnetii using the conjugated antibody labeled with Alexa-488, the spots Staphylococcus aureus, IgG and Coxiella burnetii are "lit".

  FIG. 4 represents a plate stained by the Hoescht 333-42 fluorescent dye which is excited at 350 nanometers and emits at 460 nanometers (Molecular Probes), making it possible to visualize the set of 8 deposited antigens, HL60 cells, Staphylococcus aureus, Coxiella cells Burnetii, Legionella pneumophila, Bartonella henselae, Bartonella quintana, Bartonella vinsonii subsp. berkoffii, and Bartonella elizabethae. These different antigens were deposited under the same conditions as Coxiella, described above.

Claims (21)

  A method of in vitro serological diagnosis of microbial agent by immunodetection in which the presence and, preferably, the amount of class M patient immunoglobulins specific for a microbial antigen characteristic of said microbial agent is detected in a serum sample of the test patient by detection and, preferably, quantification of an immunological reaction complex between said microbial antigen to be detected and a so-called class M specific immunoglobulin, using a first detection substance comprising a first labeling element, preferably a first labeling antibody, reacting only with said immunoglobulin of the class M patient species, characterized in that the presence of rheumatoid factor in said sample is monitored to test by contacting said test sample with a solid support on which a first anti gene corresponding to a class G non-specific immunoglobulin, preferably an immunoglobulin of the patient's species, the bringing into contact with said first detection substance, and checking whether said class G non-specific immunoglobulin is fixed on the solid support reacted with said first detection substance.   The method according to claim 1, wherein the amount of class G patient specific immunoglobulin specific for said microbial antigen is further detected and preferably quantified by detection and, preferably, quantification of an immunological reaction complex between said microbial antigen and a class G specific immunoglobulin, using a second detection substance comprising a second labeling element, preferably a second labeling antibody comprising a second screening element; different marking of said first marking element, said second detection substance only reacting with said immunoglobulin of the class G patient species, characterized in that the possible presence of antinuclear antibodies in said sample is monitored test by contacting said test sample with a solid support on which a second antigen having DNA / histone complexes, preferably comprising nuclei of nucleated cells of the patient's species, more preferably all or part of cells of origin of the continuous lineage patient's species, and it is checked whether said second substance detection agent reacted with said second antigen fixed on the solid support.   The method according to claim 1 or 2, wherein the presence and, preferably, the amount of immunoglobulin of the class G patient specific for a microbial antigen characteristic of said microbial agent is detected in a sample. of serum of the test patient by detection and, preferably, quantification of an immunological reaction complex between said microbial antigen to be detected and a said class-G specific immunoglobulin, with the aid of a second detection substance comprising a second labeling element, preferably a second labeling antibody, reacting only with a said immunoglobulin of the class G patient species, characterized in that a check is made of the presence and reactivity of said second detection substance by contacting said solid support, to which said first antigen has been attached corresponding to a said immunoglobulin of the species of the non-specific class G patient, with a solution containing a said second anti-IgG labeling antibody and not containing rheumatoid factor, and checking whether said second detection substance has reacted with said first antigen fixed on the solid support.   4. Method according to claim 3, characterized in that one verifies the reactivity of said second detection substance introduced into said serum sample to be tested, after verifying the absence of rheumatoid factor and antinuclear antibodies in said sample. .   5. Method according to one of claims 1 to 4, characterized in that a check is made of the presence and the reactivity of said first marking substance which is an anti-immunoglobulin M antibody of the species of the species. patient, by contacting a solid support to which is attached a third antigen corresponding to an immunoglobulin of the species of the non-specific patient of class M, with a solution is containing a said first marking substance and not containing rheumatoid factor and verifying whether said first substance reacts with said third antigen fixed on the solid support.   6. Method according to claim 5, characterized in that the reactivity of said first detection substance is verified after checking the absence of rheumatoid factor. Method according to one of claims 1 to 6, characterized in that it is previously checked that said tested sample does indeed contain a serum of the patient's species by detecting whether immunoglobulins of the patient's species react with a fourth antigen containing the protein A of a bacterium Staphylococcus aureus, preferably contacting said sample with a solid support to which a fourth antigen is affixed, in the presence of a third detection substance which is a substance reacting with an immunoglobulin of the patient's species and not with said fourth antigen, preferably an anti-immunoglobulin antibody of the patient's species and not reacting with protein A. 8 Method according to claim 7, characterized in that said fourth antigen is a whole Staphylococcus bacterium comprising protein A. 9. Method according to one of claims 7 or 8, characterized in that said detection substance, preferably, said third detection substance is an animal immunoglobulin, more preferably in the case of a human patient, a goat or chick immunoglobulin.   