1. A bi-epitope compound of formula (I): E1 -X-Z-Y-E2 Carrier molecule (I) wherein: El and E2, which may be identical or different, each independently represent a peptide sequence comprising at least one epitope of an analyte; and X and Y, which may be identical or different, each independently represent a linker arm, the carrier molecule is soluble and Z represents an amino acid derivative bearing a thiol function before the bonding thereof with the carrier molecule.

2. The bi-epitope compound of formula (I) as claimed in claim 1, wherein the linker arms X and Y are amino acid derivatives each forming two peptide bonds -CO-NH-, one with El or E2 and the other with Z.

3. The bi-epitope compound of formula (I) as claimed in claim 2, wherein the linker arms X and Y, which may be identical or different, each independently comprise one or more amino acid derivatives.

4. The bi-epitope compound of formula (I) as claimed in any one of the preceding claims, wherein Z is chosen from a cysteine derivative, a homocysteine derivative and a penicillamine derivative.

5. The bi-epitope compound of formula (I) as claimed in any one of the preceding claims, wherein the carrier molecule is a protein of which the molecular weight is between 20 kDa and 700 kDa, preferably between 60 kDa and 250 kDa.

6. The bi-epitope compound of formula (I) as claimed in claim 5, wherein the carrier molecule is bovine serum albumin.

7. The bi-epitope compound of formula (I) as claimed in any one of the preceding claims, wherein El and E2 are peptide sequences comprising at least one cardiac troponin I epitope or prodefensin-A6 epitope.

8. A composition containing a bi-epitope 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. The use of a compound as defined in any one of claims 1 to 7 or of a composition as defined in claim 8, as a control or standard in an immunoassay.

10. The use of a compound as defined in any one of claims 1 to 7 or of a composition as defined in claim 8, as a control or standard in a cardiac troponin I immunoassay or prodefensin-A6 immunoassay.

11. A process for detecting an analyte by immunoassay in a test sample that may contain said analyte, comprising i. an immunoassay test by bringing said test sample into contact with one or more binding partners for the analyte, ii. a test to verify the validity of the immunoassay test by bringing a bi-epitope compound of formula (I) defined in any one of claims 1 to 7 or a composition as defined in claim 8, as a positive control, into contact with said one or more binding partners for the analyte, iii. the reading of the immunoassay test if the validity verification test is positive, iv. the determination of 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.

12. The process for detecting an analyte by immunoassay as claimed in claim 11, wherein the analyte is cardiac troponin I or prodefensin-A6.

13. A process for quantifying an analyte by immunoassay in a test sample that may contain said analyte, comprising i. an immunoassay test by bringing said test sample into contact with one or more binding partners for the analyte, ii. a test to verify the validity of the immunoassay by bringing a positive control into contact with said one or more binding partners for the analyte, iii. the reading of the immunoassay test if the validity verification test is positive, and iv. the determination of the amount of said analyte in the test sample by comparison of the signal of the immunoassay test with a standard curve obtained beforehand using a bi-epitope compound of formula (I) as defined in any one of claims 1 to 7 or a composition as defined in claim 8.

14. The process for quantifying by immunoassay as claimed in claim 13, wherein the positive control is a bi-epitope compound of formula I as defined in any one of claims 1 to 7 or a composition as defined in claim 8.

15. A process for quantifying an analyte by immunoassay in a test sample that may contain said analyte, comprising i. an immunoassay test by bringing said test sample into contact with one or more binding partners for the analyte, ii. a test to verify the validity of the immunoassay test by bringing a bi-epitope 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, into contact with said one or more binding partners for the analyte, iii. the reading of the immunoassay test if the validity verification test is positive, and iv. the determination of the amount of said analyte in the test sample by comparison of the immunoassay test with a standard curve.

16. The process for quantifying by immunoassay as claimed in any one of claims 13 to 15, wherein the analyte is cardiac troponin I or prodefensin-A6.

17. A kit for implementing an immunoassay, comprising a bi-epitope compound of formula I, as defined in any one of claims 1 to 7, or a composition as defined in claim 8.

