WO2009141458A1

WO2009141458A1 - Antibody specific for propionylated/butyrylated lysine, method for obtaining same and uses thereof

Info

Publication number: WO2009141458A1
Application number: PCT/EP2009/056334
Authority: WO
Inventors: Saïd EL ALAOUI EL BALRHITI; Ismaïl CEYLAN; Vincent Thomas
Original assignee: Covalab
Priority date: 2008-05-23
Filing date: 2009-05-25
Publication date: 2009-11-26
Also published as: EP2297201A1; FR2931481A1; FR2931481B1

Abstract

The invention relates to a novel antibody or at least one functional fragment thereof, which is specific, comprising at least one lysyl unit of formula (I), in which R⁰ is H, an alkyl, an aryl, an alkylaryl or an aralkyl, R¹ is H, an alkyl, an aryl, an alkylaryl, an aralkyl, a sugar, a lipid, a nucleotide or an entity comprising at least one amino acid unit, wherein said amino acid unit is linked to the lysyl unit by a peptide bond, R² is OH, an alkyl, an aryl, an alkylaryl, an aralkyl, a sugar, a lipid, a nucleotide or an entity comprising at least one amino acid unit, wherein said amino acid unit is linked to the lysyl unit by a peptide bond, or OR⁴ where R⁴ is an alkyl, an aryl, a sugar or a lipid, and R³ is (CH₂)_n‑CH₃ where n is 1 or 2. The invention also relates to a method for producing this novel antibody and to a composition containing same. In addition, for uses in research, diagnosis and therapeutics, the invention relates to methods and kits for detection of the epitopes of formula (I) and of autoantibodies which recognize said epitopes of formula (I).

Description

PROPIONYLE / BUTYRYLEE LYSINE-SPECIFIC ANTIBODY, METHOD FOR PRODUCING THE SAME, AND APPLICATIONS THEREOF

Technical Field of the Invention

The present invention relates to the field of protein modifications called post-translational modifications and in particular the domain of acyl radical-bearing proteins. More specifically, the invention relates to novel antibodies specific for these acyl radical-bearing proteins (in particular propionyl and butyryl radicals) as well as to their method of production and their applications.

Prior art and technical problem:

Transcription and translation are pillar mechanisms for cells and post-transcriptional and post-translational modifications have fundamental impacts in cellular functioning. The proteins remaining in their native state and the proteins having undergone these post-translational chemical modifications show great differences as regards their cell localizations, their half lives, their physico-chemical properties as well as their intra or intercellular actions. . Thus, the knowledge and understanding of the mechanisms regulating these changes can be very interesting in the areas not only of basic research but also and especially the therapeutic field. It is therefore essential for this to have mastered tools to identify, detect, locate and even quantify and induce or avoid these changes. US patent application 2008/0241862 (Zhao et al.) Discloses means for identifying propionylated and butyrylated lysines in proteins. These means of identification are polyclonal antibodies which specifically recognize acetylated lysines and recognize, by cross-reaction, propionylated lysines and butyrylated lysines because of their close structure. Antibodies specific for propionylated or butyrylated lysines are obtained by purification using long and expensive techniques of exhaustion. .

Also, it therefore appears necessary to provide detection means and methods implemented more easily and directly, reliable, sensitive, reproducible and to find tools for identifying / locating these modifications (propionylation and / or butyrylation of lysyl motif). also improving the research, diagnosis, monitoring, prognosis and treatment of pathologies associated with it. Also, has the inventor developed monoclonal antibodies capable of recognizing and binding specifically to these new protein modifications without having to use purification techniques (eg depletion) to obtain their specificity.

Main objectives of the invention:

The main objective of the invention is to provide means and methods for detecting said post-translational modifications that are more appropriate, faster and more reliable than the means used in the prior art, and consequently to make it possible to elucidate the mechanisms and to improve the diagnosis, treatment, monitoring and prognosis of diseases induced by these two post-translational modifications (propionylation and / or butyrylation of lysines).

Another object of the present invention is to provide compositions comprising this more appropriate detection means for use in diagnosis, therapy and research.

Another objective of the present invention is to provide economical, high-performance, reliable and feasible diagnostic methods simply in vitro or in vivo thanks to, on the one hand, a method and a kit for detecting, in a biological sample, molecules comprising said post-translational modifications (propionylation and / or butyrylation) and secondly a method and a kit for detecting, in a biological sample, autoantibodies directed against said post-translational modifications, in vitro or in vivo.

Brief description of the invention

These objectives, among others, are achieved by the present invention which firstly relates to new antibodies or at least one of their functional fragments specific for an epitope containing at least one lysyl unit of formula (I) below:

in which R is H, alkyl, aryl, alkylaryl or aralkyl, R ¹ is H, alkyl, aryl, alkylaryl, aralkyl, sugar, lipid, nucleotide or an entity consisting of at least one amino acid unit, said amino acid unit being attached to the lysyl unit by a peptide bond,

R ² is OH, alkyl, aryl, alkylaryl, aralkyl, sugar, lipid, nucleotide, an entity consisting of at least one amino acid unit, said amino acid unit being attached to the lysyl unit by a peptide bond, or OR where R ⁴ is an alkyl, an aryl, a sugar, a lipid,

R ^{3 is} equivalent to - (CHi) _n -CH ₃ where n is 1 or 2.

These antibodies are not specific for an epitope containing at least one lysyl unit of formula (I) in which R 0, R ¹ , R ² are as defined above and R ^{3 is} equivalent to -CH ₃ .

The present invention also relates to a process for the production of said antibodies or of one of their functional fragments consisting, in the first place, of manufacturing a synthon of formula (I) above, and then to using this synthon as an epitope antigenic to allow the production of antibodies by at least one injection of at least this epitope to an appropriate animal.

The present invention also relates to a composition comprising antibodies or functional fragments of the antibodies according to the present invention, said composition being able to be used in at least one of the following applications: research, diagnosis, analysis, prognosis of pathology, therapeutic, test of efficacy or toxicity of a drug.

The present invention also relates to a method and a kit for detecting molecules containing at least the epitope of formula (I), in a biological sample, in vitro or in vivo.

The present invention also relates to a method and a kit for detecting autoantibodies against the epitope of formula (I) in a biological sample or in vivo.

Detailed description of the invention

antibody

To detect molecular modifications such as post-translational modifications in vitro and in vivo, to understand their cellular implications and possibly to have an effect on them particularly in the therapeutic context of pathologies induced by these modifications, it is particularly interesting to have specific tools , able to recognize specific targets directly. Therefore, it is the merit of the inventor to have developed isolated antibodies or at least functional fragments thereof, characterized in that they are specific for an epitope comprising at least one lysyl unit of the following formula (I):

wherein R ⁰ is H, alkyl, aryl, alkylaryl or aralkyl,

R ¹ is H, alkyl, aryl, alkylaryl, aralkyl, sugar, lipid, nucleotide or an entity consisting of at least one amino acid unit, said amino acid unit being attached to the lysyl unit by a peptide bond,

R is OH, alkyl, aryl, alkylaryl, aralkyl, sugar, lipid, nucleotide, an entity consisting of at least one amino acid unit, said amino acid unit being attached to the lysyl unit through a linkage peptide, or OR ⁴ where R ⁴ is an alkyl, an aryl, a sugar, a lipid,

R ^{3 is} equivalent to - (CH ₂ ) _U -CH ₃ where n is 1 or 2.

The invention also relates to isolated antibodies or at least one of their functional fragments, as defined above, characterized in that they are little or not specific for an epitope containing at least one lysyl unit of formula ( I) wherein R ⁰ , R, R ² are as defined above and R ^{3 is} equivalent to -CH ₃ .

