FR2775524A1 - ANTIGENIC STRUCTURE FOR USE IN THE DETECTION AND ASSAY OF ANTIBODIES AND PREPARATION METHOD - Google Patents

Abstract

The antigenic structure of the invention comprises a bridging group between a protein and a support, and the bridging group is of the S-amino-imino-alkylene type, and preferably S-amino-imino-propene (S-AIP). , where S is a support having free amino groups, obtained by covalent bonding between an aldehyde and the free amino groups of the support. The support can be constituted by microplates comprising free amino groups and the antigenic structure can be associated with a protein to constitute a set for detection and assaying of antibodies.

Description

 The present invention relates to an antigenic structure usable for the detection and the assay of antibodies, a method for the preparation of such an antigenic structure on a microplate, a set of diagnostic and assay of antibodies using such a structure, substances immunogens comprising the antigenic structure, their application to the preparation of hybrid murine cell lines, as well as the monoclonal antibodies obtained.

The detection of antibodies directed against the hapten formed by the binding of an aldehyde, such as malonic dialdehyde (MDA), with the free amino groups of the proteins, is useful for the demonstration of certain modifications which can affect the proteins, for example in living organisms. The presence of these antibodies, such as for example
AcAIP, in the human or animal organism can constitute a useful prognostic test in certain pathologies. It is known in fact that the in vivo oxidation of certain proteins, in particular low density lipoproteins (LDL), leads to the formation of dialdehyde derivatives, such as MDA, which can react with the primary amine groups of certain biological molecules to form intramolecular couplings of the 1-amino-3-imino-alkylene type, and in particular 1-amino3-imino-propene (AIP).

Thus, for example, Gonen B. et al. ["Immunogenicity of malondialdehyde-modified low density lipoproteins1 'B. Gonen,
JJ Fallon, SA Baker, Atherosclerosis, 65, 265-272 (1987)] use an ELISA technique for the determination of antibodies directed against low density lipoproteins (LDL) modified by MDA. The specificity of the antibodies is evaluated by the ELISA technique by fixing the LDL proteins modified by adsorption on the titration plate. This technique however has the drawback of the lability of the affinity bond between the plate and the protein, the large steric hindrance of the protein limiting access to the antigen, and the insufficient reproducibility of the preparations.

 FR-A-2,601,141 describes a hapten-based coupling product linked to a support molecule, such as a protein, in particular an LDL protein, via a dialdehyde such as aldehyde malonic. The products obtained, carrying amino rings, can be used to prepare antibodies directed against the -NH-hapten structure, useful in assays of specific pharmaceutical products, of the aminoquinoline type, in particular antimalarials.

Patent EP-A-484,863 describes a monoclonal antibody specific for LDL lipoproteins modified by MDA, prepared using the proteins modified by the MDA as antigens.
MDA. The assay method consists in immobilizing antibodies recognizing the LDL proteins modified by MDA on a support, in labeling reduced antibodies recognizing the LDL proteins modified by MDA, in bringing the two antibodies into contact with the test sample, and to perform an assay according to an ELISA type technique.

Y. Chancerelle et al., "Immunological relevance of MDA
Further evidence about the epitopes recognized by antibodies obtained from rabbits immunized with MDA-modified lysozyme "
Biochem. International, li, 157-163 (1991), describe a technique using lysozyme modified by malonic dialdehyde as antigen, fixed on the microplate by adsorption, after purification by exclusion-diffusion chromatography. The study shows that rabbit serum immunized with certain proteins modified by MDA, for example lysozyme, contains antibodies reacting specifically with epitopes containing the same AIP bridges as those formed in modified lysozyme.

 All this work has shown that the proteins modified by MDA are antigenic, in particular by the 1 amino-3- imino-propene (AIP) bridges formed, that the organism produces antibodies directed against these bridges, and that it is possible to detect these antibodies by means of antigens carrying these AIP sites.

 However, these techniques lack specificity with respect to the structure to be detected, and the antigens used have insufficient stability which affects the reliability of the assay methods. In particular, the reactivity of the plates and of the antigens which are adsorbed therein varies considerably from one batch to another, which makes it necessary to carry out additional experiments on homogeneous batches of plates. In addition, it is practically impossible to determine the number of reaction epitopes present on the plates.