The present invention relates to the field of diagnosis or prognosis. In particular, it relates to a synthetic bi-epitope compound that is of use during the implementation of immunoassays.
Immunoassays are commonly used in the fields of clinical, food, pharmaceutical and chemical analyses. Thus, their objective is to determine the presence of a large number of analytes, in the form of proteins (antigens/antibodies), peptides or haptens, for instance steroids or vitamins, in a sample that may contain these analytes. The immunoassay is a test widely known to those skilled in the art which involves immunological reactions between the analyte to be detected and one or more binding partner(s) for this analyte. By way of example of such immunoassays, mention may be made of methods such as ELISA (Enzyme Linked Immuno Sorbent Assay), ELFA (Enzyme Linked Fluorescent Assay) and RIA (Radio Immuno Assay) which can operate according to the "sandwich" principle, or else according to the "competition" principle, and immunodetection methods such as immunohistochemistry, immunocytochemistry, immunofluorescence, Western blot and dot blot. The "competition" methods are normally used for small molecules such as haptens, the "sandwich" methods being used for the other analytes.
These immunoassays carried out in particular in biological test laboratories require the provision, by the manufacturer, in addition to the reagents required for the test, such as the binding partners, the revealing agents or else the diluting solutions, of a positive control for the test which, used under conditions analogous to those of the test for the sample to be studied, often simultaneously, will serve to validate that the immunoassay has performed correctly. If the positive control is indeed found to be positive, the result of the test is validated and can be interpreted. If the positive control is not found to be positive, this indicates that the implementation of the immunoassay did not take place in compliance with expectations. The result of the test which is invalid should then not be interpreted and the analysis should be recommenced.
With regard to the quantification of an analyte by immunoassay in a biological sample that may contain said analyte, it requires, in addition to the abovementioned reagents required for the test and the positive control, the use of a standard curve. Said

curve is obtained i) by measuring the signal generated by standards, also called calibrators, which correspond to increasing and known 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 in plotting the curve giving the signal as a function of the amount or the concentration. Very often, it is standard practice to find a mathematical model which represents, as reliably as possible, this relationship between the signal and the amount or concentration, in order to be able to easily calculate the results of a quantitative immunoassay.
To do this, the control or standard solutions must mimic the analyte sought and be recognized in the same way by the binding partners used in the immunoassay. Thus, if the method of the immunoassay is a sandwich method, the control or standard solutions must comprise a compound which has the two epitopes for recognition of the two binding partners used. The term "bi-epitope compound" is then used.
It is not out of the question for the two epitopes of a bi-epitope compound to be 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 in detection of the sandwich immunoassay. In this case, the bi-epitope compound will contain the same epitope twice.
The control or standard solutions normally used in immunoassay tests may be of human or animal origin and may 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 storage is not suitable for fluid laboratory practice. Furthermore, these lyophilized control or standard solutions need to be redissolved in order for it to be possible for them to be used. However, in the immunoassay context, the implementation of the test must be rapid and this redissolving results in a loss of time. Furthermore, the performing of this redissolving can lead to a measurement error because of a bias due to the dilution. Ready-to-use control or standard solutions, stored in liquid form at + 2/8°C, are thus particularly recommended, this being for obvious reasons of convenience. Nevertheless, in order to be representative of the actual conditions of the assay, these ready-to-use control or standard solutions contain only low concentrations of the analyte, for example about one pg/ml, one ng/ml or

one μg/mL, depending on the range of measurement of the analyte concerned, which results in their stability at a temperature of + 2/8°C being affected. Consequently, in order to overcome this drawback, synthetic standards have been used.
European patent application EP 0 650 053A describes synthetic standards
5 containing active sites for one or more receptors, linked to one another by an
arborescent structure. This application describes more specially troponin T synthetic
standards. Nevertheless, these standards have a stability time in solution which does not
exceed 3 weeks at 4°C.
Patent application WO 98/24816, for its part, provides synthetic bi-epitope
10 compounds that can be used as a standard in “sandwich” immunoassays, for assaying troponin I, and that are stable for several months. The compounds described, of formula Σ-E1-Ȥ-E2-Ψ, comprise two peptide sequences E1 and E2 comprising a minimum troponin I epitope, each of these epitopes being bonded to one another by a linker group Ȥ which can be a central peptide comprising from 1 to 40 amino acids. Each
15 epitope can 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 drawback that the bi-epitope compounds described, in order to be sufficiently immunoreactive with the binding partners used in the immunoassay, must have a not insignificant number of amino acids, such that they are not easy to synthesize. Furthermore, by virtue of their
20 uniquely peptide nature, a large quantity of compound is required in the control or standard solution in order to have optimal immunoreactivity with the binding partners used in the immunoassay, which represents a not insignificant cost for the manufacturer of kits containing this control or this standard and thus for the laboratory which uses the kit thus produced.
25 Patent US 6 114 180 proposes, for its part, a synthetic compound that can be
used as a control or standard in immunoassays comprising two troponin I epitopes linked to one another by a carrier molecule, such as BSA, with the objective of increasing its solubility and/or its stability in solution. However, the particular construction of this compound makes it difficult to produce and raises the question of
30 the equimolarity between the two epitopes linked to the carrier molecule.
4