In the present application, the term "antibody" designates, for example, immunoglobulins (Ig) for example of the gamma (IgG) or Mu (IgM) type, preferably IgG. These antibodies may be polyclonal or monoclonal. They are derived from immunization of animals such as the rabbit, the mouse and all the animals that can be immunized and known to those skilled in the art, but they can be produced by genetic engineering. Moreover, the antibodies according to the invention may be naturally occurring or humanized animal (preferably murine) antibodies. By extension, the antibodies according to the present invention may be functional and specific fragments of said antibodies such as the Fab fragment which contains the antigen-specific recognition site or the F (ab ') ₂ fragment which contains at least the two sites of the antigen-specific recognition site. specific antigen recognition. By "specific" is meant for example that the antibodies are capable of recognizing and binding to a given epitope, namely the epitope comprising at least one lysyl unit of formula (I), via their paratope according to the known principles of immunology. In particular, the antibodies according to the present invention are, for example, capable of recognizing and binding to an epitope comprising at least one lysylpropionyl or butyrylated unit but are not suitable, for example, for recognizing and binding specifically to an epitope comprising at least one minus an acetylated lysyl motif. Indeed, the recognized antigen is defined by its particular epitope and this according to the structure or the conformation thereof. In the present application, the term "epitope" designates for example a molecule or a region thereof which can be recognized by the specific recognition part of an antibody (the paratope). An antigen is characterized by its epitopes. It is therefore in the present application a molecule containing at least one lysyl unit of formula (I) as defined above. By "peptide" forming the epitope for the generation of specific antibodies according to the invention, is meant for example a peptide comprising at least one lysyl unit substituted by a propionyl and / or butyryl radical, and more specifically a peptide sequence comprising between 2 and 50 amino acids, at least one of the amino acids being a lysyl unit substituted with a propionyl and / or butyryl radical. A peptide sequence of 2 to 25 amino acids and most preferably 2 to 15 amino acids will be preferred. By "protein" forming the epitope for the generation of specific antibodies according to the present invention is meant for example an immunogenic protein comprising at least one lysyl unit substituted with a propionyl and / or butyryl radical, and more specifically a higher peptide sequence. at least 50 amino acids, at least one of said amino acids being a lysyl unit substituted with a propionyl and / or butyryl radical. Preferably, said protein has a molecular weight of 5 kDaltons to 100 kDaltons and more particularly a molecular weight between 5 to 20 kDa.

For example, proteins such as p53 or histones, and more particularly histones H3, H4, H2A and H2B, may be mentioned. More particularly, the human H3 histone, with a molecular weight of approximately 15 kDa, may be propionylated and / or butyrylated on the lysines at the 4, 9, 14, 18, 23, and generally on at least one lysine (represented by K). Mention may also be made of human H4 histone with a molecular weight of about 11 kDa, of which at least one lysine at position 5, 8, 12, 16, 20 may be propionylated and / or butyrylated. Similarly for human H2B histone of molecular weight of about 14 kDa can be propionylated and / or butyrylated on lysines at position 5, 1 1, 12, 15, 16, .. as well as human histone H2A of weight molecular weight of about 14 kDa can be propionylated and / or butyrylated on lysines 5, 9, 13, 15, .. These propionylated and / or butyrylated lysines correspond to the formula (I) above and may thus represent antigens inducing the formation and specific recognition of the antibodies according to the invention.

According to a particular embodiment of the present invention, in the formula (1) above, the radical R ⁰ of the epitope recognized by the antibody is an H.

According to another particular embodiment of the present invention, R ⁰ is an alkyl chosen from linear, branched or cyclic alkyls, substituted or not by a heteroatom and having from 1 to 20 carbon atoms. According to yet another particular embodiment of the present invention, R ⁰ is an aryl chosen from aryls which may or may not be substituted by a heteroatom and which has 3 to 20 carbon atoms.

R ⁰ may also be an aralkyl substituted or not with a heteroatom and having 4 to 20 carbon atoms, an alkylaryl substituted or not with a heteroatom and having 4 to 20 carbon atoms.

According to a particular embodiment of the present invention, the radical R ¹ of the epitope recognized by the antibody is an H.

According to another particular embodiment of the present invention, R ¹ is an alkyl chosen from linear, branched or cyclic alkyls, substituted or unsubstituted by a heteroatom and comprising from 1 to 20 carbon atoms.

R ¹ is an aryl selected from aryls substituted or not with a heteroatom and having 3 to 20 carbon atoms.

R ¹ may also be an aralkyl substituted or not with a heteroatom and having 4 to 20 carbon atoms, an alkylaryl substituted or not with a heteroatom and having 4 to 20 carbon atoms.

In another particular embodiment of the invention, R ¹ is a sugar chosen from the saccharide, monosaccharide, disaccharide (sucrose, maltose eg) or hydrogenated polysaccharide or not, and their mixtures. In another particular embodiment of the invention, R ¹ is a lipid chosen from fatty acids, their derivatives and their esters.

In a particular embodiment of the invention, R ¹ may be a nucleotide selected from known nucleotides.

In another particular embodiment of the invention, R ¹ is an entity consisting of at least one amino acid which, via its C-terminal end, forms a peptide bond with the lysyl motif. It may thus be, for example, a simple amino acid, a peptide, preferably a peptide contained in the sequence of a histone and more particularly the histones H3, H4 or H2A, of a protein, preferably a histone protein and more particularly the histones H3, H4 or H2A, a glycoprotein, a lipoprotein ... The amino acid involved in the peptide bond with the lysyl motif is any amino acid selected from the 22 known amino acids. At least one amino acid is a natural or unnatural amino acid, D or L.

According to a particular embodiment of the present invention, the radical R ² of the epitope recognized by the antibody is an OH.

According to another particular embodiment of the present invention, R is an alkyl chosen from linear, branched or cyclic alkyls, substituted or not by a heteroatom and having from 1 to 1 to 20 carbon atoms. According to yet another particular embodiment of the present invention, R is an aryl chosen from heteroatom-substituted and non-substituted aryls having 3 to 20 carbon atoms.

R ² may also be an aralkyl substituted or not with a heteroatom and having 4 to 20 carbon atoms, an alkylaryl substituted or not with a heteroatom and having 4 to 20 carbon atoms.

In another particular embodiment of the invention, R is a sugar chosen from the saccharide, monosaccharide, disaccharide (sucrose, maltose e.g.) or hydrogenated polysaccharide or not, and their mixtures.

In another particular embodiment of the invention, R is a lipid chosen from fatty acids, their derivatives and their esters.

In a particular embodiment of the invention, R may be a nucleotide selected from known nucleotides.

In another particular embodiment of the invention, R is an entity consisting of at least one amino acid which, via its N-terminus, forms a peptide bond with the lysyl motif. It may thus be, for example, a simple amino acid, a peptide, preferably a peptide contained in the sequence of a histone and more particularly the histones H3, H4, H2A or H2B, of a protein, preferably a histone protein and more particularly the histones H3, H4, H2A or H2B, of a glycoprotein .... The amino acid involved in the peptide bond with the lysyl unit is any amino acid selected from the 22 known amino acids of which at least one amino acid is a natural or non-natural amino acid, D or L.

In a particular embodiment of the invention, R is equivalent to OR ⁴ where R ⁴ is an alkyl, an aryl, a sugar, a lipid as defined and / or exemplified above.

The epitope contains at least one propionyl or butyrylated site. Indeed, according to a particular embodiment of the present invention, the radical R ³ of the epitope recognized by the antibody according to the invention is a - (CH ₂ ) _H -CH ₃ radical where n is 1 or 2. When n is 1, the lysyl unit is substituted with a propionic acid. By convention in the In the present application, it will be possible to speak of a propionyl residue bonded to the lysyl unit, so that the epitope of formula (I) according to the present invention contains a lysyl propionyl unit. When n corresponds to 2, the lysyl unit is substituted by a butyric acid (or, by convention, a butyryl) thus forming an epitope of formula (I) containing a lysyl butyryl unit.

