EP3233896A1 - Synthetic bi-epitope compound - Google Patents

Abstract

The present invention concerns a bi-epitope compound of formula I: in which: -E1 and E2, identical or different, each separately represents a peptide sequence comprising at least one epitope of an analyte; X and Y, identical or different, each separately represents a linking arm, -the carrier molecule is soluble and -Z represents an amino acid derivative bearing a thiol function prior to the bonding of same with the carrier molecule. It also concerns the compositions containing this compound, and the use of such a compound or of the composition containing this compound as a control or standard in an immunoassay, the methods using the compound or the composition containing this compound as a control or standard, and finally the kits for implementing an immunoassay comprising the compound or the composition containing this compound.

Description

 Biepitopic synthetic compound

 The present invention relates to the field of diagnosis or prognosis. In particular, it relates to a synthetic biepitopic compound useful during the implementation of immunoassays.

 Immunoassays are widely used in the fields of clinical, food, pharmaceutical and chemical analyzes. Thus, they are intended to determine the presence of a large number of analytes, in the form of proteins (antigens / antibodies), peptides, haptens, such as steroids or vitamins, in samples that may contain these analytes. The immunoassay is a test widely known to those skilled in the art that involves immunological reactions between the analyte to be detected and one or more binding partner (s) for this analyte. Examples of such immunoassays include methods such as Enzyme Linked Linked Immuno Sorbent Assay (ELISA), Enzyme Linked Florescent Assay (ELFA) and Radio Immuno Assay (RIA) which can operate according to the "sandwich" principle. or the principle of "competition", and immunodetection methods such as immunohistochemistry, immunocytochemistry, immunofluorescence, Western-blot and Dot-blot. "Competition" methods are usually used for small molecules such as haptens, with "sandwich" methods being used for other analytes.

 These immunoassays practiced especially in the biological analysis laboratories require the supply by the manufacturer, in addition to the reagents necessary for the test, such as the binding partners, the revealing agents or the dilution solutions, a positive control of the test. This test, which is carried out under conditions similar to those of the test to be studied, often simultaneously, will be used to validate the immunoassay. If the positive control is found to be positive, the result of the test is valid and can be interpreted. If the positive control is not found positive, it indicates that the implementation of the immunoassay did not proceed as expected. The test result that is invalid should not be interpreted and the analysis repeated.

As for the quantification of an analyte by immunoassay in a biological sample that may contain said analyte, it also requires reagents necessary for the above-mentioned test and positive control, the use of a standard curve. This is obtained by: i) measuring the signal generated by standards, also called standards or calibrators, which correspond to known and increasing amounts or concentrations of the analyte or of a compound having the same antigenic reactivity as the analyte in the immunoassay used, ii) and then plotting the signal curve as a function of quantity or concentration. Very often, it is customary to find a mathematical model that represents as closely as possible this relationship between the signal and the quantity or concentration, in order to easily calculate the results of a quantitative immunoassay.

 To do this, control solutions or standards must mimic the desired analyte and be recognized in the same way by the binding partners used in the immunoassay. Thus, if the immunoassay method is a sandwich method, the control or standard solutions must include a compound that has both recognition epitopes of the two binding partners used. This is called a biepitopic compound.

 It is not excluded that the two epitopes of a biepitopic compound are identical. When the analyte to be detected or quantified is multimeric, at least dimeric, it is possible to use the same binding partner in capture and detection of the sandwich immunoassay. In this case, the biepitopic compound will contain twice the same epitope.

Control solutions or standards usually used in immunoassay tests may be of human or animal origin and contain the analyte as such in the natural state. These solutions are prepared from lyophilizate, and frozen in unit doses and stored at -20 ° C or -80 ° C. Such preservation is not suitable for a fluid laboratory practice. In addition, to be able to be used, these freeze-dried control solutions or standards need to be put back into solution. But in the context of immunoassay, the implementation of the test must be rapid and this solution in solution results in a loss of time. In addition, carrying out this redissolution can lead to a measurement error due to a bias due to dilution. Control solutions or ready-to-use standards, stored in liquid form at + 2/8 ° C, are therefore particularly recommended, for reasons of obvious convenience. Nevertheless, to be representative of the actual conditions of the assay, these control solutions or ready-to-use standards contain only low concentrations of the analyte, for example of the order of pg / ml, ng / ml or μg. / ml, depending on the measurement range of the analyte concerned, which has the effect of affecting their stability at a temperature of + 2/8 ° C. Therefore, to overcome this disadvantage, synthetic standards have been used.

 European Patent Application EP 0 650 053 A describes synthetic standards containing active sites for one or more receptors, interconnected by a tree structure. This application more specifically describes synthetic standards of troponin T. However, these standards have a stability period in solution of not more than 3 weeks at 4 ° C.

 Patent application WO98 / 24816, for its part, proposes biepitopic synthetic compounds that can be used as standard in sandwich immunoassays, for the determination of troponin I, which are stable for several months. The described compounds, of formula Σ-Ε1---Ε2-Ψ, comprise two peptide sequences E1 and E2 comprising a minimum epitope of troponin I, each of these epitopes being linked together by a linker group (linker) which may be a central peptide comprising from 1 to 40 amino acids. Each epitope may also comprise at its end a peptide sequence of 1 to 10 amino acids (Σ and Ψ). Nevertheless, the solution proposed in this patent application has the disadvantage that the described biepitopic compounds, in order to be sufficiently immunoreactive with the binding partners used in the immunoassay, must have a significant number of amino acids, so that their synthesis can not be reduced. is not easy. In addition, by their peptide-only nature, a large amount of compound is required in the control or standard solution to have optimal immunoreactivity with the binding partners used in the immunoassay, which represents a significant cost for the test manufacturer. kits containing this control or standard and therefore for the user laboratory of the kit thus produced.

US Pat. No. 6,114,180 proposes a synthetic compound that can be used as a control or standard in immunoassays comprising two epitopes of troponin I linked together by a support molecule, such as BSA, for the purpose. to increase its solubility and / or its stability in solution. However, the particular construction of this compound makes its production difficult and raises the question of the equimolarity between the two epitopes connected to the carrier molecule.

 The Applicant has surprisingly demonstrated a synthetic compound for use in "sandwich" immunoassays overcoming the disadvantages described in the prior art. Indeed, its synthesis is easy and simplified, it is stable at + 2/8 ° C and at low concentration, it is soluble and it has excellent immunoreactivity with the binding partners used in the immunoassay. In addition, it is not necessary to use a large amount, if the compound is present in the control or standard solution, to have optimal immunoreactivity with the binding partners used in the immunoassay. Finally, the construction of the compound according to the invention guarantees the equimolarity of the two epitopes. to some

 Thus, the present invention firstly relates to a biepitopic compound of formula (I):

E1 - X - Z - Y - E2

 Support molecule (I) in which:

 E1 and E2, which are identical or different, each independently represent a peptide sequence comprising at least one epitope of an analyte,

 X and Y, identical or different, each independently represent a linker,

 the carrier molecule is soluble and

 Z represents an amino acid derivative carrying a thiol function prior to its binding with the carrier molecule.

 Another subject of the invention relates to a composition containing a compound of formula I in solution in water, in a buffer or in a biological fluid.

Yet another object relates to the use of such a biepitopic compound or such a composition containing this compound as a control or standard or adjuster in an immunoassay. Yet another subject of the invention concerns immunoassay methods using as a control and / or standard or adjuster a biepitopic compound of formula I or a composition containing this compound.

 Finally, the last object of the present invention is a kit for the implementation of an immunoassay comprising a biepitopic compound of formula (I) or a composition containing such a compound.

 The Applicant has therefore developed against all expectations a synthetic biepitopic compound that overcomes all the drawbacks of the state of the art mentioned above. The compound of the invention has the following formula (I):

E1 - X - Z - Y - E2

 Support molecule (I) in which:

 E1 and E2, identical or different, each independently represent a peptide sequence comprising at least one epitope of an analyte;

X and Y, identical or different, each independently represent a linker,

 the carrier molecule is soluble and

 Z represents an amino acid derivative carrying a thiol function prior to its binding with the carrier molecule.