The applicant has demonstrated, surprisingly, a synthetic compound to be used
in “sandwich” immunoassays which overcomes the drawbacks 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 exhibits excellent immunoreactivity with the binding
5 partners used in the immunoassay. Furthermore, it is not necessary to use a large
amount thereof, 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. at certain
10 Thus, the first subject of the present invention is a bi-epitope compound of
formula (I):

in which:
- E1 and E2, which may be identical or different, each independently represent a
15 peptide sequence comprising at least one epitope of an analyte,
- X and Y, which may be identical or different, each independently represent a linker arm,
- the carrier molecule is soluble and
- Z represents an amino acid derivative bearing a thiol function before the
20 bonding thereof 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 subject relates to the use of such a bi-epitope compound or of such
a composition containing this compound as a control or standard or adjuster in an
25 immunoassay.
Yet another subject of the invention relates to the immunoassay processes using, as a control and/or standard or adjuster, a bi-epitope compound of formula I or a composition containing this compound.
5

Finally, the last subject of the present invention is a kit for implementing an immunoassay, comprising a bi-epitope compound of formula (I) or a composition containing such a compound.
The applicant has thus developed, against all expectations, a synthetic bi-
5 epitope compound which makes it possible to overcome all the drawbacks of the prior
art mentioned above. The compound of the invention has formula (I) below:

in which:
- E1 and E2, which may be identical or different, each independently represent a
10 peptide sequence comprising at least one epitope of an analyte;
- X and Y, which may be identical or different, each independently represent a linker arm,
- the carrier molecule is soluble and
- Z represents an amino acid derivative bearing a thiol function before the
15 bonding thereof with the carrier molecule.
The compound of the invention is thus a bi-epitope compound. The term “bi-epitope compound” is intended to mean a compound which comprises two epitopes of one and the same analyte in order to mimic the antigenic recognition of said analyte in a sandwich immunoassay. Of course, the synthetic bi-epitope compound in no way
20 consists of the same sequence as the analyte that it mimics. This synthetic compound
does not therefore correspond to a sequence of amino acids that exist naturally, for example a protein or a protein fragment. The compound of the invention will be referred to, in an obvious and equivalent manner throughout the application, as bi-epitope compound, non-natural bi-epitope compound, synthetic bi-epitope compound
25 or non-natural synthetic bi-epitope compound.
The prefix “immuno” in the term “immunoassay”, for example, should not 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 an antibody
6

fragment. Indeed, as is well known to those skilled in the art, this term is more widely
used to also denote tests and processes in which the binding partner is not a partner of
immunological origin/nature but consists, for example, of a receptor of the analyte that
it is desired to detect and/or quantify. Whatever its origin or its nature, the binder
5 partner concerned should be capable of binding to the analyte sought, preferably
specifically. Thus, it is known practice to use the term “ELISA assay” for assays which use binding partners that are non-immunological in the strict sense, also widely known as “ligand binding assay”, whereas the term “immuno” is included in the term in extenso corresponding to the acronym ELISA. In the interests of clarity and uniformity,
10 the term “immuno” is used in the present application to denote any biological analysis
using at least one binding partner suitable for binding to the analyte sought and detecting and/or quantifying the latter, preferably specifically, even when said binding partner is not of immunological nature or origin in the strict sense.
Sandwich-type immunoassays (or more simply “sandwich immunoassays”) use
15 a first binding partner, termed “capture binding partner”, to specifically bind the
analyte sought, and a second binding partner, termed “detection binding partner” which is labeled and is also intended to specifically bind with the analyte sought, thus revealing the binding between the capture binding partner and the analyte, and thus the presence of the analyte. In other words, the analyte sought is taken “in a sandwich”
20 between said first and second binding partners, the first (“capture”) binding partner
generally being present in excess relative to the analyte sought. The capture binding partner can, for example, be immobilized on a solid support (by covalent bonding, adsorption or any other appropriate method).
An epitope, also called antigenic determinant, is the smallest part of an antigen
25 that can be recognized by a paratope which 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.
7