As understood from the present formula (I) and from the discussion above, the epitope specifically recognized by the antibodies, or at least one of their functional fragments, according to the present invention, may consist of a simple substituted amino acid, namely lysine substituted with a propionyl radical (n = 1) or a butyryl radical (n 1), this is the case where R ⁰ , R ¹ are H and R ² is an OH but R ⁰ , R ¹ and R ² of this epitope may be the same or different. Thus, the epitope may consist of a molecule made of at least two amino acids, one of which is a propionylated or butyrylated lysine, a peptide such as the N-terminal sequence of histones, a protein such as a histone, a glycoprotein or any other molecule containing at least one lysyl motif substituted with R ³ as defined above. These proteins can be natural, cellular, tissue or plasma proteins, or artificially produced proteins, recombinant proteins produced by genetic engineering. They are preferably histone proteins and especially histones H3, H4, H2A and H2B. Preferably, the epitope of formula (I) corresponds to a polypeptide of one hundred amino acids. In the preferred embodiment of the invention, R ⁰ is H and R ¹ and R ² are any of the same or different amino acids. When R ³ is other than - (CH ₁ ) _n -CH ₃ where n is 1 or 2, the antibodies according to the invention do not bind to the molecule specifically. This is particularly the case when R is acetyl.

In a preferred embodiment of the invention, lysine-rich peptides are used to generate the antibody. It is known to one skilled in the art that an antigenic site generally contains 5 to 20 amino acids, but according to a preferred embodiment of the invention, the specific antigenic epitope recognized by the antibody may contain a lower number of acids. amino, that is to say a single amino acid, lysine substituted by a propionyl or butyryl itself, at 5 amino acids without being limited to this number. Indeed, the epitope may contain up to 10 amino acids and preferably 5 amino acids.

In its preferred form, the lysyl unit substituted with a propionyl or butyryl radical is placed as centrally as possible within said antigenic site generating the antibody according to the invention.

In a particularly preferred embodiment, the peptide or protein comprising an epitope of formula (I), contains several propionylated and / or butyrylated lysines. These peptides are made by conventional peptide synthesis methods known to those skilled in the art.

Thus, through these antibodies, the state of the propionylation and / or butyrylation of cellular proteins involved in metabolisms and cellular functions can be determined, characterized and quantified. Indeed, the antibody tool according to the present invention allows a qualitative but also quantitative detection of molecules bearing the epitope of formula (I) in biological samples, in vitro and in vivo. In addition, it is possible to obtain a cellular localization of the molecules, preferably the proteins containing said epitope of formula (I) specifically recognized by the antibodies according to the present invention, as well as a location of this epitope of formula (I) itself. even in a particular molecule, preferably a protein. This is particularly important in the understanding of cellular mechanisms and consequently in the treatment of pathologies involving the propionylation / butyrylation of molecules, preferably proteins, and particularly the propionylation of lysine, such as cancer, neurodegenerative diseases, diabetes and diabetes. 'inflammation.

The antibodies according to the present invention may be present in vivo and generated either: - naturally in the body, during pathological processes, it is then the autoantibodies exposed below, - artificially, that is to say by activation of the immune system of a given organism via the introduction of an antigen comprising said epitope of formula (I). Thus the organism produces antibodies specific for the epitope, recognized as non-self.

The antibodies according to the present invention may be defined by their immunoreactivity against epitopes selected from the group consisting of: i. epitopes comprising at least one acetylated lysyl residue (n = 0 in the definition of R ³ of formula (I)); not. epitopes comprising at least one lysyl propionyl residue (n = 1 in the definition of R ³ of formula (I)); m. epitopes comprising at least one lysyl butyrylated residue (n = 2 in the definition of R of formula (I)).

According to the present invention this immunoreactivity is reflected by a T test for measuring the immunoreactivity of sera containing the epitopes (i) and / or (ii) and / or (iii). This test T is defined eg as follows: adsorbing, on a 96-well polystyrene plate (EIA / RIA, High Binding, Corning Costar), 100 μl per well of an antigen solution (BSA-propionylated, butyrylated BSA, acetylated BSA) at 10 μg / ml in buffer 0.1 M bicarbonate pH 9.5,

incubate for 2 hours at 37 ° C., empty the wells and deposit 150 μl / well of a 0.5% BSA solution in PBS buffer to saturate the excess adsorption sites on the plastic, and incubate for one hour at 37 ° C,

deposit the sera at the indicated dilutions in PBS buffer containing 0.1% tween-20. These solutions are deposited and diluted two by two directly on the plate (1/500, 1/1000, 1/2000 ...) so as to obtain 100 μl / well of final volume,

- Incubate for one hour at 37 ° C, then wash 3 times with PBS tween,

add 100 μl / well of a peroxidase-labeled rabbit anti-IgG (H + L) sheep antibody and leave for 30 min. at 37 ° C,

washing 3 times successively to eliminate the excess of reagent, -release the peroxidase activity using the reagent oxygenated water / tetramethylbenzidine (TMB) (Covalab),

stopping the reaction with 100 μl of H ₂ SO ₄ (2N),

- measure at 450 nm absorbance on plate reader (Multiskan Ascent, Labsystems). The T test is an ELISA test.

The present invention also covers the antibodies or at least one of their functional fragments specific for an epitope containing at least one lysyl unit of formula (I) as defined above, characterized in that their immunoreactivity for BSA antigens -propionylated or BSA-butyrylated, measured in a test T defined above, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies to 1/500, the OD at 450 nm is > 1, preferably is> 1.5; for dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is> 0.5, preferably is> 0.9; for a dilution of the serum containing said antibodies at 1/2000, the OD at 450 nm is

0.5, preferably is> 0.6;

for a dilution of the serum containing said antibodies at 1/4000, the OD at 450 nm is> 0.2, preferably is>0.3; for dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is> 0.2, preferably> 0.25. The present invention also relates to antibodies or at least one of their functional fragments as defined above, characterized

In that their immunoreactivity for BSA-propionylated antigens, measured in a T test, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies at 1/500 the OD at 450 nm is greater than or equal to, in ascending order preferably 0.6; 0.8; 1.0; 1.5; 2.0; For a dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is greater than or equal to, in increasing order of preference: 0.3; 0.6; 0.8; 1.0; 1.5; 2.0; 2.2; for dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is greater than or equal to, in ascending order preferably: 0.25; 0.4; 0.6; 1.0; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is greater than or equal to, in ascending order preferably: 0.2; 0.4; 0.6; for a dilution of the serum containing said antibodies at 1/8000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.2; 0.4;

And in that their immunoreactivity for BSA-acetylated antigens, measured in a T-test, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies at 1/500 the OD at 450 nm is less than or equal to, in increasing order preferably: 1.5; 1.0; 0.9; 0.8; 0.7;

0.6; for dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is less than or equal to, in ascending order preferably: 1.2; 1.0; 0.9; 0.8; 0.5;

0.4; for dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is less than or equal to, in increasing order of preference: 1.0; 0.8; 0.6; 0.5; 0.4;

0.3; for a dilution of the serum containing said antibodies at 1/4000, the OD at 450 nm is less than or equal to, in increasing order of preference: 0.8; 0.6; 0.7; 0.5; 0.3; 0.2; for dilution of the serum containing said antibodies to 1/8000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.7, 0.6; 0.5; 0.4; 0.25;

0.15.

The present invention relates to antibodies or at least one of their functional fragments as defined above, characterized

In that their immunoreactivity for BSA-butyrylated antigens, measured in a T test, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies to 1/500, the OD at 450 nm is greater than or equal to, in increasing order of preference: 0.2; 0.4; 0.6; 1.0; 1.5; for dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.16; 0.4; 0.6; 0.9; for a dilution of the serum containing said antibodies at 1/2000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.14; 0.3; 0.5; 0.6; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.1; 0.15; 0.2; 0.3; for dilution of the serum containing said antibodies to 1/8000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.1; 0.15; 0.2; 0.25; And in that their immunoreactivity for the BSA-acetylated antigens, measured in a T test, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies at 1 / 500, the OD at 450 nm is less than or equal to, in increasing order preferably: 0.8; 0.7; 0.6; 0.4; 0.3; 0.2; for a dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.7; 0.6; 0.5; 0.3; 0.20; 0.15; for a dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.6; 0.5; 0.4; 0.3; 0.15; 0.1; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.6; 0.5; 0.4; 0.3; 0.15;

0.1; for dilution of the serum containing said antibodies to 1/8000, the OD at 450 nm is less than or equal to, in ascending order preferably 0.6; 0.5; 0.4; 0.3; 0.15; 0.1.