The compound of the invention is therefore a biepitopic compound. By biepitopic compound is meant a compound which comprises two epitopes of the same analyte in order to mimic the antigenic recognition in sandwich immunoassay of said analyte. Of course, in any case, the synthetic biepitopic compound does not consist of the same sequence as the analyte that it mimics. This synthetic compound therefore does not correspond to a sequence of amino acids existing in nature, for example a protein or a protein fragment. The compound of the invention will be called, in an obvious and equivalent manner throughout the application, a biepitopic compound, a non-natural biepitopic compound, a synthetic biepitopic compound or a non-natural synthetic biepitopic compound. The prefix "immuno" in the term "immunoassay", for example, is not to be considered in the present application as strictly indicating that the binding partner is necessarily a partner of immunological origin, such as an antibody or a fragment antibody. Indeed, as is well known to those skilled in the art, this term is more widely used to designate also tests and methods in which the binding partner is not a partner of origin / immunological nature but consists, for example, an analyte receptor that one wishes to detect and / or quantify. Regardless of its origin or nature, the binding partner concerned should be able to bind to the desired analyte, preferably in a specific manner. Thus, it is known to speak of the ELISA assay for assays that use non-immunological binding partners in the strict sense of the term, more broadly called ligand binding assay, which could be translated into French as "assay using binding. to a ligand ", while the term" immuno "is included in the verbatim title corresponding to the acronym ELISA. For the sake of clarity and uniformity, the term "immuno" is used in this application to refer to any biological assay using at least one binding partner adapted to bind to the analyte of interest and detect and / or quantify the latter. preferably, specifically, even when said binding partner is not of a strictly immunological nature or origin.

Sandwich type immunoassays (or more simply "sandwich immunoassay") use a first binding partner, called "capture binding partner", to specifically bind the desired analyte, and a second binding partner, said "Detection binding partner", labeled and also intended to bind specifically with the desired analyte, thereby revealing the binding between the capture binding partner and the analyte, and thus the presence of the analyte. In other words, the desired analyte is "sandwiched" between said first and second binding partners, the first binding partner ("capture") generally being present in excess of the desired analyte. The capture binding partner may, for example, be immobilized on a solid support (by covalent bonding, adsorption or any other suitable method). An epitope, also called an antigenic determinant, is the smallest part of an antigen that can be recognized by a paratope that is the variable part of an antibody. The structure of the epitope is complementary to the paratope of the antibody. The structure involved may be the primary structure, in the case of a linear epitope, also called sequential epitope, or the tertiary structure in the case of a conformational epitope, also called discontinuous epitope.

 The sequence of a linear epitope may include so-called conservative modifications that do not significantly change the binding between the epitope and the antibody from a specificity point of view.

 A mimotope is a macromolecule, often a peptide, that mimics the three-dimensional structure of a given epitope. An antibody that recognizes said epitope is also able to recognize and bind to the mimotope that mimics that epitope. In the case of protein antigens, peptide mimotopes, therefore linear, are often used to mimic conformational epitopes. According to the invention, the conformational epitopes can be replaced by mimotopes. Mimotopes are also included in the scope of this application by replacing the term epitope.

 When the antigen is of a protein nature, as is often the case, the linear epitopes and the mimotopes correspond to a peptide sequence of variable length. In the case of linear epitopes, it is necessary to distinguish the concepts of minimal epitope and optimal epitope. The minimal epitope corresponds to the smaller peptide sequence recognized specifically by the corresponding antibody. When an amino acid of the N-terminus or of the C-terminus of said peptide sequence is deleted, the binding of the antibody is no longer possible, the recognition is lost. A minimal epitope may contain from 3 to 20 amino acids, more often from 4 to 8 amino acids.

The optimal epitope corresponds to the specifically recognized peptide sequence that has the best possible reactivity with the corresponding antibody (the most intense signal). It is very often peptide sequences of 6 to 30 amino acids in length. The optimal epitope generally comprises the minimal epitope. In rare cases, the optimal epitope and the minimal epitope can be confused. The compound of the invention is such that it contains two peptide sequences E1 and E2 comprising at least one epitope of an analyte. By peptide sequence comprising at least one epitope of an analyte, it is meant that the peptide sequence consists of at least amino acids of the minimal epitope. According to one embodiment of the invention, the peptide sequence consists of at most amino acids of optimal epitope. Of course, a person skilled in the art knows that it is also possible to add 1, 2, 3, 4 or 5 additional amino acids to one side of the optimal epitope or both sides without greatly affecting the antigenic recognition. Such additional amino acids are also included in the definition.

The amino acids of these peptide sequences may be the amino acids naturally found in the F analyte sequence or analogous amino acids. By analogous amino acids is meant two amino acids whose substitution is a conservative substitution in nature, that is to say taking place in a family of amino acids. Specifically, amino acids are generally divided into 4 families, namely (1) acidic amino acids such as aspartate and glutamate, (2) basic amino acids such as lysine, arginine and histidine, (3) non-polar amino acids such as alanine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan and (4) polar uncharged amino acids such as glycine, asparagine , glutamine, cysteine, serine, threonine and tyrosine. Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids. For example, it can be reasonably predicted that an isolated replacement of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or a similar conservative replacement of a amino acid by another amino acid having a structural ratio, will have no major effect on biological activity or antigenicity. Those skilled in the art will readily determine the amino acid that can tolerate change by reference to Hopp / Woods and Kyte-Doolite, well known in the art. According to one embodiment, the amino acids of the peptide sequence are the amino acids naturally found in the analyte. According to another embodiment, the peptide sequence E1 or the peptide sequence E2 or both are mimotopes and therefore the amino acids of the peptide sequence are mainly different from the amino acids naturally found in the analyte.

 The term analyte refers to a substance of biological origin, contained in a sample, detected, identified and / or quantified by analysis. It must be understood in a broad sense to mean a chemical, biological or biochemical substance that is the subject of one or more analyzes. As an example of analytes, there may be mentioned a protein or a peptide.

 The analyte will be representative of a pathological state or the presence of a microorganism in a medium. By pathological condition is meant any altered health status of a patient due to diseases caused by many factors such as environmental (infectious), genetic and / or biological factors. As examples of diseases, mention may be made of infectious diseases, caused by microorganisms such as viruses, bacteria, parasites (hepatitis, sepsis, etc.), autoimmune diseases, neurodegenerative diseases, cancers (breast, prostate, colon, etc.), cardiovascular diseases (myocardial infarction, etc.), etc. Analytes are then associated with various diseases. As an example of an analyte, cardiac troponin I can be mentioned as an analyte for myocardial infarction and proDefensin-A6 as a colon cancer analyte. These analytes will be described in more detail later. According to one embodiment, E1 and E2 are peptide sequences comprising at least one cardiac troponin I epitope or prodefensin-A6.

The central radical Z is an amino acid derivative carrying a thiol function before its binding with the support molecule. By amino acid derivative is meant any molecule forming two -CO-NH- peptide bonds with the X and Y linkers to which it is attached. To do this, the corresponding amino acid comprises, before binding with the X or Y linkers, a reactive group -NH ₂ and a reactive group -COOH. After binding to the X and Y linkers, the amino acid derivative thus has an -NH- group and a -CO- group. The corresponding amino acid also has a thiol function (-SH) prior to its binding to the carrier molecule. Examples of such Z radicals include cysteine, homocysteine and penicillamine derivatives. The corresponding amino acids used to form these derivatives are of course cysteine, homocysteine and penicillamine, which are amino acids known to those skilled in the art.

The two link arms X and Y of the compound of the invention are identical or different from each other. The linking arms are characterized by their ability to form each two -CO-NH- peptide bonds, on the one hand with E1 or E2, and on the other hand with the central Z radical. The linking arms X and Y are then amino acid derivatives having a group -NH- and a group -CO-. For this purpose, the compounds used to form the linking arms comprise, before binding with the peptide sequences E1 or E2, a reactive group -NH ₂ and a reactive group -COOH. Of course, the linking arms may comprise other groups, for example lateral groups, which are not reactive as such or are not reactive because they are protected by protective groups known to those skilled in the art, for example trityl, t-butyl, t-butyl-benzyl ether or benzyl ester groups. The linking arms may be obtained from one or more compounds each having a -COOH reactive group and a -NH ₂ reactive group before their engagement in the biepitopic compound of the invention. These compounds will be referred to as "monomers useful for forming the linker arms".