The sequence of a linear epitope can comprise “conservative” modifications which do not significantly change the binding between the epitope and the antibody from a point of view of the specificity.
A mimotope is a macromolecule, often a peptide, which mimics the three-
5 dimensional structure of a given epitope. An antibody which recognizes said epitope is
also capable of recognizing and binding to the mimotope which mimics this epitope. In
the case of antigens of protein nature, peptide mimotopes, which are thus linear, are
often used to mimic conformational epitopes. According to the invention, the
conformational epitopes can be replaced with mimotopes. The mimotopes are also
10 included in the scope of the present application as a replacement for the term epitope.
When the antigen is of 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, a distinction should be made between the notions of a minimal epitope and of an optimal epitope. The minimal epitope corresponds to the peptide sequence of
15 the smallest size specifically recognized by the corresponding antibody. When an
amino acid of the N-terminal end or of the C-terminal end of said peptide sequence is deleted, the binding of the antibody is no longer possible, and the recognition is lost. A minimum epitope can contain from 3 to 20 amino acids, more often from 4 to 8 amino acids.
20 The optimal epitope corresponds to the peptide sequence which is specifically
recognized and has the best possible reactivity with respect to the corresponding antibody (the strongest signal). It very often involves peptide sequences of 6 to 30 amino acids in length. The optimal epitope as a general rule comprises the minimal epitope. In rare cases, the optimal epitope and the minimal epitope can be merged.
25 The compound of the invention is such that it contains two peptide sequences
E1 and E2 comprising at least one epitope of an analyte. The expression “peptide sequence comprising at least one epitope of an analyte” is intended to mean that the peptide sequence consists at least of the amino acids of the minimal epitope. According to one embodiment of the invention, the peptide sequence consists of at most the amino
30 acids of the optimal epitope. Of course, those skilled in the art know that it is also
possible to add 1, 2, 3, 4 or 5 additional amino acids on one side of the optimal epitope
8

or on both sides without having too great an effect on 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 sequence of the analyte or else analogous amino acids. The term
5 “analogous amino acids” is intended to mean two amino acids, the replacement of
which is a conservative substitution that occurs in nature, that is to say that takes 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) nonpolar amino acids such as
10 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 reasonably be predicted that an isolated replacement of leucine with isoleucine or valine, of an aspartate with a
15 glutamate, of a threonine with a serine, or a similar conservative replacement of an
amino acid with another amino acid which is structurally related, will have no major effect on the biological activity or the antigenicity. Those skilled in the art will easily determine the amino acid that can tolerate a change with reference to the Hopp/Woods and Kyte-Doolite plots, well known in the art. According to one embodiment, the
20 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 else both are mimotopes and consequently the amino acids of the peptide sequence are predominantly different than the amino acids naturally found in the analyte.
The term “analyte” denotes a substance of biological origin, contained in a
25 sample, detected, identified and/or quantified by an analysis. It should be understood,
in the broad sense, as denoting a chemical, biological or biochemical substance which is the subject of one or more analyses. By way of example of analytes, mention may be made of a protein or a peptide.
The analyte will be representative of a pathological condition or of the presence
30 of a microorganism in a medium. The term “pathological condition” is intended to
mean any impaired state of health of a patient, due to diseases caused by numerous
9

