FR2631125A1 - Process for the direct detection of chlamydiae, anti-chlamydiae monoclonal antibodies and their applications to the diagnosis of chlamydial infections - Google Patents

Abstract

The present invention relates to a process for the direct detection of chlamydiae, to anti-chlamydiae monoclonal antibodies and to their applications to the diagnosis of chlamydial infections. The process for detecting Chlamydia infections, especially Chlamydia trachomatis or Chlamydia psittaci by means of an immunoenzymological technique of the competitive type is characterised in that the chlamydiae which may be present in a biological sample are detected during a first stage, by placing the biological sample, treated in an appropriate manner, into contact with appropriate anti-chlamydiae monoclonal antibodies to which the chlamydiae bind if such antigens are present in the sample to be examined, then in that, during a second stage, the complex which may be obtained is incubated with the membrane lipopolysaccharide of a mutant strain of Gram negative bacteria (called ReLPS antigen) adsorbed onto an appropriate solid support, the reading of the result being visualised during a third stage by placing an appropriate conjugate in contact with the ReLPS-complexed monoclonal antibodies obtained during the second stage.

Description

The present invention relates to a method for the direct detection of chlamydiae, monoclonal anti-chlauydiae antibodies and their diagnostic applications.
tic chlamydial infections.

 The genus Chlamydia includes prokaryotic bacterial organisms, obligate parasites of eukaryotic cells. Chlamydia exhibit a life cycle of intracellular replication and extracellular life.

 Taxonomically, two species are recognized: Chlamydia trachomatis and Chlamydia psittaci.

The structure of chlamydia is reminiscent of Gram negative bacteria; in fact, chlamydiae contain internal and external membranes identical to those of gram-negative bacteria, but do not possess peptidoglycan; the chlamydial group antigen is an acid lipopolysaccharide (2-keto-3-deoxyoctane acid). The structural and antigenic similarity between a lipopolysaccharide fragment of the outer membrane of a mutant Gram negative bacterium (Salmonella ninesotta mR 595) and the membrane glycolipids of Chlamydia trachomatis and psittaci has recently been demonstrated (Nurminen et al., Infect Immun. 1985, 40.2). Moreover, since 1980 (SCHACHTER J. and HDCALDWELL), it has been well established that the bacteria of the genus Chlamydi8 possess a lipopolysaccharide group antigen known to be heat-stable.
Chlaiydiae are pathogens of humans and animals. Chlamydia trachomatis parasitizes the uro-genital, conjunctivo-ocular, synovial and respiratory mucosa of man and constitutes the first etiological agent of sexually transmitted diseases in the western world.

 Five million cases of genital chlamydiosis are recorded each year. The female genital pathology is first cervical, then endometrial and tubal and can cause sterility by tubal obstruction.

Chlamydia trachomatis is a major cause of infertility in women, and infants born to infected infants have a 50% chance of contracting conjunctivitis and / or Chlamydia trachomatis pneumonia.

 Another pathology produced by Chlamydia trachomatis and more important than the previous one is trachoma, responsible for the five hundred million cases in the world.

 Chlamydia trachomatis is also a causative agent for arthritis, perihepatitis endocarditis and male genital infections.

Chlamydia psittaci is a pathogenic bacterium for humans and other animal species. In humans,
Chlamydia psittaci is responsible for acute pneumonias (10 to 20% of pneumonia in adults) and interstitial lung disease in newborns.

 In the remainder of this description, biological sampling is understood to mean both samples of circulating fluids, and in particular, urethal, cervical, conjunctival, bronchial and perihepatic samples.

It has been shown in European Patent Application No. 98,557 that the Chlamydia trachomatis antigens cross-react with the membrane polysaccharide of mutant strains of Gram-negative bacteria, especially Salmonella ainnesota wall 595, referred to as
ReLPS.

 This European Patent Application No. 98,557 in the name of ORION Corporation Ltd discloses a preparation containing ReLPS for the diagnosis of chlamydial infections and for the production of polyclonal antibodies specific for chlamydial group antigen; weak alkaline-treated ReLPS is used as an immunogen to produce antibodies specific for chlamydial group antigen; the same molecule is used for the detection of polyclonal antibodies specific for the chlamydial group antigen.