10. Method according to one of claims 8 or 9, characterized in that said third detection substance corresponds to said first or second detection substance and, preferably, said first and second detection substances are an immunoglobulin of animal origin in the case of a human patient respectively anti IgM or anti IgG respectively marked by two different marking elements   11. Method according to one of claims 1 to 10,   characterized in that said detection substance is an antibody conjugated to a marking element which is a fluorescent substance 12. Method according to one of claims 7 to 10 and according to claim 1 1, characterized in that it is detected by fluorescence the possible presence of a reaction product complex between said fourth antigen and said third detection substance.   13. Method according to one of claims 1 to 12, characterized in that the presence is detected and, if appropriate, the amount of immunoglobulin of class M or, where appropriate, of class G specific to the said microbial antigen in said serum sample to be tested, by contacting said serum sample with said first or respectively second detection substance reacting specifically with the class M or, respectively, the immunoglobulin class G patient species specific for said microbial antigen, and in contact with a said microbial antigen fixed on solid support.   Method according to one of claims 1 to 13,   characterized in that said microbial antigen is a corpuscular antigen consisting of an inactivated whole microbe or a microbe fraction.   15. serological diagnostic method according to one of claims 1 to 14, characterized in that said microbial agent is selected from microorganisms comprising a bacterium, a virus, a parasite or a fungus.   16. Serological diagnostic method according to claim 15, characterized in that said microbial antigen is an intracellular bacterium or a virus.   17. Method of serological diagnosis according to claim 15 or 16, characterized in that said microbial antigen is selected from bacteria of the genus Rickettsia, Coxiella, Bartonella, Tropheryma, Ehrlichia, Chlamydia, Mycoplasma, Treponema, Borrelia, and Leptospira.   18. Serological diagnostic method according to claim 17, characterized in that said microbial antigen is a bacterium responsible for endocarditis.   19. Method of serological diagnosis according to claim 16, characterized in that said microbial antigen is a viral antigen selected from viruses H.I.V., C.M.V. Epstein-Barr, Measles, Rubella, Hepatitis A and B Virus 20. Method according to one of Claims 1 to 19, characterized in that a plurality of antigens selected from said first, second, third and fourth antigens and at least one said microbial antigen, preferably a plurality of microbial antigens, are attached to the same solid support.   21. Method according to claim 20, characterized in that at least one said microbial antigen and said first, second and third and, preferably, fourth antigen are fixed on the same solid support and only two detection substances are used with different labeling elements, preferably said first and second detection substances being animal immunoglobulins, preferably in the case of a human patient still a goat immunoglobulin.   22. Method according to claim 1 to 21, characterized in that a glass or plastic slide, a titration tube or a well of a plastic microtiter plate is used as a solid support.   23. Method according to one of claims 1 to 22, characterized in that the dose of said immunoglobulin of the class M patient's species is detected and, where appropriate, quantified or, where appropriate, class G, specific for said microbial antigen in the sample to be tested by automated reading using a reading device suitable for said marking elements.   24. Diagnostic kit useful for the implementation of a method according to one of claims 1 to 23, characterized in that it comprises a said solid support on which is fixed at least one said first antigen, and a said first detection substance, and, if appropriate, a second said detection substance and a said solid support on which is fixed at least one said second antigen, and if necessary a said solid support on which is fixed at least one said third antigen, and where appropriate a solid support on which is fixed at least one said fourth antigen.   25. Diagnostic kit according to claim 24, characterized in that the microbial antigen and the various so-called first, second, third and, preferably, fourth antigens are attached to the same solid support, and in that it comprises detection substance said first and second detection substance only.   26. A method of preparation of a solid support on which at least one antigen selected from a said microbial antigen, a said first and, where appropriate, a second, third or fourth antigen is fixed, allowing detection by automated reading at the same time. using a said first and, where appropriate, second detection substance, useful in a method according to claim 23 or a kit according to one of claims 24 or 25, characterized in that a preliminary washing is carried out of said solid support with a solution of an ethanol / acetone mixture, preferably at 50/50, then robotically depositing said antigens with a deposition robot preferably comprising a syringe and stabilizing the binding of said antigens by physical adsorption on said solid support by treatment with alcohol, preferably methanol or ethanol, which alcohol is subsequently removed and, preferably, the fixation of the d antigens by staining.   27. Method according to claim 26, characterized in that a said microbial antigen and, where appropriate, a said second antigen and, where appropriate, a fourth antigen, in the form of a suspension of corpuscles are deposited in a robotic manner. non-confluent cells or whole bacteria or fractions of cells or bacteria.