factors, such as environmental (infectious), genetic and/or biological factors. By way of
examples of diseases, mention may be made of infectious diseases, due to
microorganisms such as viruses, bacteria or parasites (hepatitis, sepsis, etc.),
autoimmune diseases, neurodegenerative diseases, cancers (breast, prostate, colon, etc.),
5 cardiovascular diseases (myocardial infarction, etc.), etc. The analytes are then
associated with the various diseases. By way of example of an analyte, mention may be made of cardiac troponin I which is of use as an analyte for myocardial infarction and prodefensin-A6 as a colon cancer analyte. These analytes will subsequently be described in greater detail. According to one embodiment, E1 and E2 are peptide
10 sequences comprising at least one cardiac troponin I epitope or prodefensin-A6 epitope.
The central radical Z is an amino acid derivative bearing a thiol function before
the bonding thereof with the carrier molecule. The term “amino acid derivative” is
intended to mean any molecule forming two peptide bonds –CO-NH- with the linker
arms X and Y to which it is bonded. To do this, the corresponding amino acid
15 comprises, before the bonding thereof with the linker arms X or Y, a reactive
group -NH2 and a reactive group –COOH. After bonding to the linker arms X and Y, the amino acid derivative thus comprises a group –NH- and a group –CO-. The corresponding amino acid also comprises a thiol function (–SH) before the bonding thereof with the carrier molecule. Examples of such radicals Z comprise cysteine
20 derivatives, homocysteine derivatives and penicillamine derivatives. The corresponding
amino acids, used for forming these derivatives, are of course cysteine, homocysteine and penicillamine, which are amino acids known to those skilled in the art.
The two linker arms X and Y of the compound of the invention are identical to or different than one another. The linker arms are characterized by their capacity to
25 each form two peptide bonds –CO-NH-, on the one hand with E1 or E2 and, on the
other hand, with the central radical Z. The linker arms X and Y are then amino acid derivatives comprising a group –NH- and a group –CO-. To do this, the compounds used for forming the linker arms comprise, before the bonding thereof with the peptide sequences E1 or E2, a reactive group –NH2 and a reactive group –COOH. Of course,
30 the linker arms can comprise other groups, for example side groups, which are not
reactive as such or are not reactive since they are protected by protective groups known
10

to those skilled in the art, for example trityl, t-butyl, t-butyl ether benzyl or benzyl ester groups. The linker arms can be obtained from one or more compounds which each have a reactive group -COOH and a reactive group -NH2 before they are involved in the bi-epitope compound of the invention. These compounds will be referred to as 5 “monomers of use for forming the linker arms”.
The monomers of use for forming the linker arms can be proteinogenic a-amino acids, required for the synthesis of biological proteins, which are known to those skilled in the art. These proteinogenic a-amino acids can be genetically coded; in this case, there are 22 of them. The 20 proteinogenic a-amino acids universally distributed in all
10 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-tyrosine and L-valine. The other 2 proteinogenic amino acids are much rarer: L-pyrrolysine is found only in some methanogenic archaea and L-selenocysteine is
15 present only in some enzymes of the oxidoreductase family.
In addition to these 22 genetically coded amino acids, there are several tens of other biological amino acids that can be obtained from the above by enzymatic modifications, for instance L-citrulline, L-pyroglutamic acid, L-ornithine, L-3,4-dihydroxyphenylalanine, y-aminobutyric acid and domoic acid.
20 The monomers that are of use for forming the linker arms can also be pseudo
amino acids, also known as artificial amino acids, that is to say non-biological amino acids. In this case, the only requirement is that the compound comprises two free functions, -COOH and -NH2.
According to one particular embodiment of the invention, the linker arms X and
25 Y, which may be identical or different, each comprise one or more amino acid derivatives (such as proteinogenic a-amino acids, and/or biological amino acids and/or pseudo amino acids). The linker arms can be prepared from one to six amino acids, preferably from one to four amino acids. For example, the linker arms can have the sequence GGGS, or the sequence SGGG, or the sequence GSGSGS or else the
30 sequence SGSGSG
11

According to one embodiment of the invention, X and Y in formula (I), that is
to say after formation of the peptide bonds with E1/E2/Z, exhibit one or more
derivatives of monomers of formula (II) below:
-NH-R-CO- (II)
5 in which R is a radical consisting of one or more groups chosen independently from the
groups (-C(R’)H-C(R’’)H, (-C(R’)H-C(R’’)H-O-), (-C(R’)H-) and (-C(R’)H-O-), R’ and R’’ being chosen independently from hydrogen, a hydroxyl group and C1-C5 alkyl groups. According to one embodiment, R consists of one or more groups chosen independently from (-CH2-CH2-O-), (-CH2-O-) and (-CH2-). According to one
10 particular embodiment, the radical R may comprise from one to six groups
(-CH2-CH2-O-), preferably from one to four groups (-CH2-CH2-O-). By way of nonlimiting examples of a radical R, mention may be made of pentaoxaoctadecanoyl, tetraoxapentadecanoyl, trioxadodecanoyl, trioxatridecanoyl, dioxaoctanoyl, oxapentoyl and hexaoxaheneicosanoyl and derivatives thereof. By way of example of a compound
15 that is of use for giving a monomer derivative of formula (II), mention may be made of
8-amino-3,6-dioxaoctanoic acid (CAS No.: 134978-97-5), which is a known pseudo amino acid. For example, the linker arm may be a dimer or a trimer of 8-amino-3,6-dioxaoctanoic acid, (Ado)2 or (Ado)3.
By way of other example of groups of the radical R, mention may be made of
20 the biological amino acid derivatives mentioned above, in residue form, that is to say
without their –COOH and –NH2 group but comprising an –NH- group and a –CO-group. Thus for example, if the compound forming the linker arm is leucine, of formula (CH3)2CH-CH2-CH(NH2)-COOH, then R of the compound of formula (II) will be