 The monomers useful for forming the linking arms may be protein-forming α-amino acids necessary for the synthesis of biological proteins, which are known to those skilled in the art. These proteinogenic α-amino acids can be genetically encoded; in this case, they are 22 in number. The 20 protein-amino acids universally distributed in all living beings are: L-Alanine, L-Arginine, L-Asparagine, L-Aspartate, L-Cysteine, L-Glutamate , L-Glutamine, L-Glycine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L -Thyrosine and L-Valine. The 2 other proteinogenic amino acids are much rarer: L-Pyrrolysine is only found in certain methanogenic archaea and L-Selenocysteine is present in only a few enzymes of the family of oxidoreductases.

In addition to these 22 genetically encoded amino acids, there are several dozen other biological amino acids that can be obtained from the previous ones by enzymatic modifications, such as, for example, L-Citrulline, L-pyroglutamic acid, L-Ornithine, L-3,4-dihydroxyphenylalanine, γ-amino butyric acid and domoic acid.

Monomers useful for forming the linking arms may also be pseudo amino acids, also called artificial amino acids, i.e. non-biological amino acids. In this case, the only condition is that the compound comprises two free functions, -COOH and -NH ₂ .

 According to a particular embodiment of the invention, the X and Y linking arms, which are identical or different, each comprise one or more amino acid derivatives (such as protein-forming α-amino acids and / or amino acids and / or or pseudo amino acids). Linkers may be prepared from one to six amino acids, preferably from one to four amino acids. For example, the linkers may have the GGGS sequence, or the SGGG sequence, or the GSGSGS sequence or the SGSGSG sequence.

 According to one embodiment of the invention, X and Y in formula (I), that is to say after formation of peptide bonds with E1 / E2 / Z, has one or more monomer derivatives of formula (II). ) next :

 -NH-R-CO- (II)

in which R is a radical consisting of one or more groups chosen independently from the groups -C (R ') HC (R ") H, (-C (R') HC (R") H-O-), (-C (R ') H-) and (-C (R') HO-), R 'and R "being independently selected from hydrogen, hydroxyl group and C 1 -C 5 alkyl groups. R is composed of one or more groups independently selected from (-CH ₂ -CH ₂ -O-), (-CH ₂ -O-) and (-CH ₂ -). According to a particular embodiment, the radical R can comprise from one to six groups (-CH ₂ -CH ₂ -O-), preferably from one to four groups (-CH ₂ -CH ₂ -O-). R, mention may be made of pentaoxaoctadecanoyl, tetraoxapentadecanoyl, trioxadodecanoyl, trioxatridecanoyl, dioxaoctanoyl, oxapentoyl and hexaoxaheneicosanoyl and their derivatives, as an example of a compound that is useful for obtaining a monomer derivative of formula (II) there may be mentioned 8-amino acid -3,6-dioxaoctanoic acid (CAS No. 134978-97-5), which is a known pseudo amino acid. By for example, the linking arms may be a dimer or trimer of 8-amino-3,6-dioxaoctanoic acid, (Ado) ₂ or (Ado) ₃ .

By way of another example of groups of the radical R, mention may be made of the biological amino acid derivatives mentioned above, in the form of a residue, that is to say without their group -COOH and -NH _2, but comprising an -NH- group and a -CO- group. For example, if the compound forming the linking arm is leucine, of formula (CH ₃ ) ₂ CH-CH ₂ -CH (NH ₂ ) -COOH, then R of the compound of formula (II) will be

ÇHÎCH _{S £} CH

 Each linking arm has, between the -CO or -NH groups of both ends, a size of between 10 and 60 Å, preferably between 20 and 30 Å, which constitutes a particular embodiment of the invention.

Since the monomers useful for forming the X and Y linking arms have -COOH and -NH ₂ reactive groups before their engagement in the biepitopic compound of the invention and allow the formation of peptide bonds, the sequence E1-XZY-E2 can be considered as a peptide, hereinafter referred to as a biepitopic peptide, so that it can be produced by peptide synthesis, which has the advantage

 an easy, fast, standardized, reproducible and simplified synthesis, the production of an equimolar compound with respect to E1 and E2.

The biepitopic peptide is obtained according to the procedures well known to those skilled in the art such as the solid phase peptide synthesis described by Merrifïeld, 1963. The improvements of this technique have been reviewed and discussed by Fields and Noble, 1990. Such a synthesis uses a solid phase on which is fixed the first C-terminal amino acid. In this context, each -NH ₂ group of the new amino acid, added to form the peptide, is protected by a protective group of Fmoc (9-fluoremethyloxycarbonyl) or Boc (t-butoxycarbonyl) type, to promote reaction between the group -NH ₂ presented by the solid phase and the group - COOH of the new amino acid added, as well known to those skilled in the art.

 According to the invention, the length of the biepitopic peptide does not exceed a length corresponding to 100 amino acids, preferably it does not exceed a corresponding length of 20 to 30 amino acids and more preferably it does not exceed a corresponding length of 25 to 27 amino acids.

 The biepitopic peptide is linked via the radical Z to a support molecule. The support molecule serves to stabilize the biepitopic compound and makes it possible to make the peptide sequences E1 and E2, comprising at least one epitope of the analyte of said biepitopic compound, more available, while maintaining the equimolarity between E1 and E2. .

 The term carrier molecule means any soluble molecule that can be coupled to a peptide. As a soluble molecule, mention may be made of proteins such as bovine serum albumin, immunoglobulin G and thyroglobulin, and polymers such as polylysines. According to one embodiment, the carrier molecule is a protein whose molecular weight is between 20kDa and 700kDa, preferably between 60kDa and 250kDa.

 Polylysines are polymers known to those skilled in the art. They are available for example at Sigma-Aldrich.

 Bovine serum albumin and thyroglobulin are known to those skilled in the art. For immunoglobulin G, it should be chosen so that it does not come from the species used to obtain the antibodies of the immunoassay, nor from the species from which the sample to be analyzed, to avoid interference problems. By way of example, mention may be made of immunoglobulins G from rabbits, mice, horses, goats and pigs (non-exhaustive list).

 According to one embodiment, the carrier molecule is bovine serum albumin.

According to another embodiment, the carrier molecule is a rabbit immunoglobulin G in an immunoassay whose antibodies originate from the mouse and whose sample to be analyzed comes from humans. The coupling between the biepitopic peptide and the carrier molecule of protein nature at the Z radical can be done covalently, according to methods well known to those skilled in the art. The sulfhydryl group (-SH) present in the side chain of the radical Z is reactive with respect to the maleimide groups, haloacetyl groups and pyridyl disulfides. Thus, in a first step, it is necessary to activate the support molecule, by reaction with a molar excess of a crosslinking agent (crosslinker in English) which will be able to react at the level of amino groups (-NH ₂ ) accessible of the carrier molecule and thus to introduce on the surface of the carrier molecule reactive groups chosen from maleimides or haloacetyls. These groups are preferred because they make it possible to obtain a coupling by a thioether bond which is stable. Among the crosslinking agents which make it possible to introduce maleimide groups, mention may be made, in a non-exhaustive manner, of N- (8-Maleimidocaproyloxy) succinimide ester, m-Maleimidobenzoyl-N-hydroxysuccinimide ester and succinimidyl 4- (N- maleimidomethyl) cyclohexane-1-carboxylate or the sulfo succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate. Among the crosslinking agents which make it possible to introduce haloacetyl groups, non-exhaustive mention may be made of N-succinimidyl (4-iodoacetyl) aminobenzoate and sulphosuccinimidyl (4-iodoacetyl) aminobenzoate. Then, the activated support molecule is purified by desalting, for example by gel filtration chromatography, or by dialysis, in order to remove the excess of the crosslinking agent and the by-products. Finally, the activated support molecule is placed in the presence of the biepitopic peptide comprising the radical Z in a relatively central position. The maleimide or haloacetyl groups react with the sulfhydryl group (-SH) of the Z radical of the biepitopic peptide to form a stable covalent thioether bond. The reaction between maleimide and sulfhydryl groups should be carried out under conditions close to neutral pH (pH 6.5 to 7.5) and the exclusion of foreign thiols, for example most reducing agents, from the composition of the reaction buffer to avoid competition for coupling sites. The reaction between haloacetyl and sulfhydryl groups should be carried out at pH 7.2 to 9. To limit the generation of free iodine which is likely to react with tyrosine, histidine and tryptophan, it is necessary to better to carry out the reaction in the dark. Such methods, known to those skilled in the art, are described for example in Shan S. Wong's Chemistry of Protein Conjugation and Cross-linking, CRC Press Inc., Boca Raton, Florida, USA, 1991.