The present invention relates to an antibody as defined above, characterized in that in a T test, its immunoreactivity with respect to the BSA-acetylated (BA) or the propionylated BSA (BP) is such that the measurement of OD at 450 nm corresponds to one of the following characteristics:

The present invention relates to an antibody as defined above, characterized in that in a T test, its immunoreactivity with BSA-acetylated (BA) or butyrylated BSA (BB) is such that the measurement of the OD at 450 nm corresponds to one of the following characteristics:

Method of making the antibody

The residues of the epitope of the formula (I), namely R ⁰ , R ¹ , R ² and R ³ , are grafted to the lysyl residue either naturally, for example in vivo, or artificially in vitro by simple reaction of the residues or molecules together or by forced reactions known to those skilled in the art. Thus, it is possible to produce antibodies by endogenous antigens carrying the epitope of formula (I) or by the synthesis of these antigens. The present invention thus relates to a method of manufacturing the antibodies or their functional fragments as defined above, characterized in that it consists in: a) implementing a synthon of formula (I), b) coupling said synthon to a carrier molecule, c) recovering plasma / serum from said animal to which the synthon-carrier molecule cut has been previously injected, d) selecting the specific antibodies of said synthon used in step a) using one or more epitopes of formula (I) as defined above, e) optionally purifying the antibodies by the known methods of skilled in the art.

The synthon corresponds to the formula (I). More precisely, said synthon corresponds to at least one substituted amino acid: a propionylated lysine or a butyrylated lysine. The synthon according to the present invention and as defined above is made by the method described by M. Bashir Uddin Surfraz et al in J. Med. Chem. 2007, 50, 1418-1422 and by Takeshi M. et al in Biomacromolecules, 2007, 8, 318-321.

Such a synthon is particularly interesting in the production of the antibodies according to the invention and particularly in the production of the antigen constituted by said synthon or comprising it. Once the synthon produced and is coupled directly to a carrier protein, or inserted into a peptide sequence, it can be used in the manufacture of specific antibodies directed against it or stored, preferably frozen at -20 ^° C. for later use. . By the term "coupled" is meant for example bound or conjugated. This binding or conjugation can be done chemically, for example by a covalent bond, adsorption or another method of attachment known to those skilled in the art. Preferably, the coupling is done by a coupling agent such as cardodiimide or glutaraldehyde. The carrier molecule to which the synthon is coupled is a molecule of molecular weight large enough to induce an immune reaction and the production of circulating antibodies. This molecule can be a protein, a polypeptide, a natural or synthetic polymer. It is preferably a protein and in particular a protein of molecular weight greater than or equal to 5 kDaltons such as albumin or keyhole limpet hemocyanin (KLH for Keyhole Limpet Haemocyanin). In a preferred form of the invention, KLH is used as the carrier protein. The synthon-carrier molecule pair is injected into an animal to be immunized. It is preferably in suspension and mixed with a solution of Freund's adjuvant (complete or incomplete). The animal to which is injected said synthon-carrier molecule in suspension can be the rabbit, the mouse, the rat, the goat, the sheep, the horse or the guinea pig, preferably the rabbit, the rat and the mouse. The injection of said pair can be done in one or more times according to conventional and known methods of immunization. The main routes of administration are subcutaneous, intradermal, or intramuscular injections; intravenous and intraperitoneal injections are only used in special cases. If there are several injections of the synthon-carrier molecule pair, the types of antibodies produced, specific to the synthon, are different. Indeed, it may be type G or M immunoglobulins. Thus, approximately 1 ml of said antigenic couple is injected intramuscularly into a rabbit and about 100 μl into a mouse. The concentration of synthon in the injection mixture is about 50 μg / ml.

The blood of the animal containing, among others, said antibodies specific for the injected synthon is recovered by sampling or by exsanguination. Preferably, plasma or serum also containing, inter alia, antibodies specific for the synthon as defined above. The serum containing the desired antibodies, directed against said synthon, normally contains several different antibody species directed against several epitopes of the same antigen, this polyclonal preparation is called. Techniques can also be employed to isolate and clone lymphocytes that produce only one molecular species (i.e. clonotype) of antibodies: antibodies that recognize only a single epitope of the antigen.

The step of selecting antibodies specific for said synthon as defined above is done using the ELISA (Enzyme-Linked Immuno Sorbent Assay) technique. This selection technique is widely described in the literature. To summarize, this principle known to those skilled in the art, is carried out as follows: the synthon described above or an epitope of formula (I) is attached to a carrier conventionally used in ELISA techniques at least a washing is carried out, the plasma or serum of the animal which has been immunized is added to the support, at least one washing is carried out, a solution containing antibodies, preferably of goat or sheep, specifically antibodies derived from the animal having been immunized, preferably rabbit, rat or mouse, said added antibodies being coupled to a molecule useful for their detection, preferably a substrate-degrading enzyme such as TMB degrading peroxidase or DAB at least one wash is carried out, the substrate of the molecule useful for detection (TMB or DAB) is added, the optical density is read, indicating the presence of the desired antibodies, and selected insi. In this selection technique, the propionylated and / or butyrylated BSA as defined in the present application is preferably used but the synthon as defined above coupled to a carrier protein or a peptide containing at least one lysylpropionylated unit and / or butyryl coupled to a carrier protein may also be used. When a step of purification of the antibodies is carried out, the latter may be made in the liquid or solid phase and in different ways namely affinity chromatography using either separation columns, magnetic beads or resins bearing said synthon in as a ligand. A solid support such as a sepharose column on which the synthons have been attached is preferably preferred, preferably by covalent bonding.

The present invention also relates to isolated antibodies or at least one of the functional fragments thereof, characterized in that they are obtained from an antigen consisting of a synthon of formula (I) in which R ⁰ equals at H, alkyl, aryl, alkylaryl or aralkyl, R ¹ is H, alkyl, aryl, alkylaryl, aralkyl, sugar, lipid, nucleotide, R is OH, alkyl, aryl, alkylaryl, aralkyl, sugar, lipid, nucleotide or OR ⁴ wherein R ⁴ is alkyl, aryl, sugar, lipid, R is - (CH ₂ ) _H -CH ₃ where n is 1 or 2.

Thus, the antibodies according to the present invention, obtained from said synthon, are specific for their epitope directly after their production, without requiring the use of other techniques such as the depletion of polyclonal antibodies to obtain their high specificity vis- with respect to propionylated or butyrylated lysines. This presents a major advantage. In an alternative embodiment, the invention also relates to a method for manufacturing the antibodies or a functional fragment thereof as defined above, characterized in that it consists of: a) reacting a molecule of formula CH ₃ - (CH 2) _n -CO-O-CO- (CH ₂ ) _n -CH ₃ where n is 1 or 2 with a carrier molecule in order to substitute the lysyl units of said carrier molecule, bl) recovering plasma / serum said animal to which said carrier molecule thus substituted has been injected beforehand; cl) selecting the antibodies specific for the substituted lysyl units produced in step a1) using one or more epitopes of formula (I) as defined above; above, dl) optionally purify the antibodies according to conventional methods.

Step a1 is carried out as follows: The carrier molecule, preferably BSA or KLH, is reacted with propionic or butyric anhydride, of formula CH ₃ - (CH ₂ ) _H -CO-O-CO- (CHi) _n -CH ₃ where n is 1 or 2 respectively, so that the lysines of the carrier molecule react to form a molecule containing substituted lysines and more specifically propionylées lysines and / or butyrylées. This carrier molecule, the lysines of which are substituted, is used as an immunogen comprising the epitopes of formula (I) described above.