Each linker arm has, between the –CO and –NH groups of the two ends, a size of between 10 and 60 Å, preferably between 20 and 30 Å, which constitutes one particular embodiment of the invention.
12

Since the monomers that are of use for forming the linker arms X and Y have
reactive groups –COOH and –NH2 before they are involved in the bi-epitope
compound of the invention and which allow the formation of peptide bonds, the
sequence E1-X-Z-Y-E2 can be considered to be a peptide, hereinafter referred to as bi-
5 epitope peptide, such that it can be produced by peptide synthesis, which has the
advantages of
- easy, rapid standardized, reproducible and simplified synthesis,
- the production of a compound which is equimolar with respect to E1 and E2.
10 The bi-epitope peptide is obtained according to the procedures well known to
those skilled in the art, such as the solid-phase peptide synthesis described by Merrifield, 1963. The improvements to this technique have been reviewed and discussed by Fields and Noble, 1990. Such a synthesis uses a solid phase to which the first C-terminal amino acid is attached, In this context, each -NH2 group of the new
15 amino acid, added so as to form the peptide, is protected with a protective group of
Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (t-butoxycarbonyl) type, in order to
promote the reaction between the –NH2 group presented by the solid phase and the –
COOH group of the new amino acid added, as is well known to those skilled in the art.
According to the invention, the length of the bi-epitope peptide does not exceed
20 a length corresponding to 100 amino acids, preferably it does not exceed a length
corresponding to 20 to 30 amino acids and more preferentially it does not exceed a length corresponding to 25 to 27 amino acids.
The bi-epitope peptide is bonded, via the radical Z, to a carrier molecule. The carrier molecule has the role of stabilizing the bi-epitope compound and makes it
25 possible to make the peptide sequences E1 and E2, comprising at least one epitope of
the analyte of said bi-epitope compound, more available, this being while at the same time preserving equimolarity between E1 and E2.
The term “carrier molecule” is intended to mean any soluble molecule which can be coupled to a peptide. By way of soluble molecule, mention may be made of
30 proteins such as bovine serum albumin, immunoglobulin G and thyroglobulin, and
polymers such as polylysines. According to one embodiment, the carrier molecule is a
13

protein of which the molecular weight is between 20 kDa and 700 kDa, preferably between 60 kDa and 250 kDa.
Polylysines are polymers known to those skilled in the art. They are available
for example from Sigma-Aldrich.
5 Bovine serum albumin and thyroglobulin are known to those skilled in the art.
For the immunoglobulin G, it should be chosen such that it comes neither from the species used to obtain the antibodies of the immunoassay, nor from the species from which the sample to be analyzed comes, in order to avoid interference problems. By way of example, mention may be made of rabbit, mouse, horse, goat, pig, etc.,
10 immunoglobulins G (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 in which the antibodies come from mice and in which the sample to be analyzed comes from human beings.
15 The coupling between the bi-epitope peptide and the carrier molecule of protein
nature at the level of the radical Z can take place by covalence, according to methods well known to those skilled in the art. The sulfhydril group (-SH) present in the side chain of the radical Z is reactive with respect to maleimide, haloacetyl and pyridyl disulfide groups. Thus, in a first step, the carrier molecule should be activated by
20 reaction with a molar excess of a crosslinker which will be capable of reacting at the level of the accessible amine groups (-NH2) of the carrier molecule and of thus introducing, at the surface of the carrier molecule, reactive groups chosen from maleimides or haloacetyls. These groups are preferred because they make it possible to obtain coupling via a thioether bond which is stable. Among the crosslinkers which
25 make it possible to introduce maleimide groups, mention may be made, non-exhaustively, of N-(s-maleimidocaproyloxy)succinimide ester, m-maleimidobenzoyl-N-hydroxysuccinimide ester, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate or else sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. Among the crosslinkers which make it possible to introduce haloacetyl
30 groups, mention may be made, non-exhaustively, of N-succinimidyl(4-iodoacetyl)aminobenzoate and sulfosuccinimidyl(4-iodoacetyl)aminobenzoate. The
14