 In order to promote the coupling of the biepitopic peptide on the Z radical, if the X / Y binding arms and / or the E1 / E2 peptide sequences contain amino acid derivatives bearing a thiol function, it is appropriate to use, for the formation of said peptide, amino acid derivatives protected at this thiol function by a stable protecting group during the synthesis steps of said peptide as well as during the step of coupling with the support molecule. Thus, only the amino acid used to give the radical Z will carry a reactive thiol function. To avoid such a step of protecting the thiol functions of the X / Y arms and / or the E1 / E2 peptide sequences, neither X, nor Y, nor E1 nor E2 should contain a functional amino acid derivative. thiol, which constitutes a particular embodiment of the invention.

 Amino acids protected in their thiol function are available for example from NOVABIOCHEM®.

 After coupling the biepitopic peptide with the support molecule, the thiol functions will or will not be deprotected according to techniques known to those skilled in the art. As protecting group, mention may be made of the t-butylthio group which is easily removed in aqueous medium (0.1 M ammonium bicarbonate) in the presence of DTT (dithiothreitol). When only the X / Y coupling arms contain amino acid derivatives carrying a thiol function, such deprotection will not be necessary and will be discouraged. When the E1 / E2 peptide sequences contain amino acid derivatives carrying a thiol function, such deprotection will be necessary if this affects the epitope recognition.

 It is also possible that the coupling arms and / or the peptide sequences do not contain any amino acid derivative carrying a thiol function. Thus, the biepitopic compound of the invention comprises one or more of the following characteristics:

linker X does not contain a thiol-functional amino acid derivative, linker Y does not contain an amino acid derivative bearing a thiol function,

 the peptide sequence E1 does not contain an amino acid derivative bearing a thiol function, and

 the peptide sequence E2 does not contain an amino acid derivative carrying a thiol function.

 According to a particular embodiment, the peptide sequences E1 and E2 do not comprise any amino acid derivative bearing a thiol function. According to yet another embodiment, none of X, Y, E1 and E2 comprises an amino acid derivative bearing a thiol function.

 The compound of the invention, for its implementation in an immunoassay, may be contained in a composition which comprises or contains said compound of formula (I) in solution in water, in a buffer or in a biological fluid, which constitutes another object of the invention.

 A composition containing the biepitopic compound of formula (I) in solution in water is a liquid and clear solution obtained by completely dissolving said compound and whose majority solvent is water, representing at least 50% by volume relative to the volume total of the solution. The amount of solvent is, of course, a function of the analyte in question and will be easily determined by those skilled in the art.

 The compound of formula (I) may also be in solution in a buffer. The buffers to be used are widely known to those skilled in the art and are a function of the analyte concerned. As an example of buffers, mention may be made of buffers such as PBS, HEPES and TRIS-HC1 buffers.

 When the composition contains a biological fluid, it may correspond to the sample that we want to test. By way of examples, mention may be made of whole blood or its derivatives, for example serum or plasma, urine, saliva and effusions.

The amount of compound of formula (I) in the composition of the invention depends on the analyte concerned and the corresponding measurement range. It will be easily determined by the skilled person. Thus, it may be of the order of pg / mL, ng / mL or μg / mL. The same characteristics and preferences described above, in particular as regards the choice of El, E2, X, Y, Z, carrier molecule and analyte also apply to the compositions of the invention.

 Of course, the compositions of the invention may comprise other compounds, such as salts, filler proteins such as BSA or synthetic polymers such as dextran or polyethylene glycol, detergents, which are well known to those skilled in the art.

 As indicated above, the compounds and compositions of the invention are particularly advantageous because they are easily synthesized, are stable at + 2/8 ° C and are soluble under the conditions of an immunoassay. Furthermore, the compounds of the invention have, against all odds, an immunoreactivity with the binding partners used in the particularly high immunoassay, ie the binding partners recognize them particularly well, so that, if wants to use them as a control, standard and / or adjuster, they can be contained in control solutions, standard and adjuster in small quantities, while being stable under immunoassay conditions.

 Thus, another object of the invention is the use of such a biepitopic compound or such a composition containing this compound as a control or standard or adjuster in an immunoassay.

 By use as a control of the compound of formula (I) or the composition containing this compound, its use is meant for, inter alia, to verify that the immunoassay functions according to expectations (also called a control or positive control) and that the detection of the analyte in the test sample is not falsely negative.

By use as a standard of the compound of formula (I) or the composition containing this compound is understood to mean its use for the establishment of a standard range. The establishment of the standard range, necessary step to perform quantification of an analyte, is a step widely known to those skilled in the art as described above. It consists in measuring the signal generated by increasing or known quantities or concentrations of the analyte, in plotting the curve giving the signal as a function of the quantity or the concentration and in finding a mathematical model. who represents this relationship as faithfully as possible. To do this, several aqueous compositions of the invention are used, each containing a different concentration of analyte. The mathematical model will be used to extrapolate the unknown quantities or concentrations of analyte contained in the test sample.

 By use as calibrator, also called calibrator, of the compound of formula (I) or the composition containing this compound, which is a particular standard, its use to adjust the measurement of the immunoassay of the analyte. In this case, the analyte concentration is fixed and known. The signal generated during use in the immunoassay by the adjuster is also known. The adjuster is used to verify that the measurement (signal) produced during the implementation of the immunoassay corresponds to the expected value. If this is not the case, the adjuster is used to measure the drift which may be corrected mathematically or by physical intervention on the measuring instrument (adjustment). For convenience, the term standard will include in this application the term adjuster.

 As indicated above, the analyte is any substance of biological, chemical or biochemical origin contained in a sample, detected, identified and / or quantified by analysis.

 According to a first embodiment, the analyte is cardiac troponin I and the compound of formula (I) or a composition containing it is used as a control, standard or adjuster in a cardiac troponin I immunoassay.

Troponin is known to be a myofibrillar protein complex, consisting of three proteins, the troponins I, T and C. This protein complex makes it possible to contribute to the regulation of the muscle contraction by the Ca ²⁺ ion, by interacting with myosin and actin.

 Cardiac troponin I (accession No. Uniprot PI 9429) is the subunit of troponin responsible for the inhibition of binding between myosin and actin.

 The epitopes and mimotopes of cardiac Troponin I are known to those skilled in the art. In a particular embodiment of the invention, the peptide sequences E1 and E2 are chosen from the following sequences:

Sequence 1: ATEPHAKK Sequence 2: AGLGFAELQDL

Sequence 3: KISASRKLQLKT

 The peptide sequences derived from said peptide sequences by substitution, deletion or insertion of an amino acid also belong to the field of the invention, insofar as they retain the capacity to be recognized by the antibody concerned.

 According to another embodiment, the compound of formula (I) or a composition containing it are used as a control, standard or adjuster in an immunoassay of Prodéfensine-A6.

 Defensins are a family of antimicrobial peptides involved in host defense against microbial attack. In the mature form, they consist of 30 to 40 amino acids and have the property of selectively disaggregating the membranes. Like other eukaryotic proteins, Defensins may be present not only as a mature protein but also as a precursor. We then speak of Prodéfensine. Prodéfensine-A6 (Accession No Uniprot Q01524), has been described as being useful as a marker in the context of cancer and in particular colorectal cancer, in particular in the patent application WO2010 / 112777 of the Applicant.