 This Application No. 98,557 specifies in particular that it is a two-way cross reaction, i.e., that ReLPS reacts with anti-chlamydial antibodies and vice versa chlamydial antigens (group antigens ) react with anti-ReLPS antibodies.

 European Patent Application No. 183,383, in the name of IQ (Bio) Limitez, describes a method for qualitatively and quantitatively determining the presence of Chlamydia trachomatis using a conventional immunoenzymatic method, said method comprising the preparation of a sample, obtained from a starting material (biological sample) by a process including heating said starting material at a temperature between 70 and 110 C, for a time ranging from 5 to 40 inutesf so as to make the determination more specific.

 In Ann. Biol. Clin., 1985, 43, 603, the inventor has described monoclonal antibodies specific for Chlamydia trachomatis for rapid diagnosis by indirect immunofluorescence, obtained from cultures of two hybridomas (clones 12C6 and 2F12).

The European Patent Application on behalf of The
United States of America, No. 193,431 discloses onoclonal antibodies against chlamydial group antigens, obtained from a monoclonal antibody producing hybridoma culture that specifically recognize chlamydial group antigen.

The inventor published in J. Immunol. nethods, 1986, 94, 153-59, an article which describes an anti-chlamydial antibody detection immunoenzyme reaction, which takes advantage of cross-reactivity between the antigen
ReLPS (produced from a mutant strain of Salmonella thyphimurium, S. ninnesota mR 595) and lipopolysaccharide antigen of all strains of chlamydiae.

 ELISA and microimmunofluorescence techniques for the serological detection of chlamydia are compared in this paper. They get 78 correlation Z for the sera tested.

 This immunoenzymatic method is a useful tool for the detection of all serotypes of chlamydial infections; nevertheless, it is a detection of anti-chlamydial antibodies. This method does not allow the direct diagnosis of the presence of chlamydia in a biological specimen (mucosa, tissue for example), which would present, in clinical interest.

It therefore appears that the tests described in the prior art mainly detect the presence of anti-chlamydial antibodies in a biological sample, either by using the antigen-group reaction of chlamydia-anti-chlamydial antibodies, or by using the antigen reaction ReLPS anti-chlamydial antibody; the revelation of the reaction is carried out either by indirect fluorescence or by an enzymatic reaction. The
European patent application 183,383 detects the presence of
Chlamydia in a sample, but the treatment conditions as described in this application do not make this detection sensitive enough and specific.

 Usually, the detection of chlamydia can only be performed by cell culture of a specimen. However, the detection of Chlamydia trachomatis requires specific cultures, which require several days, are difficult to implement, expensive and performed in specialized laboratories.

 It has therefore been the object of the present invention to provide a method for detecting chlamydia directly in a biological specimen.

 Such a method allows the unambiguous diagnosis of an evolving chlamydial infection.

 It is also an object of the invention to provide hybrid cell lines producing monoclonal antibodies suitable for use in the direct diagnosis of chlamydial infections.

 It is also an object of the invention to provide a kit or cabinet for the implementation of said detection method.

 The subject of the present invention is a method for detecting Chlamydia infections, especially Chlamydia trachomatis or Chlamydia psittaci infections, using a competitive immunoenzymological technique, characterized in that the chlamydiae possibly present in a biological sample are detected in a first step by contacting the biological sample, suitably treated, with appropriate anti-chlamydial monoclonal antibodies, to which chlamydia attach, if such antigens are present in the sample to be controlled, and in that, in a second step, the complex optionally obtained is incubated with the membrane lipopolysaccharide of a mutant strain of Gram-negative bacteria (hereinafter referred to as ReLPS antigen) adsorbed on a solid support appropriate, the reading of the result being revealed in a third step, by contacting a conjugate app used with the monoclonal antibodies complexed with ReLPS obtained during the second step.