Steps b1) to d1) are identical to steps c) to e) of the method of manufacturing antibodies or a functional fragment thereof described above. The present invention also relates to a method for manufacturing the antibodies, or a functional fragment thereof, as defined above, characterized in that it consists in: a2) implementing an epitope of formula (I) in which at least one of the residues R ⁰ _, R ¹ is different from H or R ² is different from OH, b2) couple said epitope to a carrier molecule, c2) recovering plasma / serum from said animal to which said epitope-carrier molecule pair has has been previously injected, d2) select the specific antibodies of said epitope used in step a) using one or more epitopes as defined in claim 1, e2) optionally purify the antibodies according to conventional methods.

Steps b2) to e2) are identical to steps b) to e) described above in the method of making antibodies specific to the synthon itself and will therefore not be recalled. The implementation of an epitope as defined above is carried out by the production, the extemporaneous manufacture of said epitope or by the use of an epitope corresponding to the formula (I) already synthesized according to known conventional synthetic methods. preferably peptide or protein syntheses.

Composition

The present invention also relates to a composition comprising antibodies, or at least one of their functional fragments, as defined above, or obtained according to the process as defined above.

Said composition may advantageously comprise reagents such as labeled or non-labeled secondary antibodies and revealing or visualization solutions making it possible to demonstrate the antibody-antigen reactions. Said composition may also comprise excipients of different types of type conventionally used in compositions containing antibodies.

Said composition, just like the antibodies according to the invention, can advantageously be used in the field of research, to explain biological phenomena and to test the efficacy of said antibodies or of the composition as defined above. Moreover, this composition comprising at least antibodies specific for the epitope of formula (I) exposed above, can be used in at least one of the following applications: research, diagnosis, analysis, prognosis of pathology, therapeutic, test for efficacy or toxicity of a drug. Among the pathologies related to the propionylation and / or butyrylation of lysine residues, there may be mentioned, for example, cancer, neurodegenerative diseases, diabetes and inflammatory pathologies. Method and device for detecting epitopes

According to another embodiment, the present invention relates to a method for detecting, in a biological sample, in vitro or in vivo, the epitope of formula (I) as defined above, characterized in that it comprises the steps following: A) optionally fix on a suitable support the antibodies or at least one of their functional fragments as defined above or included in a composition as defined above,

B) contacting a biological sample potentially containing said epitope of formula (I) with the antibodies or at least one of their functional fragments as defined above or as included in a composition as defined above,

C) optionally perform at least one wash,

D) detecting / revealing the antibody-epitope complexes of formula (I).

Preferably, beforehand, the antibodies according to the present invention are fixed on a substantially solid support or any reaction support making it possible to carry out an immunological test. The attachment to the support is preferably carried out by adsorption or covalent bonding. A possible washing step makes it possible to eliminate, after said fixation, the antibodies not fixed to said support, if appropriate. The biological sample concerned, to be used with the antibodies according to the invention, may be any substance capable of containing said epitope in question of formula (I) such as a solid or liquid substance, such as for example an animal, a complete organ or not, tissue cells, nuclear extracts of given cells, cytoplasmic extracts of given cells, blood, plasma, serum, lymph, urine, saliva. Preferably, serum is used. When the sample contains molecules containing at least one substituted lysyl unit corresponding to formula (I) and constituting an epitope, these molecules via this epitope will bind specifically to the antibodies previously fixed on a suitable support. A possible wash will eliminate non-fixed antibodies to the support and non-specific antibody-molecule complexes. According to methods known to those skilled in the art, the specific antibody may be used in the liquid phase, in solution.

In the context of the method and the kit for detecting the epitopes of formula (I) of the present application, the immunological reaction supports used may be of a different nature and are chosen from: slides, microplates, fluorescent latices, and latices. Magnetic membranes, immuno-filtration membranes or immuno-migration membranes, biochips, beads, fins, tubes but also liposomes, lipid vesicles, biological microparticles or obtained from polymers, emulsions .... and the detection method of step D) can be divided into several technologies such as ELISA, immunoturbidimetry, slide agglutination, nephelometry, turbidimetry, turbidimetry or latex particle amplified nephelometry, immunofluorescence (on slide, microplate, fluorescent latex, magnetic latex, test, on membrane, biochips etc .. and in general can be used any method for identifying and / or quantifying a reaction between an antigen and more particularly an epitope and an antibody, whatever the isotype.

Alternatively, techniques for the direct detection of antibodies as antibody-epitope complexes such as refractometry, diffraction of light rays by the reaction surface, methods for modifying electric conductivity or magnetic field can be used. etc ...

Preferably, the immunological reaction, namely the recognition of the antibody or at least one of its functional fragments according to the invention with the epitope of formula (I), is followed by an indicator reaction which allows the photometric detection of the enzymatic activity linked to the antibody-epitope complex of formula (I).

These methods for detecting the antibody-epitope complex of formula (I) make it possible not only to detect the epitopes of formula (I) present in a given biological sample in vitro or in vivo, but they also make it possible to locate and / or quantify said epitope at subcellular or molecular level as well as to estimate the potential biological roles of these detected post-translational modifications.

The present invention also relates to a detection kit, in a biological sample in vitro or in vivo, of a molecule containing at least the epitope of formula (I) as defined above, characterized in that it comprises: at least an antibody or at least one of its functional fragments as defined above, preferably fixed on a support and the appropriate material and necessary to detect the antibody-epitope complexes of formula (I). This kit makes it possible to quickly and reliably detect the presence or absence of a molecule comprising at least one epitope of formula (I)

Method and kit for detecting autoantibodies

These post-translational modifications of propionylation and / or butyrylation of lysines could be observed in vitro. Thanks to the present invention, it is now possible to observe these modifications in vivo. They can cause pathologies such as inflammatory diseases, cancer, neurodegenerative diseases and diabetes. Inadequate propionylation / butyrylation of a lysine residue can become an antigen in vivo and therefore induce an immune reaction, the defective protein being identified as non-self. The body then develops antibodies against its own body called autoantibodies. Indeed, in the present application, the term "autoantibody" means for example an immunoglobulin directed against a constituent of the body that produces the immunoglobulin (or antibody) in question. According to the present invention, it is an antibody directed against an epitope of formula (I) as defined above. These autoantibodies thus induce autoimmune pathologies and it may prove to be very important to be able to rapidly diagnose the presence or absence of autoantibodies in vivo in a patient, to be able to perform an analysis, preferably a serological follow-up. and a prognosis for pathologies. The invention therefore also relates to a method for detecting specific autoantibodies of the epitopes of formula (I) as defined above, in a biological sample in vitro or in vivo, preferably a plasma or a serum, characterized in that what it includes the following steps:

Al) optionally fixing on a suitable support at least one epitope of formula (I),

B1) contacting said epitope of formula (I), preferably fixed on a suitable support with a biological sample,

Cl) optionally carry out at least one wash,

D1) detecting / revealing said (optional) autoantibody-epitope complexes of formula (I) by appropriate detection means.

Preferably, the support is solid but it may be any reaction support for performing an immunological test. The epitope of formula (I) is attached to a suitable support. As stated above, preferably the attachment is by adsorption but it can also be performed by co val valent bond on a suitable support. It will be ensured that the amount of fixed epitope of formula (I) is not a limiting factor so that all the autoantibodies of the sample to be tested can thus be specifically attached thereto.

The different supports that can be used are identical to those usable and detailed above for implementing the method and the kit for detecting epitopes of formula (I).

The biological sample to be tested may be any biological substance capable of containing said epitope-specific autoantibodies (I) such as cells of the tissues, blood, plasma, serum, urine and saliva. Preferably, the sample to be tested is plasma or serum, diluted or not, of a patient potentially containing autoantibodies directed against the epitope of formula (I). The possible washes are intended to eliminate on the one hand the epitopes not fixed on the support, but also and especially to eliminate the free autoantibodies or non-specifically fixed to their specific epitope. According to the present invention, the different detection methods used are qualitative and / or quantitative. These methods for detecting autoantibody-epitope complexes are known to those skilled in the art. They can be broken down into several technologies such as those set out above for the process and the kit for detecting epitopes of formula (1).