 Epitopes and mimotopes of the proDefensin-A6 protein are known and are described for example in the patent application WO2010 / 112777. In a particular embodiment of the invention, the peptide sequences E1 and E2 are chosen independently in the following groups of sequences, knowing that if E1 is selected from a group, E2 is chosen from another group:

Group 1:

 - Sequence 4: NYVTPPWAIFRH

 - Sequence 5: WTGVLSPTQEYR

 - Sequence 6: SHLTPPWMDYRV

 - Sequence 7: VMAVTCSTCDSR

 - Sequence 8: LTPPTEDLRPPD

Group 2:

- Sequence 9: YGNHSCTHIGHC Sequence 10: GPSYTCLHFGHC

 Sequence 11: TEREVHNWFPFH

 Group 3

 Sequence 12 YPHPWSMHVIRA

 Sequence 13 TTTPHPWALFAV

 Sequence 14 TPHPWQRWVVYS

 Sequence 15 EDVLRWHPEWPG

 Group 4

 Sequence 16: YHETWPPKSAQL

 Sequence 17: YHDNWPQPSRSW

 Sequence 18: QHNHQRHGAMGA

 Sequence 19: YHDMWPMSGRMA

 Sequence 20: YHDNWPPLNGAR

 Sequence 21: YHDMWPAIQLSP

 Sequence 22: YHEKFPGPVVLP

 Group 5

 - Sequence 23: QAEDDPLQAK

 These controls, standards and / or adjusters are particularly suitable for their implementation in the methods of detecting and / or quantifying an analyte by immunoassay in a test sample capable of containing said analyte.

 Also, another subject of the invention relates to a method for detecting an analyte by immunoassay in a test sample capable of containing said analyte comprising

 i. an immunoassay test by contacting said test sample with one or more analyte binding partners,

 ii. a test for checking the validity of the immunoassay test by contacting a biepitopic compound of formula I as defined previously or of a composition as defined above, as a positive control, with said one or more partners analyte binding,

iii. reading the immunoassay test if the validity check test is positive, iv. determining the presence of said analyte in the test sample when the signal obtained by the immunoassay test of step i is greater than the detection threshold of the immunoassay test.

 The test sample in the context of the invention may be of various origins, for example of food, environmental, biological, veterinary, clinical, pharmaceutical or cosmetic origin.

 Examples of food-based samples include, but are not limited to, a sample of milk products (yogurt, cheese, etc.), meat, fish, egg, fruit, vegetable, water, drink (milk, fruit juice, soda, etc.). Of course, these food-based samples may also come from sauces or more elaborate dishes or unprocessed or partially processed raw materials. A food sample may also be derived from a feed intended for animals, such as cakes, animal meal. All these samples, if they are not liquid, are previously treated to be in liquid form.

 As indicated above, the sample may be of environmental origin and may consist of, for example, surface sampling, water, etc.

 The sample may also consist of a biological sample, of human or animal origin, which may correspond to samples of biological fluid (urine, whole blood or derivatives such as serum or plasma, saliva, pus, cerebrospinal fluid, etc.). ), stool (eg, cholera diarrhea), nose, throat, skin, wound, organ, tissue or isolated cells, swab specimens. This list is obviously not exhaustive.

In general, the term "sample" refers to a part or quantity, more specifically a small part or a small quantity, taken from one or more entities for analysis. This sample may possibly have undergone prior treatment, involving for example mixing, dilution or grinding steps, in particular if the starting entity is in the solid state. The sample analyzed is, in general, capable of - or suspected of - containing at least one analyte representative of the presence of microorganisms or a disease to be detected, characterized or monitored.

 The steps of this method of detecting an analyte by immunoassay are steps widely known to those skilled in the art which have been described previously. In particular, the first step consists of contacting the test sample with one or more analyte binding partners, preferably two binding partners for a sandwich test. As previously described, one of the two partners may be coupled to a label to form a conjugate or a tracer. The other binding partner can be captured on a solid support as known to those skilled in the art. This is called capture partner for the latter and detection partner for the first.

 The measured signal emitted by the conjugate is then proportional to the amount of analyte of the biological sample.

 The binding partners to the analyte of interest are any molecule capable of binding to the analyte. By way of example of analyte binding partners, mention may be made of binding partners of immunological nature or origin, such as antibodies (monoclonal or polyclonal) and antibody fragments, which are well known to the patient. Those skilled in the art, as well as binding partners that are not of nature or immunological origin, such as nanofitins, analyte receptors if they exist, aptamers, DARPins or any other molecule that is known to have an interaction with said analyte.

 Nanofitins (trade name) are small proteins that, like antibodies, are able to bind to a biological target that can detect, capture or simply target within an organism.

Aptamers are oligonucleotides, usually RNA or DNA, identified in libraries containing up to 10 ¹⁵ different sequences, by a combinatorial method for selecting in vitro called SELEX "Systematic Evolution of Ligands by Exponential Enrichment" (Ellington AD and Szostak JW ., 1990). Most aptamers are composed of RNA, because of the ability of 1 RNA to adopt various and complex structures, which makes it possible to create cavities on its surface. various geometries, making it possible to fix various ligands. These are biochemical tools of interest that can be used in biotechnological, diagnostic or therapeutic applications. Their selectivity and ligand binding properties are comparable to that of antibodies.

 "DARPins" for Designed Ankyrin Repeat ProteINS (Boersma YL and Plutckthun A, 2011) are another class of proteins that mimic antibodies and can bind with high affinity and selectivity to target proteins. They derive from the family of ankyrin proteins that are adapter proteins that make it possible to bind integral membrane proteins to the spectrin / actin network that constitutes the "spine" of the cellular plasma membrane. The structure of the ankyrins is based on the repetition of a pattern of about 33 amino acids and so are the DARPins. Each pattern has a secondary structure of helix-turn-helix type. The DARPins contain at least three, preferably four to five repeating units and are obtained by "screening" combinatorial libraries.

 Marker means, in particular, any molecule containing a group reactive with a group of the binding partner, directly without chemical modification, or after chemical modification to include such a group, which molecule is capable of directly or indirectly generating a detectable signal. A non-limiting list of these direct detection markers consists of:

 enzymes which produce a detectable signal for example by colorimetry, fluorescence, luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, chromophores such as fluorescent compounds, luminescent compounds, dyes,

 32 35 125

• radioactive molecules such as ^JZ P, ^JJ S or

fluorescent molecules such as Alexa or phycocyanins, and electrochemiluminescent salts such as organometallic derivatives based on acridinium or ruthenium. Indirect detection systems can also be used, such as, for example, ligands capable of reacting with an anti-ligand. The ligand then corresponds to the marker to form, with the binding partner, the conjugate.

 The ligand / anti-ligand pairs are well known to the person skilled in the art, which is the case, for example, of the following pairs: biotin / streptavidin, hapten / antibody, antigen / antibody, peptide / antibody, sugar / lectin, polynucleotide / complementary polynucleotide.

 The anti-ligand can then be detectable directly by the direct detection markers described above or be itself detectable by another ligand / antiligand pair, and so on.

 These indirect detection systems can lead, under certain conditions, to amplification of the signal. This signal amplification technique is well known to those skilled in the art, and reference may be made to prior patent applications FR 2781802 or WO 95/08000 of the Applicant.

 Depending on the type of labeling used, those skilled in the art will add reagents allowing the visualization of the marking or the emission of a detectable signal by any type of suitable measuring device, such as for example a spectrophotometer, a spectrofluorimeter, a densitometer or a high definition camera.

 The test step of checking the validity of the immunoassay is as described above.

 The immunoassay test i) and the control test ii) can be implemented in any order, simultaneously or successively, on the same solid support or not.

 The reading of the immunoassay test is also a step widely known to those skilled in the art that depends on the test used.

 Finally, the last step consists in determining the presence of said analyte in the test sample when the signal obtained by the immunoassay test of step i is greater than the detection threshold of the immunoassay test. This step is also widely known to those skilled in the art.

In addition to detection, the compounds of the invention are also suitable for quantification of analyte in a test sample. So another object of the invention relates to a method for quantifying an analyte by immunoassay in a test sample capable of containing said analyte, comprising

 i. an immunoassay test by contacting said test sample with one or more analyte binding partners,

 ii. a test for checking the validity of the immunoassay test by contacting a positive control with said one or more analyte binding partners,

 iii. reading the immunoassay test if the validity check test is positive, and

 iv. determining the amount of said analyte in the test sample by comparison of the immunoassay test signal with a standard curve previously obtained using a biepitopic compound of formula (I) as defined previously or a composition as defined previously.