 The present invention therefore relates to a defined antigenic structure, comprising a bifunctional aldehyde bridging group between an amino structure and a support, as well as a process for its preparation.

 Another subject of the invention is antibody detection and assay plates providing excellent selectivity of the antibodies, as well as improved stability and reliability compared to known conventional techniques.

 The present invention also relates to the immunogenic substances comprising the above antigenic structures, a process for obtaining hybrid murine cell lines by means of said immunogenic substances, as well as the monoclonal antibodies obtained directed against the amino-imino alkylene bridge, and in particular the amino-imino-propene bridge.

The antigenic structure according to the present invention is distinguished in that it comprises a group of the S-amino-imino-alkylene type, and preferably S-amino-imino-propene (S
AIP), where S represents a support comprising free amino groups, obtained by covalent bond between an aldehyde and the free amino groups of support S. The support is preferably a microplate comprising at least one free amino group, but it can be more generally consisting of any suitable substance containing free amino groups, for example a protein.

Thus, the antigenic structure according to the invention can be represented by a formula of the type
S - N = CH - A - R where S is the support carrying an amino group, A is a group derived from an aldehyde, R is a hydrogen atom or a group - CH = N - P, and P is a residue of an amino group molecule, such as a protein or amino acid.

 When P represents an amino acid residue, it may for example be a simple amino acid or a diamine acid. In the case of simple amino acids, the attachment to the rest of the antigenic structure is made by the N-terminal group, while in the case of diamine acids, the attachment is made by one of the amino groups and the second group can advantageously be blocked by an appropriate group, for example an acetyl group. As an example of diamined acid of this type, mention may in particular be made of acetyllysine.

 The preparation process according to the present invention consists in reacting a compound containing at least one aldehyde function with a support, for example a plate or a microplate, comprising at least one free amino group.

 The microplates used in the invention, comprising free amino groups, are generally commercially available (for example the microplates Ref. 3390 from the company Costar). These microplates are characterized by the presence of an amino group attached to a more or less long carbon chain constituting a spacer group. The microplates can be of the usual type in the art, based on various plastics, and preferably based on polystyrene.

 The aldehyde used for the coupling can be chosen from mono- or poly-aldehydes such as formalin, glyoxal, glutaraldehyde, malonic dialdehyde (MDA), 4-hydroxynonenal (4HNE), etc.

 Thus, in accordance with the present invention, by controlling the parameters of the chemical synthesis reaction of the antigenic structure, it therefore becomes possible to fix and precisely determine the number of reactive sites present on the plate. On the other hand, the covalent bond between the antigenic structure and the plate is much more stable than in the known techniques using adsorption.

 As indicated above, the immunogenic substance according to the invention comprises an antigenic structure of the aminoimino-alkylene type, and preferably amino-imino-propene (AIP), associated with an amino structure and in particular a protein such as albumin , e.g. mouse albumin.

 The method for obtaining hybrid murine anti-hapten amino-imino-alkylene, and in particular amino-imino-propene (AIP) cell lines, of the invention consists in carrying out the fusion of splenocytes of mice immunized with an immunogenic substance comprising the antigenic structure according to the invention, with mouse myeloma cells. The immunogenic substance can in particular be mouse albumin modified with malonic dialdehyde (MDA) as indicated above.

The process for obtaining cell lines is carried out according to conventional techniques. More specifically, the method of the invention comprises the following steps
a) mice are immunized with an immunogenic substance in accordance with the invention, preferably in the presence of a Freund's adjuvant
b) the spleen of the immunized mice is removed;
c) the splenocytes are separated and fused with mouse myeloma cells, in the presence of a fusion promoter
d) the hybrid cells obtained are cultured in a selective medium in which the non-fused myeloma cells do not develop
e) the cells producing the desired antibody are selected by conventional techniques
f) if necessary, the selected cells are cloned.

The immunogenic substance used in the process of the invention is preferably mouse albumin modified with
MDA or 4HNE. For example, MDA can be obtained by acetic hydrolysis of 1,1,3,3-tetraethoxypropane, and the MDA solution obtained is brought into contact with a solution of mouse albumin, then the mixture is incubated for a period generally between 2 and 4 weeks so as to obtain the immunogenic substance comprising the antigenic structure of the invention associated with albumin as the carrier structure. The immunogen can be administered by subcutaneous or intraperitoneal injection, in one or, preferably, several injections spaced several days apart.