activated carrier molecule is then purified by desalting, for example by gel filtration
chromatography, or else by dialysis, in order to remove the excess crosslinker and the
by-products. Finally, the activated carrier molecule is placed in the presence of the
bi-epitope peptide comprising the radical Z in relatively central position. The
5 maleimide or haloacetyl groups react with the sulfhydryl group (-SH) of the radical Z
of the bi-epitope peptide so as to form a stable covalent thioether bond. The reaction
between the maleimide and sulfhydryl groups should be carried out under conditions
close to neutral pH (pH 6.5 to 7.5) and should exclude foreign thiols, for example most
of the reducing agents, from the composition of the reaction buffer in order to avoid
10 competition for the coupling sites. The reaction between the haloacetyl and the
sulfhydryl groups should be carried out at pH 7.2 to 9. In order to limit the generation
of free iodine which is capable of reacting with tyrosine, histidine and tryptophan, it is
preferable to carry out the reaction in the dark. Such processes, known to those skilled
in the art, are described for example in “Chemistry of Protein Conjugation and Cross-
15 linking” by Shan S. Wong, CRC Press Inc., Boca Raton, Florida, United States, 1991.
In order to promote the coupling of the bi-epitope peptide on the radical Z, if the
linker arms X/Y and/or the peptide sequences E1/E2 contain amino acid derivatives
bearing a thiol function, it is advisable to use, for the formation of said peptide, amino
acid derivatives that are protected at the level of this thiol function with a protective
20 group which is stable during the steps of synthesis of said peptide and also during the
step of coupling with the carrier molecule. Thus, only the amino acid used to give the
radical Z will bear a reactive thiol function. In order to avoid such a step of protecting
the thiol functions of the arms X/Y and/or of the peptide sequences E1/E2, it is
advisable for neither X, nor Y, nor E1 nor E2 to contain an amino acid derivative
25 bearing a thiol function, this constituting one particular embodiment of the invention.
Amino acids protected at the level of their thiol function are available for example from Novabiochem®.
After coupling of the bi-epitope peptide with the carrier molecule, the thiol
functions will be optionally deprotected according to techniques known to those skilled
30 in the art. As protective group, mention may be made of the t-butylthio group which is
easily removed in an aqueous medium (0.1M ammonium bicarbonate) in the presence
15

of DTT (dithiothreitol). When only the coupling arms X/Y contain amino acid
derivatives bearing a thiol function, such a deprotection will not be necessary and will
not be advised. When the peptide sequences E1/E2 contain amino acid derivatives
bearing a thiol function, such a deprotection will be necessary if this affects the epitope
5 recognition.
It is also possible for the coupling arms and/or the peptide sequences to contain no amino acid derivative bearing a thiol function. Thus, the bi-epitope compound of the invention comprises one or more of the following characteristics:
- the linker arm X does not comprise an amino acid derivative bearing a thiol
10 function,
- the linker arm Y does not comprise an amino acid derivative bearing a thiol function,
- the peptide sequence E1 does not comprise an amino acid derivative bearing a thiol function, and
15 - the peptide sequence E2 does not comprise an amino acid derivative bearing
a thiol function.
According to one particular embodiment, the peptide sequences E1 and E2
comprise no amino acid derivative bearing a thiol function. According to yet another
embodiment, none of the elements among X, Y, E1 and E2 comprises an amino acid
20 derivative bearing a thiol function.
The compound of the invention, for implementation thereof in an immunoassay, can 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, this constituting another subject of the invention.
25 A composition containing the bi-epitope compound of formula (I) in solution in
water is a clear liquid solution obtained by complete dissolution of said compound and the major solvent of which is water, representing at least 50% by volume relative to the total volume of the solution. The amount of solvent depends of course on the analyte involved and will be easily determined by the person skilled in the art.
30 The compound of formula (I) can also be in solution in a buffer. The buffers to
be used are widely known to those skilled in the art and depend on the analyte involved.
16