 According to an advantageous embodiment of said method, the conjugate is a suitable mammalian anti-immunoglobulin antibody, in particular of the mouse, conjugated to a suitable marker.

The marker may be any known marker such as enzymes, fluorescers, radioactive substances. It can also be a specific signal amplification system such as biotin
N-hydroxy.

 According to another embodiment of said method, the conjugate is the anti-chlamydial monoclonal antibody coupled to a suitable marker.

 The ReLPS antigen is adsorbed onto a solid support selected from the group consisting of microtiter plates, latex beads, nitrocellulose paper disks.

The ReLPS antigen is produced, in a manner known per se, from a mutant strain of bacteria
Gram negative and in particular Salmonella typhimurinni (Salmonella minnesota mR 595).

 According to another advantageous embodiment, the biological sample is heated at a temperature of between 115 ° C. and 125 ° C. for 2 to 4 minutes.

 The subject of the present invention is also cell lines producing monoclonal anti-chlamydial antibodies, characterized in that they are obtained by immunization of suitable mammals, in particular mice, with a strain of Chlamydia trachomatis, followed by a fusion between spleen cells taken from said immunized mammals and SP20 mouse hybrid cells (HGPRT negative), determination of clones that produce anti-chlamydial antibodies, selection of suitable clones that recognize both most strains of chlamydia and ReLPS antigen and culturing said clones for the production of the desired monoclonal antibodies.

 According to an advantageous embodiment of the invention, the clones are selected by their ability to produce antibodies which have a high affinity of at least 101 H-1, vis-à-vis the ReLPS.

The cell lines obtained according to the invention were deposited with the National Collection of Cultures of Microorganisms held by the INSTITUT
PASTOR.

 Line 4B21 was deposited under No. 73B, dated March 15, 1988.

 Line 7B21 was filed as I-739, dated March 15, 1988.

 Line 12C6 was deposited under No. I-740, dated March 15, 1988.

 Line A8 was filed as No. 753, dated May 3, 1988.

 The present invention also relates to anti-chlamydial monoclonal antibodies produced by the hybrid cell lines according to the invention.

 According to a preferred embodiment, said antibodies are used in admixture at appropriate concentrations.

 The production of antichlamydial monoclonal antibodies, by clones different from those in accordance with the present invention, is known in particular from European Patent Application No. 192,033 and Ann. Biol.

Clin. 1985, 603; however, the monoclonal antibodies produced by the clones according to the invention have advantageous properties, in the context of the detection method according to the invention, in particular in that they are capable of recognizing all the strains of chlamydiae and the lipopolysaccharide ReLPS of a mutant strain of Gram-negative bacteria, including Salmonella typhimurium.

The present invention further relates to a ready-to-use kit for the detection of chlamydia in a biological sample to be tested, characterized in that it comprises
appropriate doses of monoclonal antibodies directed against chlamydia according to the invention, optionally conjugated to an appropriate marker;
- a biological sample called positive control, containing chlamydiae
- a biological sample called negative control, not containing chlamydiae;
- Appropriate doses of ReLPS of a mutant strain of Gram-negative bacteria
- appropriate quantities of a suitable disclosure system
useful amounts of buffers suitable for carrying out said reaction
a suitable solid support capable of being associated with the ReLPS antigen.

In a variant, the kit is characterized in that it comprises
appropriate doses of monoclonal antibodies directed against chlamydia, according to the invention
- Appropriate doses of ReLPS of a mutant strain of Gram-negative bacteria
appropriate doses of a suitable mammalian anti-immunoglobulin antibody, in particular of the mouse, conjugated to an appropriate marker
- appropriate quantities of a suitable disclosure system
useful amounts of buffers suitable for carrying out said reaction
- a biological sample called positive control, containing chlamydiae
- a biological sample called negative control, not containing chlamydiae
a suitable solid support capable of being associated with the ReLPS antigen.

 The marker may be any known marker such as enzymes, fluorescers, radioactive substances. It may also be a specific signal amplification system such as biotin N-hydroxysuccinimide associated with streptavidin peroxidase.