The present invention also relates to a kit for detecting autoantibodies specific for the epitopes of formula (I) as defined above, in a biological sample in vitro or in vivo, preferably a plasma or a serum, characterized in that that he understands:

at least one epitope of formula (I) as defined above and the appropriate material necessary to detect / reveal the antibody-epitope complexes of formula (I).

In the context of the implementation of the method and the autoantibody detection kit according to the invention, one of the different detection methods mentioned above is used in the context of the implementation of the method or kit of detection of epitopes of formula (I).

Thanks to said auto-antibody detection kit, it is thus simpler, easier and faster to establish a diagnosis of possible pathologies induced by lysines substituted with a propionyl or butyryl residue, to carry out monitoring and analysis of the pathology and especially the serology of the patient to monitor, evaluate the efficacy and toxicity of a drug or a composition targeting for example these autoantibodies or use these autoantibodies in research.

Examples

The following examples illustrate the present application.

The invention will be better understood with reference to the accompanying figures in which: Figure 1 shows two titration curves of rabbit anti-lysine propionylated serum. Figure 2 shows two titration curves of rabbit anti-lysine butyrylated serum. FIG. 3 represents a photograph of a Western blot analysis of the acetylation reaction of histones H3 and H4 revealed by means of anti-acetyl antibodies. FIG. 4 represents a photograph of a western blot analysis of the propionylation reaction of the H3 and H4 histones revealed by the antibodies that are the subject of the invention.

FIG. 5 represents a photograph of a western blot analysis of the propionylation reaction of histones H3 and H4 in the presence or absence of various co-factors and revealed by means of the antibodies which are the subject of the invention.

FIG. 6 represents a photograph of a Western blot analysis of the detection of the natural propionylation of H2A histones revealed by the antibodies that are the subject of the invention.

Example 1 Process for the Preparation of Polyclonal Antibodies Specific for the Epitope of Formula (I)

1.1 Preparation of epitopes of formula (I) (step a1) for the production of immunogens and antigens.

1.1. a / Preparation of immunogens (KLH) containing lysyl acylated units (propionyl and / or butyrylated) for the immunization of animals.

This preparation has been described in the following article: Thomas V et al, in J Immunol

Methods. 2004 Sep; 292 (1-2): 83-95.

Briefly, the propionylated KLH and butyrylated KLH antigens were prepared as follows:

Propionylated KLH: 2 mg of KLH solubilized in 1 ml of 0.1 M bicarbonate buffer pH 9.5 were brought into contact with 0.7 mmol of propionic anhydride (CH ₃ - (CHa) -CO-O-

CO- (CHo) -CH ₃ ). After incubation for 2 hours at 22 ° C., the modified protein, substituted with propionyl radicals on the lysyl units, is dialysed 3 times against 2 liters of buffer.

PBS.

KLH butyrylated .2 mg of KLH solubilized in 1 ml of 0.1 M bicarbonate buffer pH 9.5 were brought into contact with 0.7 mmol of butyric anhydride (CH ₃ - (CH ₂ ) a-CO-O-CO- (CH ₃ ) ) ₂ - CH ₃ ). After incubation for 2 hours at 22 ° C., the modified protein substituted with butyryl radicals on the lysyl units is dialyzed 3 times against 2 liters of PBS.

1.1. b / Preparation of antigens (BSA) containing lysyl acylated units (propionyl and / or butyrylated) for the ELISA test.

To obtain the propionylated BSA protein (BP), the same procedure and the same proportions as for propionylated KHL above are used. To obtain the butyrylated BSA protein (BB), the same procedure and the same proportions as for butyrylated KLH above are used.

To obtain the acetylated BSA protein (BA), the same procedure and the same proportions as described in Thomas V. et al in J Immunol Methods. 2004 Sep; 292 (1-2): 83-95 are used.

1.1. c / Control of the degree of acylation by spectrophotometric assay of the primary amines.

To 50 μl of solutions of native BSA and of acylated BSA (BP, BB) (2 mg / ml) are added 50 μl of a solution of saturated sodium tetraborate and 50 μl of 2,4,6-trinitrobenzene sulphonic acid ( 0.ImM). 2,4,6-trinitrobenzene sulphonic acid is a reagent that allows the colorimetric assay of primary amines. The acylation consists of forming amide bonds on the primary amines and in particular on the εNH ₂ residues of the lysine of the BSA. The amidated residues are not reactive with 2,4,6-trinitrobenzene sulphonic acid, it follows that a decrease in absorbance is proportional to the degree of acylation, this assay technique therefore makes it possible to calculate the percentage of acylated amines. After 30 minutes of reaction, the absorbance at 420 nm is measured spectrophotometrically. The decrease in absorbance compared to that obtained with the native BSA is expressed as a percentage.

Table 1: Degree of acylation of proteins by spectrophotometric assay of primary amines.

Table 1 shows that for the 2 synthesized antigens (propionylated BSA and butyrylated BSA), the acylation is almost complete, it follows that almost all of the accessible εNH ₂ groups of lysine present on the BSA are acylated.

1.2 Immunization of Animals 4 New Zealand female rabbits weighing 2.5 kg received 4 injections intradermally and subcutaneously on days JO, D14, D28 and J74 of ImI of a mixture (50% / 50%). a solution containing 100 μg of the immunogen KLH-propionyl (2 rabbits) and KLH-butyryl (2 rabbits) and Freund's adjuvant according to the immunization protocol in the table below. Regular blood samples (at days 53, 74 and 90) were taken to control the immune response (step c1). This blood is allowed to coagulate for 30 minutes and then is centrifuged for 10 minutes at 2500 rpm to recover the serum and test the immuno-reactivated antibodies (step b1).

1.3- Selection of antibodies specific for the epitope in the sera (step c)

The antibodies specific for the substituted lysyl units were selected in the serum with the epitope of formula (I) produced above by determining the immunoreactivity of the rabbit serum by the ELISA technique (step c1).

BP or BB antigens are adsorbed on 96-well polystyrene plates. To do this, 100 μl of a solution 10 μg / ml of each antigen in 0.1 M bicarbonate buffer pH 9.5 are distributed per well. After incubation for 2 hours at 37 ° C., the wells are emptied and a deposit of 150 μl / well of a 0.5% BSA solution in PBS buffer is prepared in order to saturate the excess adsorption sites on the surface. plastic. This saturation step requires an incubation of one hour at 37 ° C. At this stage, the plates are ready for the deposition of the sera. For this test, the sera are used starting from the 1/500 dilution in PBS buffer containing 0.1% tween-20. These solutions are deposited and then diluted two by two directly on the plate (1/500, 1/1000, 1/2000 ...) so as to obtain 100 μl / well of final volume.

After one hour of incubation at 37 ° C., the plates are washed 3 times with PBS tween. An anti-rabbit IgG (H + L) sheep antibody labeled with peroxidase and deposited at a rate of 100 μl / well and left for 30 min. at 37 ^° C. The plates are then washed as previously by 3 successive washes to remove the excess reagent. The peroxidase activity is revealed using the hydrogen peroxide / tetramethylbenzidine (TMB) reagent (Covalab). The reaction is stopped with 100 μl of H ₂ SO ₄ (2N). The absorbance is measured on a plate reader at 450 nm. 1.4-purification of the antibodies (step el)

The immuno-purification steps of the antibodies are carried out according to the protocols described in the publication Thomas V et al in J. Immunol Methods. 2004 Sep; 292 (1-2): 83-95.

1.5- Results: Determination of the immunoreactivity of rabbit sera by ELISA

5 .1- Selection of anti-N-ε-propionyl-L-Lysine rabbit antibodies with acetylated BSA (BA) or propionylated BSA (BP).