 The fusion of splenocytes with mouse myeloma cells is carried out in the presence of a fusion promoter, a polyethylene glycol such as PEG 1000 or PEG 1500, preferably using one myeloma cell for four or five cells of splenocytes.

 The culture of the hybrid cells is carried out in a conventional manner on culture plates, by supplementing the medium with appropriate nutrients.

 The selection of the cells producing the desired antibody can be carried out by immunoenzymatic technique on microplate, by a specific ELISA method, making it possible to determine that the antibodies produced are directed against the AIP structure.

 It is thus possible, in accordance with the present invention, to develop a kit or assembly for detection and assay of antibodies, in particular AcAIP, present in biological media, on the principle of ELISA tests. The detection and assay assembly comprises the above-mentioned antigenic structure combined with a protein or an amino acid. The assay can be done by measuring the optical density of the sample, or by the assay technique by competition allowing precise quantification of the level of specific immunoglobulins, by means of corresponding monoclonal antibodies. Since the amino acid or protein used is not recognized by the antibody, its nature is indifferent.

 The antigenic structure according to the invention as well as the microplates comprising them can be used for the detection and the assay of specific antibodies in biological fluids, in immunobiology and in immunopathology, in particular in certain human or animal pathologies involving modifications of proteins, in particular LDL lipoproteins, for example in the case of atherosclerosis.

 More particularly, the monoclonal antibodies obtained in accordance with the invention can be used, directly or after labeling, to visualize the corresponding antigenic structures on a cell, a tissue, an organ or internal components of the cell (nucleus, mitochondria, membranes) , as well as on a section of tissues or organs, by immunocytochemistry, immunofluorescence or flow cytometry techniques, or by other known techniques of visualization. According to another example of application of the invention, the monoclonal antibodies can be used in immunocompetition techniques vis-à-vis the antigen or corresponding polyclonal antibodies.

 The invention can also find application in the detection and the assay of various components, such as for example the detection and the assay of aldehydes in polluted effluents by a technique of competition vis-à-vis a monoclonal antibody.

 The following examples illustrate the invention in more detail without limiting its scope.

Example 1
Preparation of plates
Polystyrene-based plates having free amino groups of the Costar-NH2 ref type are used. 3390. The
2 surface of the wells of the plate is approximately 3 cm, for a height of 1 cm and a radius of 0.4 cm.

 300 IU of the 10-3 M malonic dianhydride reagent in solution in 0.5% acetic acid are poured into each well of the plates. This reagent is prepared by adding the precursor tetraethoxypropane (250 μl) in 1,000 ml of 0.5 W aqueous acetic acid solution, allowing the hydrolysis to take place overnight at a temperature of approximately 40 ° C.

 Cover and incubate for 24 hours in the dark.

The wells are then emptied and rinsed several times with a saline solution of phosphate buffer at pH 7.4. This solution is prepared by diluting the solution having the following composition ten times in water.
NaCl 80.0 g
KH2PO4 2.0 g
Na2HPO4 11.5 g
KCl 2.0 g
Water to make up to: 1000 ml
300 μl of reagent consisting of 10 M Na-acetyl-lysine are then poured into each well (obtained by adding 188 mg of Na-acetyl-lysine in 1000 ml of a 0.5% aqueous acetic acid solution) , then incubated for 24 hours at 370C in the dark.

 The wells are rinsed several times using the same saline solution of phosphate buffer as above.

 The plates thus prepared are stored dry at a temperature of about 40C.

Example 2
AcAIP antibody assay
in human or animal serum
Plates prepared as indicated in Example 1 are used, which are rehydrated by carrying out three washes using the saline solution of phosphate buffer described above.

300 ssl of reagent consisting of the blocking solution having the following composition are distributed in each well
Phosphate buffer - Tween
(containing 0.05 of Tween 20) it
BSA (bovine serum albumin - fraction V) 2 g
Fish gelatin (W / V) 12 g
The plates are incubated for 1 hour at 370C and then the wells are emptied.

 200 Hl of an appropriate dilution of the serum to be studied are poured into each well, with the exception of the first two columns which receive an equivalent volume of phosphate buffer solution as a reference. It is then left to incubate for 2 hours at 370C.