 In the case where the marker is an enzyme, the appropriate revelation systems include, but are not limited to: paranitrophenyl phosphate (for alkaline phosphatase), 1,2-phenylenediamine dihydrochloride (OPD) for peroxidase.

 Solid supports capable of being associated with the ReLPS antigen include microtiter plates, latex beads, nitrocellulose paper disks.

 In addition to the foregoing, the invention further comprises other arrangements, which will become apparent from the following description, which refers to examples of implementation of the present invention.

 It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

Example 1 Preparation of the ReLPS antigen
This preparation is such as that described in the article published in J. Immunol. Methods, 1986, 94, 153-159, in the name of the Inventor.

 The mutant bacteria are incubated for 18 hours at 37 ° C. on agar-Salmonella plates and harvested by washing with distilled water.

The washed and dried bacteria are treated with a mixture of phenol (90 g of phenol in 11 ml of water), chloroform and petroleum ether in a ratio of 2: 5: 8 respectively. After homogenization of the bacterial suspension, the bacteria are centrifuged (5000 rpm for 15 minutes), the solvents are evaporated and the lipopolysaccharide (LPS) is precipitated with water. The
LPS is washed three times with 80 S phenol, washed again with ether and finally the pellet is taken up in distilled water at 45 ° C., centrifuged at 100,000 g for 4 hours and stored at room temperature after lyophilization.

 Example 2 Preparation and Selection of Non-Clonal Antibodies Recognizing ReLPS and Chlamydiae

 The antibodies were obtained by fusion of spleen cells of BALB / C mice immunized with Chlamydia trachomatis strain LB1 with non-secretory SP20 hybridoma cells. After cloning, carried out by limiting dilution, the secretory hybridomas were selected.

 The reactivity of the monoclonal antibodies 4B21, 12C6, A8 and 7B21 was studied, with respect to the different strains of Chlamydia trachomatis and Chlamydia psittaci, as against the ReLPS antigen.

Polystyrene microtiter plates are coated with ReLPS (150 ng / 100 μl / well) diluted in PBS pH 7.4 containing 0.025% gluraraldehyde, sealed with paraffin, and incubated overnight at 3-7 ° C. with gentle agitation; after three washes with
PBS-Tween 20 at 0.05 S, the plates are incubated at 37 ° C. for one hour with 200 μl of 0.1 M ethanolamine.

 Monoclonal anti-chlamydial antibodies at increasing concentrations ranging from 125 to 2000 ng are incubated at 37 ° C for 90 minutes.

The reaction is revealed by the introduction of alkaline phosphatase conjugated anti-mouse IgG antibodies, which are conveniently diluted with PBS.
Tween 20 at 4 X, added to each well and incubated at 37 ° C for 90 minutes After three washes with PBS
Tween 20 at 0.05% and a final wash with 0.1 M diethanolamine, 100 μl of substrate (paranitrophenyl phosphate) is added, followed by incubation for 30 minutes at 37 ° C. The absorbance is measured at 405 nm.

 Figure 1 shows recognition of the ReLPS antigen by the monoclonal antibodies produced by clones 4B21, 7B21, 12C6 and A8. The abscissa axis corresponds to the monoclonal antibody concentrations (ng). The ordinate axis corresponds to the optical densities. The different curves represent this recognition as a function of the concentration of antibodies, (*) for the monoclonal antibodies produced by the clone A8, (r) for the monoclonal antibodies produced by the clone 12C6, (1) for the monoclonal antibodies produced by the clone 7B21, (+) for the monoclonal antibodies produced by clone 4B2 1.

 Table I below specifies the chlamydial strains recognized by the monoclonal antibodies produced by clones 4B21, 7B21, A8 and 12C6.