 The steps of the quantization method are as defined above. In particular, the immunoassay test i) and the control test ii) can be implemented in any order, simultaneously or successively, on the same solid support or not.

 In this quantification method, the positive control may be any compound useful as a control, which has an antigenic reactivity comparable to the analyte in the immunoassay used. According to one embodiment, the positive control is a biepitopic compound or a composition as described above.

 The standard curve is prepared with the compound or composition of the invention. Nevertheless, any other appropriate standard solution can be used. Thus, another subject of the invention relates to a method of quantifying an analyte by immunoassay in a test sample capable of containing said analyte comprising

 i. an immunoassay test by contacting said test sample with one or more analyte binding partners,

ii. a test for checking the validity of the immunoassay test by bringing into contact a biepitopic compound of formula I as defined above or a composition as defined above, as a positive control, with said one or more binding partners to the analyte,

 iii. reading the immunoassay test if the validity check test is positive, and

 iv. determining the amount of said analyte in the test sample by comparing the signal of the immunoassay test with a standard curve.

 The same characteristics and preferences described above, especially as regards the choice of particular compounds and analytes, at the various stages of the immunoassay also apply to the detection and quantification methods of the invention.

 In particular, in all these detection and quantification methods, the analyte may be cardiac troponin I or prodefensin-A6.

 The immunoassay methods of the invention involve the implementation of diagnostic kits comprising the compounds or compositions of the invention, which constitutes another object of the invention.

 In addition to the compounds or compositions of the invention as described above, the kits according to the invention may also contain the compounds necessary for the implementation of a method for the detection or quantification by immunoassay of the presence of a analyte of interest, for example by "sandwich" immunoassay, such as binding partners and all compounds necessary for demonstrating the reaction between the binding partner (s) and the analyte of interest.

 The invention will be better understood with the aid of the following examples, which are given by way of illustration and without limitation, and with the aid of the figures, in which:

 FIG. 1 is a graph giving the RFV fluorescence signal, determined by the VIDAS® automaton, emitted by a biepitopic compound according to the prior art (Compound REF), a biepitopic compound according to the invention (Compound 1) and a biepitopic peptide corresponding to the biepitopic compound 1 of the invention, but not coupled to a carrier molecule (uncoupled peptide 1), depending on their concentration;

FIG. 2 is a graph giving the RFV fluorescence signal, determined by the VIDAS® automaton, emitted by the biepitopic compound according to the prior art. (Compound REF) and biepitopic compounds according to the invention (Compounds 1 and 3), depending on their concentration.

 FIG. 3 is a graph giving the RFV fluorescence signal, determined by the VIDAS® automaton, emitted by the biepitopic compound according to the prior art (REF Compound) and biepitopic compounds according to the invention (Compounds 1 and 4) , depending on their concentration.

EXAMPLES

 Example 1 Synthesis of Peptides

 Peptide syntheses were performed using either the ABI 433A synthesizer from Applied Biosystems (Foster City, CA, USA) or the Liberty synthesizer from CEM Corporation (Matthews, NC, USA). Rink Amide MBHA resin (Cat No. 855003, Novabiochem®, Merck Millipore, Molsheim, France) was used as a polymeric solid support.

 At the end of chemical synthesis, the peptides were deprotected and cleaved from the polymer in the presence of a mixture of trifluoroacetic acid-ethanedithiol-triisopropylsilane-water (94 / 2.5 / 1 / 2.5 V / V / V / V ) for about 2 hours. After removal of the polymer by filtration, the peptides were isolated by precipitation in diethyl ether at 0 ° C.

 In order to increase their degree of purity, the peptides were purified by reverse phase preparative high performance liquid chromatography (HPLC) on a VYNAC DENALI ™ 120 C18 column, 10 μιη (Mandel Scientific Company Inc., Guelph, Ontario, Canada). . Each peptide was eluted with a step gradient of acetonitrile (from 0 to 95%) in aqueous solution containing 0.1% of trifluoroacetic acid, the percentage of acetonitrile of the bearings having been chosen so as to optimize the isolation. of the peak which corresponds to the peptide of interest. After this final step, two different analysis techniques were implemented to control and characterize the peptides obtained.

For each peptide, an analytical HPLC profile was generated on a Chromo lith® High Resolution RP-18 encapped reverse phase column (Merck Millipore, Molsheim, France). Elution was performed by a linear gradient of acetonitrile (from 0 to 100%) in aqueous solution containing 0.1% trifluoroacetic acid and monitored by measuring the absorbance at 214 nm. This analysis makes it possible to determine the level of purity of the peptide.

 Each peptide was also analyzed by liquid chromatography-mass spectrometry (LC / MS) on a ZORBAX Eclipse Plus Cl 8 R HD 2 column, 1 × 50 mm, particle size 1, 8μιη (Agilent Technologies, Santa Clara, CA. USA) coupled to a Q-TOF LC / MS 6540UHD mass spectrometer Accurate-Mass (Agilent Technologies). This analysis makes it possible to determine the molar mass of the peptide.

 Sequences of synthesized peptides as well as characterization results are shown in Table 1.

Table 1. Sequences and characteristics of synthesized peptides.

The abbreviation Tn1 corresponds to cardiac Troponin I and PDEF-A6 to proefense-A6. The abbreviation Ado corresponds to 8-amino-3,6-dioxaoctanoic acid (CAS No. 134978-97-5). Table 2. Summary of the biepitopic compounds according to the invention obtained (formula I) and tested in immunoreactivity.

. _| LFL _* is liras Molecule

1 half rhinl Anah tc Kpitopc K l Kpilop * 1: 1

 Y support

Compound 1 Tnl ATEPHAKKK AGLGF AELQDL (Teen) ₂ (Teen) ₂ BSA

Compound 2 Tnl KISASRKLQLKT AGLGF AELQDL (Teen) ₂ (Teen) ₂ BSA

Compound 3 Tnl ATEPHAKKK AGLGF AELQDL (Ado) ₂ (Ado) ₂ IgG

Compound 4 Tnl ATEPHAKKK AGLGF AELQDL GGGS SGGG BSA

 PDFF-

Compound 5 _A6 QAEDDPLQAK WTGVL .SPTQEYR (Teen) ₂ (Teen) ₂ BSA

The abbreviation Tn1 corresponds to cardiac Troponin I and PDEFA6 to proDefensin-A6. The abbreviation Ado corresponds to 8-amino-3,6-dioxaoctanoic acid (CAS No. 134978-97-5). The abbreviation BSA corresponds to Serum Bovine Albumin. IgG is rabbit immunoglobulin G.

Example 2 Preparation of the Biepitopic Compounds

 The biepitopic compounds were obtained by carrying out covalent couplings between the peptides obtained in Example 1 on the one hand and support molecules on the other. Table 2 presents in detail the different biepitopic compounds according to the invention prepared. All these compounds satisfy formula I. Table 3 summarizes all the couplings carried out, specifying the peptide and support molecule pairs.

 The mode of operation of the couplings is as follows:

Initially, the protein selected as a carrier molecule was activated in the presence of an excess of Sulfo-SMCC (Sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, CAS No. 92921-24- 9, Cat No. 22322, Pierce, Thermo Scientifïc, Villebon sur Yvette, France). For Bovine Serum Albumin (BSA, Proliant Health & Biologicals, Ankeny, IA, USA), we chose a molar ratio of BSA / SMCC of 1/20. Thus, the BSA was diluted to 10 mg / mL in PBS pH 7.2 (phosphate buffered saline) and 53 of a 25 mg / mL solution of sulfo-SMCC in water, prepared extemporaneously, was added dropwise. . After an incubation of 1 hour ± 5 minutes at 30 ° C. ± 2 ° C. in a water bath, with gentle magnetic stirring, the BSA-SMCC was dialyzed against a 50 mM phosphate buffer, 150 mM NaCl pH 6.8 in a dialysis tubing with a cutoff of 12 to 14 kDa. Dialysis was performed at room temperature and the dialysis bath was changed every hour, 3 times. After dialysis, the protein concentration of the BSA-SMCC solution was determined by measuring the absorbance at 280 nm and this concentration was adjusted to 5 mg / mL in a 50 mM phosphate buffer, 150 mM NaCl pH 6 8. This step allows the modification of the surface of the carrier molecule which now carries several reactive groups of maleimide type.