Table 2: Optical density measurements according to the dilutions of the sera. Table 2 reflects the immunoreactivity of rabbit sera containing antibodies specific for propionylated and / or butyrylated lysines, vis-à-vis acetylated BSA and propionylated BSA. Propionylated BSA forms a complex with antibodies specific for propionylated lysine (high absorbance).

Figure 1 illustrates the immunoreactivity of sera against two antigens: acetylated BSA and propionylated BSA. The ordinate axis represents optical density values measured at 450 nm and the x-axis represents the different dilutions of the serum. This figure 1 illustrates that the sera contain antibodies specific for propionylated lysines. In fact, the antibodies obtained and contained in the serum react strongly with the lysylpropionyl unit contained in the BP antigen (absorbance of 1.647 at the 1/2000 dilution of the serum), whereas they do not react or very little with the motif. acetylated lysyl contained in BA antigen (absorbance of 0.250 at 1/2000 dilution of serum). These antibodies make it possible to differentiate N-ε-propionyl-L-lysine from N-ε-acetyl-L-lysine and are therefore highly specific for propionylated lysines. 5.2-Selection of anti-N-ε-butyryl-L-lysine rabbit antibodies with acetylated BSA (BA) or butyrylated BSA (BB).

Table 3: Optical density measurements according to the dilutions of the sera. Table 3 reflects the immunoreactivity of rabbit sera containing antibodies specific for propionylated and / or butyrylated lysines against acetylated BSA and butyrylated BSA. Butyrylated BSA forms a complex with antibodies specific for butyrylated lysine (high absorbance).

Figure 2 illustrates the immunoreactivity of sera against two antigens: acetylated BSA and butyrylated BSA. The ordinate axis represents optical density values measured at 450 nm and the x-axis represents the different dilutions of the serum. This figure 2 illustrates that the sera contain antibodies specific for butyrylated lysines. In fact, the antibodies obtained and contained in the serum react with the lysyl butyryl unit contained in the BB antigen (absorbance of 1.558 at the 1/500 dilution of the serum) whereas they do not react with the acetylated lysyl motif contained in BA antigen (absorbance of 0.158 at 1/500 dilution of serum). These antibodies make it possible to differentiate N-ε-butyryl-L-Lysine from N-ε-acetyl-L-lysine and are therefore highly specific for butyrylated lysines.

Example 2. Method of producing the antibodies

The synthon is obtained according to the methods described by M. Bashir Uddin Surfraz et al in J. Med. Chem. 2007, 50, 1418-1422 and by Takeshi M. et al in Biomacromolecules, 2007, 8, 318-321.

5 mg of lysylpropionylated and / or butyrylated synthon is coupled to 2 mg of BSA or KLH using carbodiimide as coupling agent to conjugate the synthons by the alpha carboxylic acid domains left free for carrying out said coupling according to the technique described by Thomas V et al in J Immunol Methods. 2004 Sep; 292 (1-2): 83-95. The immunogen thus prepared is injected into the animals under the same conditions and procedures described in Example 1.

Example 3 Study of the Specificity of the Anti-Propionyl and Anti-Butyryl Antibodies on the In Vitro and In Vivo Modified Histones and the Method for Detecting the Epitope of the Formula (I)

Preparation of histones, polyacrylamide gel electrophoresis and western blots were carried out according to the method referenced in Bannister AJ and Kouzarides T, Nature. 1996 Dec 19-26; 384 (6610): 641-3. The appropriate antibodies used are the anti-acetyl lysine monoclonal antibody (HA1 clone mab0019-Covalab) and the anti-priopionyl and anti-butyryl rabbit polyclonal antibodies (object of the invention). The enzymatic reactions of the histones acetyl tranferases P / CAF and PCB were carried out according to the method described by Bannister AJ and Kouzarides T in Nature. 1996 Dec 19-26; 384 (6610): 641-3 by adding to the reaction medium the acetyl CoA, propionyl CoA or final butyryl-CoA cofactors of 100 μM.

Treatment of HL60 cells: HL60 cells were incubated with 1.5 μM Trichostatin A (TSA) a histone deacetylase inhibitor for 5 hours. The cells are then washed 3 times with PBS buffer and centrifuged at 13,000 g. The cell pellet is then resuspended in IPH buffer (50mM Tris pH 8, 15OmM NaCl, 5mM EDTA, 5% NP-40, 1.5uM TSA) and the suspension is left for 15 min at 4 ° C. The tubes are then centrifuged at 13,000 g for 10 min. The chromatin pellet is then taken up in the electrophoresis buffer as described previously to carry out the western blot.

Results:

FIG. 3 illustrates the acetylation reaction of the histones H3 and H4 of the HL60 cells by the Histone Acetyl Transferases HATs (P / CAF & CBP) in the presence (+) or not (-) of the TSA inhibitor. The revelation is carried out with the anti-acetyl lysine monoclonal antibody (mab0019). Conclusion: The control reaction to verify the model of posttranslational histone modification in vitro clearly shows significant acetylation of H3 and H4 histones. The addition of TSA exerts an inhibitory effect on the HDAC enzyme since the intensity of labeling is greater. These results are in perfect agreement with the literature and validate the system used. FIG. 4 illustrates the propionylation reaction of the histones H3 and H4 of the HL60 cells by the histones acetyl transferases HATs (P / CAF & CBP) in the presence (+) or not (-) of the TSA inhibitor. The revelation is carried out with the polyclonal rabbit anti-lysine propionylated antibody (object of the invention). Conclusion: In the post-translational modification conditions of the HL60 cells demonstrated in FIG. 3, these results clearly show a propionylation of the histones H3 and H4. The addition of TSA shows that the HDCA enzyme has an effect on propionylation, namely a depropionylation of histone lysines. FIG. 5 illustrates the propionylation reaction of the histones H3 and H4 of the HL60 cells by the histones acetyl transferases HATs (P / CAF & CBP) in the presence of: acetyl-CoA (Ac), propionyl-CoA (Pr), butyryl-CoA (Bu) or without cofactor (-). The revelation is carried out with the polyclonal rabbit anti-lysine propionylated antibody (object of the invention). Conclusion: Anti-lysine propionylated polyclonal antibodies specifically recognize H3 and H4 histone propionylated by the action of HATs (P / CAF and CBP). There is no cross reaction of anti-lysine propionylated antibodies with acetylated or butyrylated histones.

FIG. 6 illustrates the detection of naturally propionylated H2A histones of HL60 cells in the (+) or non (-) presence of the TSA inhibitor. The revelation is carried out with the polyclonal rabbit anti-lysine propionylated antibody (object of the invention).

Conclusion: The polyclonal rabbit anti-lysine propionylated antibody detects a natural propionylation of histone H2A in HL60 cells. Propionylation is slightly affected by the action of HDAC as shown by the result obtained with the addition of TSA. The antibodies of the invention can also be used in immunohistochemistry or immunocytology for the detection of epitopes of formula (I) according to the technique described in Roch et al. 1991 May 10; 48 (2): 215-20.

Example 4 Method for Detecting Specific Autoantibodies of the Epitopes of Formula (I)

The same method described in Example 1.3 above is used to detect autoantibodies in a sample and in particular in a serum. BP or BB antigens are adsorbed on 96-well polystyrene plates. To do this, 100 μl of a solution 10 μg / ml of each antigen in 0.1 M bicarbonate buffer pH 9.5 are distributed per well. After incubation for 2 hours at 37 ^° C., the wells are emptied and a deposit of 150 μl / well of a solution of 0.5% BSA in PBS buffer is prepared in order to saturate the excess adsorption sites on the surface. plastic. This saturation step requires an incubation of one hour at 37 ° C. At this stage, the plates are ready for depositing the serum to be tested. For this test, the serum to be tested is a human serum taken and then used at different dilutions in PBS tween 0.1%. This solution is deposited and then diluted two by two directly on the plate (1 / 50.1 / 100, 1/200,) so as to obtain 100 .mu.l / well of final volume. After one hour of incubation at 37 ° C., the plates are washed 3 times with PBS containing 0.1% tween-20. An anti-human IgG (H + L) sheep antibody labeled with peroxidase and deposited at a rate of 100 μl / well and left for 30 min. at 37 ° C. The plates are then washed with 3 successive washes to remove the excess reagent (sheep antibody). Said possible epitope-autoantibody complexes are revealed thanks to the substrate of peroxidase. The peroxidase activity is revealed using the hydrogen peroxide / tetramethylbenzidine (TMB) reagent (Covalab). The reaction is stopped with 100 μl of H ₂ SO ₄ (2N). The absorbance is measured on a plate reader at 450 nm.