TABLE I

<tb><SEP> clones <SEP> 4B21 <SEP> 7B21 <SEP> A8 <SEP> 12C6
<tb><SEP> strain
<tb><SEP> A <SEP> 8A1 <SEP> +++ <SEP> ++ <SEP> + <SEP> +
<tb><SEP> B <SEP> TW51 <SEP> +++ <SEP> + <SEP> + <SEP> +
<tb><SEP> D <SEP> LB1 <SEP> ++ <SEP> ++ <SEP> + <SEP> +
<tb> E <SEP> E <SEP> DX20 <SEP> ++ <SEP> ++ <SEP> + <SEP> +
<tb><SEP> F <SEP> MRS <SEP> 301 <SEP> ++ <SEP> + <SEP> + <SEP> +
<tb><SEP> G <SEP> IOL <SEP> 238 <SEP> j <SEP> + <SEP> + <SEP> + <SEP> +
<tb><SEP> HNS <SEP> 4 <SEP> +++ <SEP> ++ <SEP> + <SEP> +
<tb><SEP> I '<SEP> - <SEP> - <SEP> + <SEP> +
<tb><SEP> JUW <SEP> 36 <SEP> +++ <SEP> ++ <SEP> - <SEP> + <SEP> +
<tb><SEP> K '<SEP> + <SEP> - <SEP> + <SEP> +
<tb><SEP> L <SEP> 1 <SEP> 440 <SEP> L <SEP> ++ <SEP> + <SEP> + <SEP> +
<tb><SEP> L <SEP> 2 <SEP> 434 <SEP> B4 <SEP> +++ <SEP> - <SEP> ++ <SEP> + <SEP> +
<tb><SEP> L <SEP> 3 <SEP> 404 <SEP> B4 <SEP> ++ <SEP> ++ <SEP> + <SEP> +
<tb><SEP> PSITTACI <SEP> 1 / 6BC <SEP> | <SEP> - <SEP> - <SEP> + <SEP> +
<Tb>
Example 3 Detection of chlamydiae in a cell suspension by the monoclonal antibody 4B21

 Table II shows that the addition of increasing amounts of chlamydia suspension results in inhibition of 1.5 μg / ml recognition of ReLPS by monoclonal antibody 4B21.

 EXAMPLE 4 Detection of Chlamydiae in a Cellular Suspension by the 7B21 Monoclonal Antibody

 Table II shows that the addition of increasing amounts of chlamydial suspension results in an inhibition of the recognition of the ReLPS antigen by the monoclonal antibody 7B21.

 Example 5 Detection of chlaaydiae in a cell suspension by the monoclonal antibody A8

 Table II shows that the addition of increasing amounts of chlamydial suspension results in an inhibition of the recognition of the ReLPS antigen by the A8 monoclonal antibody.

TABLE II
INHIBITION OF MONOCLONAL ANTIBODIES
ANTI-CHLAMYDIAE BY THE LGV2 STRAIN
CHLAMYDIA TRACHOHATIS,
ELISA ReLPS 1.5 pg / ml

<tb><SEP> Concentrations <SEP> in <SEP> Chlamydia <SEP> trachomatis
<tb><SEP> 104 <SEP> 105 <SEP> 106 <SEP> 107 <SEP> 108 <SEP> 109
<tb><SEP> Hybridomas
<tb><SEP> 4 <SEP> B21 <SEP> 920 <SEP> 850 <SEP> 810 <SEQ> 702 <SEQ> 512 <SEP> 93
<tb> 7 <SEP> B21 <SEP> 820 <SEP> 760 <SEP> 694 <SEP> 530 <SEQ> 280 <SEP> 125
<tb><SEP> A8 <SEP> 512 <SE> 530 <SE> 418 <SE> 346 <SE> 228 <SE> 63
<Tb>
Example 6 Detection of chlaaydiae, by the method according to the invention, in which the monoclonal antibody is labeled with alkaline phosphatase.

First step: Incubation of the heated biological sample at 120 ° C for three minutes just before the reaction with the mixture of monoclonal antibodies an-t-chlamydiae
Appropriate amounts of a mixture of alkaline phosphatase conjugated monoclonal antibodies 7B21, 4B21 and A8 are incubated for 90 minutes with treated biological samples, optionally containing chlamydiae.