 The peptide to be coupled was dissolved at 5 mg / ml in 50 mM phosphate buffer, 150 mM NaCl, 5 mM EDTA pH 6.8, taking into account the purity. We chose a molar ratio of BSA / peptide of 1/10. Thus, 2.35 mg of BSA-SMCC at a concentration of 5 mg / mL (0.47 mL) was added to 1 mg of peptide at a concentration of 5 mg / mL (200 μM). This mixture was incubated for 16 hours minimum at 2/8 ° C on a wheel. The reaction was then quenched by addition of 0.1 M 2-mercaptoethylamine (CAS No. 60-23-1, cysteamine) in 50 mM phosphate buffer, 150 mM NaCl, pH 6.8, prepared extemporaneously. After incubation for 20 ± 5 minutes on a wheel at laboratory temperature, the peptide-BSA conjugate was dialyzed against PBS pH 7.2 in a dialysis tubing with a cut-off of 12 to 14 kDa. Dialysis was continued for 16 hours minimum at 2/8 ° C. After dialysis, the protein concentration was adjusted to the theoretical 2 mg / mL of BSA in pH 7.2 PBS buffer. The concentration of peptide-BSA conjugate was then determined by measuring the absorbance at 280 nm. This step allows the reaction between the maleimide groups and the sulfhydryl (-SH) groups of the peptide, at the level of the terminal or median cysteine according to the peptide sequence to be coupled, in order to form thioether bonds.

Compounds 1, 2, 4 and 5 were all obtained by applying the procedure described above. For the compound REF, which corresponds to the biepitopic compound as described in US Pat. No. 6,114,180, the same procedure was also applied except that 2 peptides (peptide 2 and peptide 3) were brought into the presence of BSA-SMCC simultaneously. and each peptide was coupled to a theoretical BSA / peptide molar ratio of 1/10. For compound 3, peptide 1 was coupled to rabbit polyclonal immunoglobulin G (bioMérieux). The procedure was identical except that the BSA was replaced by another support molecule. The theoretical molar ratio of support molecule / peptide was 1/10.

Table 3. Summary of peptide-molecule support couplings.

Example 3 Study of the Immunoreactivity of the Biepitopic Compound 1 According to the Invention

 The study of the biepitopic character of the compounds was carried out by an immunoassay of cardiac Troponin I using the VIDAS® immunoassay automaton (bioMérieux). The disposable cone serves both as a solid phase for the reaction and as a pipetting system. The cartridge is composed of 10 wells (X0 to X9) sealed with a sealed and labeled aluminum foil.The first well (X0) has a pre-cut part to facilitate the introduction of the sample.The last well (X9) is an optical cuvette in which the fluorescence of the substrate is measured The different reagents necessary for the analysis are contained in the intermediate wells All the steps of the test are performed automatically by the instrument They consist of a succession of cycles of The immunoassay of the cardiac troponin I was carried out by a one-step sandwich test.

 a) Sensitization and passivation of cones

The characteristics and the suppliers of the antibodies used are presented in Table 4. The cones were sensitized with 300 of a solution of the monoclonal antibodies 19C7 and B90 each diluted to 2.5 μg / ml in PBS buffer pH 6.2. After approximately 20 hours of incubation at +18 / 25 ° C. with the sensitization solution, the cones were emptied. Then, 300 of this same solution containing 10 g / l of bovine albumin are added. Passivation continues at + 18/25 ° C overnight. The cones are emptied, dried and stored at + 4 ° C until use, protected from moisture.

Table 4. Antibodies used for immunoassay of cardiac Troponin I.

 the abbreviation TnC corresponds to cardiac troponin C. The abbreviation Cat. No. corresponds to the supplier's catalog reference. b) Procedure of the immunoassay

 The compounds to be tested were diluted in PBS-BSA buffer at different concentrations and assayed as a sample.

 As soon as the VIDAS® cone is in contact with the sample, the immunological reaction begins because the capture antibodies are immobilized on this cone. The automaton mixes the sample to be tested (135 μί) with 270 of the conjugate solution. This solution contains the 2 monoclonal antibodies, 3D5F7 and 7B9, in the form of Fab 'fragments coupled to alkaline phosphatase. These conjugates were diluted to approximately 0.75 μg / ml in 100 mM phosphate buffer pH 6.4, also containing 150 mM NaCl and filler proteins.

The incubation lasts 6.8 minutes at 37 ° C. and allows specific binding of cardiac Troponin I, or cardiac TnI biepitopic compounds, or cardiac TnI peptides, to antibodies adsorbed on the cone on the one hand, and conjugates of the other. Then unbound components are removed by 3 washes with a buffer Tris 200 mM H 7.8, 300 mM NaCl, Triton X-100 0.2%. During the final revelation step, the 4-methylumbelliferyl phosphate substrate is sucked and then forced back into the cone; the conjugate enzyme catalyzes the hydrolysis reaction of this substrate to 4-methylombelliferone whose emitted fluorescence is measured at 450 nm. The value of the fluorescence signal (RFV = relative fluorescence value) is proportional to the concentration of the antigen present in the sample.

Table 5 summarizes the fluorescence signals (RFV = relative fluorescence value) determined by the VIDAS® automaton when comparing the immunoreactivity of the biepitopic compound REF (the prior art), the biepitopic compound 1 according to the invention and uncoupled peptide 1 (Example 1). Figure 1 shows these same data graphically. As a reminder, the peptide 1 comprises 2 epitopes of the cardiac TnI, one recognized by the B90 capture antibody of the immunoassay described above, and the other by the 3D5F7 detection antibody. In the compound 1 according to the invention, the peptide 1 is coupled via the median cysteine to the BSA in order to ensure better antigen presentation and improved stability. The compound REF has the same 2 epitopes of Tnl coupled also on the BSA. Unlike compound 1, each of the 2 epitopes is in the form of an individual peptide (peptides 2 and 3) which has been coupled to BSA at the terminal cysteine. Compound 1, compound REF, which corresponds to a biepitopic compound as described in US Pat. No. 6,114,180, and peptide 1, which corresponds to a biepitopic synthetic compound as described in patent application WO98 / 24816, are all reagents in the immunoassay of cardiac TnI but their levels of reactivity are very different. Thus, to obtain a signal of about 1000 RFV, it takes 21 μΜ of compound 1 against 1118 μΜ of the REF compound, about 50 times less. Compound 1 therefore has a much better immunoreactivity than the compound REF. Moreover, the good dynamics of the VIDAS® signal obtained with the compound 1 is not reproduced with the compound REF, even by testing much higher concentrations of this compound. The uncoupled peptide 1 is much less well recognized than the two biepitopic compounds coupled to BSA. Table 5. Immunoreactivity of the Bitepitopic Compounds 1, 3, 4, REF and Cardiac Peptide 1 Tn1.

 The abbreviation [c] corresponds to the μ en concentration of the compound. The abbreviation S corresponds to the signal in RFV.

Example 4 Comparison of the Immunoreactivity of the Biepitopic Compounds According to the Invention Using Different Supporting Molecules

In this example, peptide 1 which comprises 2 different epitopes of cardiac TnI was coupled on 2 different support molecules: BSA (compound 1) and rabbit immunoglobulin G (compound 3). Obtaining these biepitopic compounds is described in Example 2. Comparison of the immunoreactivity of these compounds in the immunoassay of cardiac TnI was performed as described in Example 3 and the results are shown in Table 5 above and FIG. 2, which represents a graph giving the RFV fluorescence signals emitted by the various compounds, a biepitopic compound according to the prior art (compound REF) and biepitopic compounds according to the invention (Compounds 1 and 3), depending on their concentration. The results show that compounds 1 and 3 are both reactive in the immunoassay of cardiac TnI and have a comparable reactivity, much greater than that observed for the compound REF.