Claims

1. Isolated antibodies or at least one of their functional fragments, characterized in that they are specific for an epitope comprising at least one lysyl unit of formula (I) below:

wherein R ⁰ is H, alkyl, aryl, alkylaryl or aralkyl,

R ^{3 is} equivalent to - (CH ₂ ) _O -CH ₃ where n is 1 or 2.

2. Isolated antibodies or at least one of their functional fragments, according to claim 1 characterized in that they are little or not specific for an epitope containing at least one lysyl unit of formula (I) in which R ⁰ , R ¹ , R ² are as defined above and R ^{3 is} equivalent to -CH ₃ .

3. Antibody or at least one of their functional fragments according to claim 1 or 2, characterized in that their immunoreactivity for BSA-propionylated antigens, measured in a T test, is such that it responds to least one of the following characteristics: for a dilution of the serum containing said antibodies to 1/500, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.6; 0.8; 1.0; 1.5; 2.0; 2 5 '2 7 - for dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is greater than or equal to, in increasing order of preference: 0.3; 0.6; 0.8; 1.0;

1.5; 2.0; 2.2; for dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is greater than or equal to, in ascending order preferably: 0.25; 0.4; 0.6; 1.0; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is greater than or equal to, in ascending order preferably: 0.2; 0.4; 0.6; for a dilution of the serum containing said antibodies at 1 / 8,000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.2; 0.4; And in that their immunoreactivity for BSA-acetylated antigens, measured in a T-test, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies at 1/500 the OD at 450 nm is less than or equal to, in increasing order preferably: 1.5; 1.0; 0.9; 0.8; 0.7;

0.6; for a dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is less than or equal to, in increasing order preferably: 1.2; 1.0; 0.9; 0.8; 0.5;

0.4; for a dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is less than or equal to, in ascending order preferably: 1.0; 0.8; 0.6; 0.5; 0.4; 0.3; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.8; 0.6; 0.7; 0.5; 0.3;

0.2; for dilution of the serum containing said antibodies to 1/8000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.7, 0.6; 0.5; 0.4; 0.25;

0.15.

4. Antibody or at least one of their functional fragments according to any one of Claims 1 to 3, characterized in that their immunoreactivity for BSA-butyrylated antigens, measured in a T test, is such that it corresponds to at least one of the following characteristics: for a dilution of the serum containing said antibodies at 1/500, the OD at 450 nm is greater than or equal to, in increasing order of preference: 0.2; 0.4; 0.6; 1.0; 1.5; for dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.16; 0.4; 0.6; 0.9; for a dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.14; 0.3; 0.5; 0.6; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.1; 0.15; 0.2; 0.3; for dilution of the serum containing said antibodies to 1/8000, the OD at 450 nm is greater than or equal to, in increasing order preferably: 0.1; 0.15; 0.2; 0.25; And in that their immunoreactivity for BSA-acetylated antigens, measured in a T-test, is such that it meets at least one of the following characteristics: for a dilution of the serum containing said antibodies at 1/500 the OD at 450 nm is less than or equal to, in ascending order preferably 0.8; 0.7; 0.6; 0.4; 0.3;

0.2; for a dilution of the serum containing said antibodies to 1/1000, the OD at 450 nm is less than or equal to, in increasing order of preference: 0.7; 0.6; 0.5; 0.3; 0.20;

0.15; for a dilution of the serum containing said antibodies to 1/2000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.6; 0.5; 0.4; 0.3; 0.15; 0.1; for a dilution of the serum containing said antibodies to 1/4000, the OD at 450 nm is less than or equal to, in ascending order preferably: 0.6; 0.5; 0.4; 0.3; 0.15;

0.1; for dilution of the serum containing said antibodies to 1/8000, the OD at 450 nm is less than or equal to, in ascending order preferably 0.6; 0.5; 0.4; 0.3; 0.15;

0.1.

5. A process for producing the antibodies or at least one of their functional fragments as defined in one of claims 1 to 4, characterized in that it consists in: a) implementing a synthon of formula ( I), b) coupling said synthon to a carrier molecule, c) recovering plasma / serum from said animal to which said synthon-carrier molecule pair has been previously injected, d) selecting antibodies specific for said synthon used in step a) to using one or more epitopes of formula (I) as defined in claim I, e) optionally purify the antibodies according to conventional methods.

6. A process for producing the antibodies or at least one of their functional fragments as defined in one of claims 1 to 4, characterized in that consists of: a) reacting a molecule of the formula CH ₃ - (CH ₂ ) _n -CO-O-CO- (CH ₂ ) _n -CH ₃ where n is 1 or 2 with a carrier molecule in order to substitute the lysyl units of the carrier molecule, bl) recovering the plasma / serum of said animal to which said carrier molecule thus substituted has been previously injected, cl) selecting the antibodies specific for the substituted lysyl units carried out in step a1) using one or several epitopes of formula (I) as defined in claim 1, dl) optionally purify the antibodies according to conventional methods.

7. Process for the manufacture of antibodies or at least one of their functional fragments as defined in one of Claims 1 to 4, characterized in that it consists in: a2) implementing an epitope of formula ( I) wherein at least one of the residues R ⁰ R ¹ is different from H or R ² is different from OH, b2) coupling said epitope to a carrier molecule, c2) recovering plasma / serum from said animal to which said epitope-molecule pair carrier has been previously injected, d2) select the specific antibodies of said epitope used in step a2) using one or more epitopes of formula (I) as defined in claim 1, e2) optionally purify the antibodies according to classical methods.

8. Composition comprising antibodies or at least one of their functional fragments as defined in one of Claims 1 to 4, or obtained according to the process as defined in one of Claims 5 to 7.

9. Composition according to claim 8, characterized in that it is used in at least one of the following applications: research, diagnosis, analysis, prognosis of pathology, therapeutic, test of efficacy or toxicity of a drug.

10. A method for detecting, in a biological sample, the epitope of formula (I) as defined in claim 1, characterized in that it comprises the following steps: A) optionally fixing on an appropriate support the antibodies or one of their functional fragments as defined in one of claims 1 to 4 or included in a composition as defined in claim 8, B) contacting a sample potentially containing said epitope of formula (I) with the antibodies or at least one of their functional fragments as defined in one of claims 1 to 4 or included in a composition as defined in claim 8, C) optionally performing at least one wash,

D) detecting / revealing the antibody-epitope complexes of formula (I).

11. A kit for detecting, in a biological sample, a molecule containing at least the epitope of formula (I) as defined in claim 1, characterized in that it comprises: at least one antibody as defined in the claim 1, preferably attached to a support and the appropriate material and necessary to detect antibody-epitope complexes of formula (I).

12. A method for detecting specific autoantibodies of the epitopes of formula (I) as defined in claim 1, in a biological sample, preferably a plasma or a serum, characterized in that it comprises the following steps: ) optionally fixing on a suitable support at least one epitope of formula

(I)

B1) contacting said epitope of formula (I), preferably fixed on a suitable support, with a sample,

C 1) optionally carry out at least one washing, Dl) detecting / revealing said (optional) autoantibody-epitope complexes of formula (I) by appropriate detecting means.

13. A kit for detecting specific autoantibodies of the epitopes of formula (I) as defined in claim 1, in a biological sample, preferably a plasma or a serum, characterized in that it comprises:

at least one epitope of formula (I) as defined in claim 1 and

the appropriate and necessary material for detecting / revealing the antibody-epitope complexes of formula (I).