Step 2: The complexes formed in the first step are incubated at 371C for 80 minutes on polystyrene plates coated with ReLPS. These microtiter plates were pre-coated with ReLPS (1.5 μg / ml) sonicated and diluted in PBS pH 7.4 containing 0.025% glutaraldehyde; the plates are paraffin-sealed and incubated at 37 ° C overnight with gentle shaking; after three washes with 0.05% of
Tween 20-PBS, the plates are incubated at 37 ° C. for one hour with 200 μl of 0.1 M ethanolamine and remain ready for use.

 The anti-Chlamydia-ReLPS antibody reaction is revealed by the introduction of 100 μl of substrate and chromogen and the measurement of light absorption is made at 405 nm, which is decreased when there is chlamydiae. in the sample compared to the control without chlamydiae.

 The labeling of said monoclonal antibodies with alkaline phosphatase does not alter the recognition properties of the monoclonal antibody with respect to chlamydia on the one hand and, on the other hand, of the ReLPS adsorbed on polystyrene. Figure 2 shows inhibition of ReLPS binding - anti-chlamydial monoclonal antibodies conjugated to alkaline phosphatase in the presence of increasing amounts of Chlamydia trachomati.s.The ordinate axis corresponds to optical densities, the histogram (A) is obtained in the absence of chlamydia: Column (g) corresponds to the revelation of the anti-chlamydial alkaline phosphatase-conjugated monoclonal anti-Chlamydia binding, column () corresponds to a control without fixed ReLPS; the histogram (B) is obtained in the presence of chlamydiae and shows the inhibition of the alkaline phosphatase-conjugated anti-chlamydial monoclonal antibody-associated ReLPS (column ()), column () being a control.

The amount of ReLPS fixed is 150 ng.

 Table III shows the inhibition of the binding of monoclonal antibodies B21 and B21 to Chlamydia trachomatis on ReLPS.

TABLE III
MONOCLONAL ANTIBODIES CONJUGATED TO
ALKALINE PHOSPHATASE 4B21 AND 7B21
(500 ng / ml)

<tb><SEP> Concentrations <SEP> in <SEP> Chlamydia <SEP> trachomatis
<tb><SEP> 104 <SEP> 105 <SEP> 106 <SEP> 107 <SEP> 108 <SEP> 109
<tb><SEP> Hybridomas
<tb><SEP> (DO)
<tb><SEP> 7 <SEP> 7 <SEP> B21
<tb> i <SEP> 1250 <SE> 1003 <SE> 840 <SE> 620 <SE> 318 <SE> 120
<tb><SEP> 4 <SEP> B21
<Tb>
Example 7: Detection of chlaaydiae by the method according to the invention, in which the monoclonal antibodies are conjugated to the biotin N-hydroxysuccinimide
The protocol is similar to that of Example 6; however, the anti-chlamydial monoclonal antibodies are coupled to the biotin N-hydroxysuccinimide, complexed with streptavidin-peroxidase, and the detection of the complex, is carried out by incubation with the substrate and the appropriate chromogen which in the present embodiment is 1,2-phenylenediamine dihydrochloride.

 Examples 6 and 7 are examples of a direct method; indeed, we mean by direct method.

a method in which the monoclonal antibody is coupled to an enzymatic or radioactive tracer or to a specific signal amplification system and complexed to an enzymatic label.

 Example 8 Detection of Chlarydiae by the method according to the invention, wherein the conjugate is an anti-mouse Ig antibody coupled to an enzyme.

 In the first step, as described in Example 6, the anti-chlamydial monoclonal antibodies are not conjugated; in the second step, the revelation is carried out by introducing anti-mouse IgG conjugated to an enzyme or biotin and diluted in PBS-Tween at 4 X, incubated at 37 ° C for 90 minutes. After washing three times with PBS Tween 20, 0.05%, the substrate and the chromogen are added and after a variable incubation depending on the enzyme system used the absorbance is measured at the wavelength corresponding to the atomic absorption of the product used.

 In the present nonlimiting embodiment, the anti-IgG antibodies are conjugated to alkaline phosphatase, and after addition of 100 μl of paranitrophenyl phosphate, the incubation is 30 minutes at 37 ° C.