Example 5 Comparison of the Immunoreactivity of the Biepitopic Compounds According to the Invention Using Different Spacer Arms

 In this example, the compared biepitopic compounds differ only at the spacer arm. In the case of compound 1, the two spacing arms are identical, it is a dimer of the artificial amino acid Ado. Compound 4, for its part, contains the GGGS sequence as X-arm and the SGGG sequence as Y-arm. As a reminder, the two compounds have 2 cardiac TnI epitopes and the support molecule is BSA. Obtaining these biepitopic compounds is described in Example 2. Comparison of the immunoreactivity of these compounds in the immunoassay of cardiac TnI was performed as described in Example 3 and the results are shown in Table 5 above and FIG. 3, which represents a graph giving the RFV fluorescence signals emitted by the various compounds, a biepitopic compound according to the prior art (compound REF) and biepitopic compounds according to the invention (Compounds 1 and 4), according to their concentration. The results show that compound 4 comprising GGGS and SGGG arms is less well recognized than compound 1 comprising arms (Ado) but is much greater than compound REF.

Example 6: Stability Tests

Compounds 1 and 2 were diluted to 3.75 ng / ml in the various buffers indicated in Table 6. A first assay was performed on day 0, the day of preparation of the solutions. The values obtained served as a reference for the monitoring of the stabilities. Dilute solutions of biépitopiques compounds were stored at + 2/8 ° C and assayed on day 7 (7 ^th day after preparation. Table 6 below shows the variation of RFV signal immunoassay between D0 and D7 (Signal J7 / Signal J0 x 100). Compounds 1 and 2 are stable and their antigenic properties are preserved when stored at + 2/8 ° C for 1 week.

Table 6. Stability of compounds 1 and 2 at + 2/8 ° C for 7 days.

In a second step, a longer duration stability study was carried out for compound 1 only, diluted in PBS pH 6.2, BSA 50 g / L. Preserved at + 2/8 ° C, compound 1 is stable in diluted solution: 97% of the signal of the immunoassay is found at 1 month of preservation, 94% at 3 months and 90%> at 6 months. Preserved at + 18/25 ° C, the compound 1 is stable in dilute solution for about 1 month (88% of the signal is found). It is also important to note that Compound 1 is able to withstand at least 3 freeze / thaw cycles at -20 ° C without any degradation of its antigenic properties. We did not test a larger number of freeze / thaw cycles.

 All of these results demonstrate the excellent stability of the solutions of compound 1 according to the invention.

Example 7 Study of the Immunoreactivity of the Biepitopic Compound According to the Invention Comprising a Mimotope

The biepitopic compound 5 has been designed to function as a control and / or standard and / or adjuster during an immunoassay of Prodéfensine A6. Compound 5 combines a linear epitope (QAEDDPLQAKL) and a mimotope (WTGVLSPTQEYR). The immunoassay for Prodéfensine A6 was carried out using the VIDAS® immunoassay automaton (bioMérieux), following the protocol described in the application WO2010 / 112777, namely using, as capture antibody, the clone 12H4E1 ( bioMérieux), which recognizes the linear minimal epitope of sequence EDDPLQ, and, as detection antibody, clone 1H8C9 (bioMérieux), whose epitope is not linear but is a mimotope of sequence WTGVLSPTQEYR. These epitopes / mimotopes are those found in compound 5.

 Table 7 below summarizes the fluorescence signals (RFV = relative fluorescence value) determined by the VIDAS® automaton when testing different concentrations of the compound 5 (molecular molar mass: 98155 Daltons). Compound 5 is well reactive in the immunoassay of Prodéfensine A6.

Table 7. Immunoreactivity of the biepitopic compound 5.

Bibliographical References

- Boersma YL and Plûtckthun A, 2011, Curr. Opin. Biotechnol, 22: 849-857

 Ellington AD and Szostak JW., 1990, Nature, 346: 818-822

 - Fields and Noble, 1990, Int J Pept Protein Res., 35: 161-214

 - Merrifield 1963, J Am Chem Soc. 85: 2149-2154

 - Shan S. Wong, 1991, CRC Press Inc., Boca Raton, Florida, USA, Chemistry of Protein Conjugation and Cross-Linking

Claims

1. Biepitopic compound of formula (I):

E1 - X - Z - Y - E2

 Support molecule (I) in which:

 E1 and E2, identical or different, each independently represent a peptide sequence comprising at least one epitope of an analyte; and

X and Y, identical or different, each independently represent a linker,

 the carrier molecule is so faded and

 Z represents an amino acid derivative carrying a thiol function prior to its binding with the carrier molecule.

2. A biepitopic compound of formula (I) according to claim 1, wherein the X and Y linking arms are amino acid derivatives each forming two -CO-NH- peptide bonds, one with E1 or E2 and one other with Z.

3. The biepitopic compound of formula (I) according to claim 2, wherein the X and Y linking arms, which are identical or different, each independently comprise one or more amino acid derivatives.

4. A biepitopic compound of formula (I) according to any one of the preceding claims, wherein Z is selected from a cysteine derivative, a homocysteine derivative and a penicillamine derivative.

5. A biepitopic compound of formula (I) according to any one of the preceding claims, wherein the carrier molecule is a protein whose molecular weight is between 20 kDa and 700 kDa, preferably between 60 kDa and 250 kDa.

6. The biepitopic compound of formula (I) according to claim 5, wherein the carrier molecule is bovine serum albumin.

7. A biepitopic compound of formula (I) according to any one of the preceding claims, wherein E1 and E2 are peptide sequences comprising at least one cardiac troponin I epitope or prodefensin-A6.

8. Composition containing a biepitopic compound of formula (I) as defined in any one of claims 1 to 7 in solution in water, in a buffer or in a biological fluid.

9. Use of a compound as defined in any one of claims 1 to 7 or a composition as defined in claim 8 as a control or standard in an immunoassay.

Use of a compound as defined in any one of claims 1 to 7 or a composition as defined in claim 8 as a control or standard in an immunoassay of cardiac troponin I or proefensin-A6. .

11. A method of detecting an analyte by immunoassay in a test sample capable of containing said analyte comprising

 i. an immunoassay test by contacting said test sample with one or more analyte binding partners,

ii. a test for checking the validity of the immunoassay test by bringing into contact a biepitopic compound of formula (I) as defined in any one of Claims 1 to 7 or of a composition as defined in the claim 8, as a positive control, with said one or more binding partners to the analyte, iii. reading the immunoassay test if the validity check is positive, iv. determining the presence of said analyte in the test sample when the signal obtained by the immunoassay test of step i is greater than the detection threshold of the immunoassay test.

A method of detecting an analyte by immunoassay according to claim 11, wherein the analyte is cardiac troponin I or prodefensin-A6.

A method of quantifying an analyte by immunoassay in a test sample capable of containing said analyte comprising

 i. an immunoassay test by contacting said test sample with one or more analyte binding partners,

 ii. a test for checking the validity of the immunoassay by contacting a positive control with said one or more binding partners to the analyte, iii. reading the immunoassay test if the validity check test is positive, and

 iv. determining the amount of said analyte in the test sample by comparing the signal of the immunoassay test with a standard curve previously obtained using a biepitopic compound of formula (I) as defined in any one of the claims 1 to 7 or a composition as defined in claim 8.

14. An immunoassay quantification method according to claim 13, wherein the positive control is a biepitopic compound of formula I as defined in any one of claims 1 to 7 or a composition as defined in claim 8.

A method of quantifying an analyte by immunoassay in a test sample capable of containing said analyte comprising

 i. an immunoassay test by contacting said test sample with one or more analyte binding partners,

ii. a test for checking the validity of the immunoassay test by contacting a biepitopic compound of formula (I) as defined in any one of claims 1 to 7 or a composition as defined in claim 8, as a positive control, with said one or more analyte binding partners,

 iii. reading the immunoassay test if the validity check test is positive, and

 iv. determining the amount of said analyte in the test sample by comparing the signal of the immunoassay test with a standard curve.

The immunoassay quantification method according to any one of claims 13 to 15, wherein the analyte is cardiac troponin I or prodefensin-A6.

A kit for carrying out an immunoassay comprising a biepitopic compound of formula I as defined in any one of claims 1 to 7, or a composition as defined in claim 8.