 The plates are then rinsed with the phosphate buffer solution to remove the unbound antibodies.

 200 μl of an appropriate dilution of specific anti-Ig antibody, labeled with peroxidase, are then added, with the exception of the 1st column which receives an equivalent volume of buffer, then the mixture is left to incubate for 2 hours at 370C.

 The plates are rinsed again with the phosphate buffer solution to remove the unbound antibodies. Then 200 ssl of chromogenic substrate specific for peroxidase are added to all the wells.

 The reaction is stopped by adding approximately 50 μl of 2.5 M sulfuric acid to all the wells at the end of a previously determined incubation time (approximately 3 min.) In order to be able to obtain a sufficient optical density.

 The optical density is read on a microplate reader of the usual type comprising a filter adjusted to the wavelength characteristic of the chromogen (that is to say 492 nm). The values in the 1st column represent the reference ("white reagent") while the values in the 2nd column represent the values of the non-specific binding of the labeled immunoglobulin used.

 The titration curve, giving the optical density as a function of the dilution, is shown in Figure 1.

Example 3
Preparation of monoclonal antibodies
A. Preparation of an immunogen
An immunogen consisting of mouse albumin modified with malonic dialdehyde (MDA) is first prepared by the following method.

 Malonic dialdehyde (MDA) is obtained by hydrolyzing 125 μl of 1,1,3,3-tetraethoxypropane (TEP) in 50 ml of 1 W acetic acid in aqueous solution. 1 ml of this solution is brought into contact with 1 ml of mouse albumin solution (concentration 10 mg / ml) and the mixture is kept under incubation for 21 days in order to develop the aminoimino-propene (AIP) sites on the albumin.

 At the end of incubation, the antigen is conditioned to a concentration of 1 mg / ml.

B. Immunization of mice
6 week old female Balb / C mice are immunized by intraperitoneal injection of 25 µg of antigen per mouse.

Two new injections are made on D + 21 and D + 46.

 The first immunization is carried out in the presence of complete Freund's adjuvant, and the following in the presence of incomplete adjuvant. On days D + 3, D + 37 and D + 73, a blood sample is taken from the retro-orbital sinus using sterile Pasteur pipettes. The blood thus collected is centrifuged twice and the aliquot serum diluted 1/10 in PBS.

 The immunization evaluation is made by testing the sera collected in dilution on microtiter plates by a specific ELISA technique. According to this technique, the antigen (AIP bridge) is produced directly on the plate by chemical means as indicated in Example 1, which makes it possible to selectively fix the corresponding antibodies.

 These tests show the positivity of the sera.

C. Cell fusion
Three days before the fusion, 10 μl of antigen are added to 150 μl of sterile PBS and are injected into a mouse chosen according to the ELISA test above. After sacrifice of the animal, the spleen is removed and a strong splenomegaly is observed with activation nodules. Two myelomas are used for the fusion (SP2 / O-AG14 and X63Ag8 / 653).

Cell counts after washing by the usual techniques give the following results 6
mouse splenocytes: 140 x 106 living cells
6
myeloma SP2 / O-AG14: 40 x 10 living cells
myeloma X63Ag8 / 653: 28 x 106 living cells
In all cases, the viability rate is greater than 95%.

Lymphocyte hybridization is carried out in the presence of
PEG 1500 (Sigma) at 40% with a splenocyte / myeloma ratio of 5/1. 24 96-well plates were prepared in the presence of 10,000 Balb / C mouse peritoneal macrophages per well. The distribution of the cells is carried out by varying the quantity of splenocytes per well between three groups of plates (2.5 x 104, 5 x 104, and 105 respectively) for fusion with the myeloma SP2 / O-AG14, while it is 105 splenocytes per well for fusion with myeloma
X63Ag8 / 653.

 The medium used is as follows: RPMI 1640 (Biowhittaker), 2 mM glutamian (Biomérieux), 1 mM sodium pyruvate (Sigma); HAT (Sigma): 100 mM hypoxanthine, 0.4 mM aminopterin, 16 mM thymidine, 20% decomplemented fetal calf serum (USDA), penicillin 100U / ml, streptomycin 100 mg / ml (Sigma).