 Absorbance is measured at -405 nm. This reaction makes it possible to visualize the binding of monoclonal antibodies against ReLPS / chlamydiae on the ReLPS or the inhibition of said binding in the presence of chlamydiae.

 Example 8 is an example of an indirect method; indeed, the antibody revealing the reaction is different from the specific monoclonal antibody directed against it.

 As is apparent from the above, the invention is not limited to those of its modes of implementation, and application that have been described more explicitly; on the contrary, it encompasses all variants that may come to the mind of the technician in the field, without departing from the scope or scope of the present invention.

Claims (10)

 1.) A method for detecting Chlamydia infections, in particular Chlamydia trachomatis or Chlamydia psittaci, using a competitive immunoenzymological technique, characterized in that chlamydia that may be present in a biological sample are detected during in a first step, by contacting the biological sample, suitably treated, with appropriate anti-chlamydial monoclonal antibodies. ReLPS, obtained during the second stage. to which chlamydia are attached, if such antigens are present in the sample to be monitored, and in that in a second step, the resulting complex is incubated with the membrane lipopolysaccharide of a mutant strain of bacteria Gram-negative (hereinafter referred to as ReLPS antigen) adsorbed on a suitable solid support, the reading of the result being revealed in a third step, by bringing into contact an appropriate conjugate with the monoclonal antibodies complexed with  2) The method according to claim 1, wherein the conjugate is a suitable mammalian anti-immunoglobulin antibody, especially of the mouse, coupled to a suitable marker.  3 ') Method according to claim 1, characterized in that the conjugate is an anti-chlamydia monoclonal antibody coupled to a suitable marker.  4 ') Process according to any one of claims 1 to 3, characterized in that the biological sample is heated at a temperature between 115'C and 125-C for 2 to 4 minutes.  5)) Cell lines producing anti-chlamydial monoclonal antibodies, characterized in that they are obtained by immunization of suitable mammals, especially mice, with a suitable Chlamydia trachomatis strain, followed by fusion between the spleens collected from said immune mammals and SP2O mouse hybrid cells, determination of clones which produce anti-chlamydial antibodies, selection of suitable clones which recognize both most chlamydial strains and the ReLPS antigen and culture of said clones for the production of the desired monoclonal antibodies.  Cell lines according to Claim 5, characterized in that the clones are selected for their ability to produce antibodies which have a high affinity of at least 107 μl with respect to ReLPS. Micro-organisms held by INSTITUT PASTEUR.  7) Cell lines according to claim 5 or claim 6, characterized in that they have been deposited under nI-738, dated March 15, 1988, under No. 739, dated March 15, 1988 , under No. 740, dated March 15, 1988, under No. 753, dated May 3, 1988, from the National Collection of Cultures of Canada.  8) Anti-chlamydial monoclonal antibodies produced by the hybrid cell lines according to claims 5 to 7.  9) Antibody according to claim 8, characterized in that they are used in admixture at appropriate concentrations.  a suitable solid support capable of being associated with the ReLPS antigen.  useful amounts of buffers suitable for carrying out said reaction  - appropriate quantities of a suitable disclosure system  - Appropriate doses of ReLPS of a mutant strain of Gram-negative bacteria  - a biological sample called negative control, not containing chlamydiae  - a biological sample called positive control, containing chlamydiae  appropriate doses of monoclonal antibodies directed against the chlamydia according to the invention, optionally coupled to a suitable marker  10.) Ready-to-use kit for the detection of chlamydia in a biological sample to be controlled, characterized in that it comprises  a suitable solid support capable of being associated with the ReLPS antigen.  - a biological sample called negative control, not containing chlamydiae  - a biological sample called positive control, containing chlamydiae  useful amounts of buffers suitable for carrying out said reaction  - appropriate quantities of a suitable disclosure system  appropriate doses of a suitable mammalian immunoglobulin antibody; including mouse, conjugated to a suitable marker  - Appropriate doses of ReLPS of a mutant strain of Gram-negative bacteria  appropriate doses of monoclonal antibodies directed against chlamydia, according to the invention  1 ready-to-use kit, characterized in that it comprises