 The observation of the evolution is accompanied by the following observations: D + 3: appearance of the first clones; D + 7 change of medium in all wells; D + ll: addition of 5,000 macrophages per well.

 After microscopic observation, the wells in which a single clone has grown are removed for screening.

D. Screening of Fusion Hybridoma Supernatants
600 hybridoma supernatants (used pure) are tested in ELISA using the same technique as in point B. above.

At the end of the screening 45 hybridomas are selected for the reactivity of their supernatant on the AIP antigen in
ELISA. The antibodies produced in the 45 hybridoma supernatants are isotyped using anti-isotype mouse reagents from the Cliniscience kit (SBA Clonotyping II, n 5060-05).

Only the antibodies secreting antibodies of the IgG type are cultured in order to ensure their preservation and to provide a sufficient quantity of supernatant. Thus, 15 hybridomas are selected and tested on a specific ELISA plate, and by competitive technique against an antibody rich in specific antibodies.

 We can thus select the clones having the best responses towards the antigen. Two clones are tested (competition with respect to a serum) and developed, and the antibodies obtained are purified and reduced to the concentration of 0.5 mg / ml, then ELISA tests in dilution on specific AIP plates are performed.

 The tests show that the antibody obtained as indicated above is specifically directed against the AIP bridge, and this independently of the structure of the molecule (albumin or lysozyme) to which the antigen is linked.

Example 4
This example describes the application of the antibodies obtained in the previous example in immunocytometry to reveal AIP antigenic sites on endothelial cells in culture.

 A series of dilutions of the biotinylated antibody are made. The working dilution selected is the last dilution giving a clear fluorescence.

Birth control (non-peroxidized cells)
Endothelial cells in subculture 2 and not peroxidized are labeled with the antibody. In immunofluorescence, a diffuse, slightly marked fluorescence is observed, revealing the presence of native AIP sites on the cells.

Positive labeling (peroxidized cells)
Peroxidized cells have a very positive labeling of their cell membrane, thus demonstrating the development of specific antigenic sites on the surface of cells.

Claims (10)

 1. Antigenic structure of the type comprising a bridging group between a protein and a support, characterized in that it comprises a bridging group of the S-aminoimino-alkylene type, where S represents a support comprising free amino groups, obtained by covalent bond between an aldehyde and the free amino groups of the support S.  2. Antigenic structure according to claim 1, characterized in that the bridging group is of the S-amino-imino-propene type, where S is the support comprising free amino groups.  3. Antigenic structure according to claim 1, characterized in that it is represented by the formula  S - N = CH - A - R where S is the support carrying an amino group, A is a group derived from an aldehyde, R is a hydrogen atom or a group - CH = N - P, and P is a remains with an amino group, such as a protein or amino acid.  4. Antigenic structure according to claim 1, characterized in that the support S is a microplate comprising free amino groups.  5. Method for preparing an antigenic structure according to any one of the preceding claims, characterized in that it consists in reacting a compound containing at least one aldehyde function with a support comprising at least one free amino group  6. Method according to claim 5, characterized in that the aldehyde is chosen from formalin, glyoxal, glutaraldehyde, malonic dialdehyde (MDA), 4-hydroxynonenal (4HNE).  7. Antibody detection and assay assembly, characterized in that it comprises an antigenic structure according to any one of claims 1 to 5, combined with a protein.  8. Immunogenic substance for obtaining monoclonal antibodies directed against the amino-imino-alkylene bridge, and in particular the amino-imino-propene bridge, characterized in that it comprises an antigenic source according to any one of the claims 1 to 3.  9. Method for obtaining hybrid murine cell lines, characterized in that it comprises the following steps  a) mice are immunized with an immunogenic substance according to claim 8, preferably in the presence of a Freund's adjuvant  b) the spleen of the immunized mice is removed  c) the splenocytes are separated and fused with mouse myeloma cells, in the presence of a fusion promoter  d) the hybrid cells obtained are cultured in a selective medium in which the non-fused myeloma cells do not develop  e) the cells producing the desired antibody are selected by conventional techniques  f) if necessary, the selected cells are cloned.  10. Monoclonal antibodies directed against the aminoiminoalkylene bridge, and in particular the aminoiminopropene bridge, obtained by the method according to claim 9.