By way of example of buffers, mention may be made of buffers such as the PBS,
HEPES and Tris-HCl buffers.
When the composition contains a biological fluid, said fluid can correspond to
the sample that it is desired to test. By way of examples, mention may be made of total 5 blood or derivatives thereof, 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 involved and on the corresponding measurement range. It will
easily be determined by those skilled in the art. Thus, it can be about one pg/ml, one
ng/ml or one μg/ml.
10 The same characteristics and preferences described previously, in particular
with regard to the choice of E1, E2, X, Y, Z, carrier molecule and analyte, also apply to
the compositions of the invention.
Of course, the compositions of the invention can comprise other compounds,
such as salts, filler proteins such as BSA or synthetic polymers of dextran or 15 polyethylene glycol type, or detergents, well known to those skilled in the art.
As previously indicated, the compounds and the compositions of the invention
are particularly advantageous since they are easily synthesized, are stable at + 2/8°C
and are soluble under the conditions of an immunoassay. Moreover, the compounds of
the invention have, against all expectations, an immunoreactivity to the binding 20 partners used in the immunoassay that is particularly high, that is to say that the binding
partners recognize them particularly well, such that, if it is desired to use them as a
control, standard and/or adjuster, they can be contained in the control, standard and
adjuster solutions in a low amount, while at the same time being stable under the
immunoassay conditions.
25 Thus, another subject of the invention relates to the use of such a bi-epitope
compound or of such a composition containing this compound, as a control or standard
or adjuster in an immunoassay.
The term “use as a control of the compound of formula (I) or the composition
containing this compound” is intended to mean the use thereof for, inter alia, verifying 30 that the immunoassay operates according to expectations (also called positive control)
and that the detection of the analyte in the test sample is not falsely negative.
17

The expression “use as a standard of the compound of formula (I) or the
composition containing this compound” is intended to mean the use thereof for
establishing a standard range. The establishment of the standard range, which is a
necessary step to be able to carry out a quantification of an analyte, is a step widely
5 known to those skilled in the art as previously described. It consists in measuring the
signal generated by increasing and known amounts or concentrations of the analyte, in plotting the curve giving the signal as a function of the amount or of the concentration and in finding a mathematical model which represents this relationship as reliably as possible. To do this, several aqueous compositions of the invention are used, each
10 containing a different concentration of analyte. The mathematical model will be used to
determine by extrapolation the unknown amounts or concentrations of analyte contained in the test sample.
The expression “use as an adjuster, also called calibrator, of the compound of formula (I) or the composition containing this compound, which is a particular
15 standard” is intended to mean the use thereof for adjusting the measurement of the
immunoassay of the analyte. In this case, the concentration of analyte is fixed and known. The signal generated during the use in the immunoassay by the adjuster is also known. The adjuster serves to verify that the measurement (signal) produced during the implementation of the immunoassay indeed corresponds to the expected value. If this is
20 not the case, the adjuster serves to measure the derivation that it will be possible, where
appropriate, to mathematically correct or to correct by physical intervention on the measuring instrument (adjustment). For convenience, the term “standard” will, in the present application, comprise the term “adjuster”.
As previously indicated, the analyte is any substance of biological, chemical or
25 biochemical origin contained in a sample, that is detected, identified and/or quantified
by an 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.
30 It is known that troponin is a myofibrillar protein complex consisting of three
proteins, troponins I, T and C. This protein complex makes it possible to contribute to
18

the regulation of Ca2+ ion-mediated muscle contraction, by interacting with myosin and
actin.
Cardiac troponin I (Uniprot accession No. P19429) is the troponin subunit
responsible for the inhibition of binding between myosin and actin.
5 Cardiac troponin I epitopes and mimotopes are known to those skilled in the art.
In one particular mode of the invention, the peptide sequences E1 and E2 are chosen
from the following sequences:
Sequence 1: ATEPHAKKK
Sequence 2: AGLGFAELQDL
10 Sequence 3: KISASRKLQLKT
The peptide sequences deriving 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 in question.
According to another embodiment, the compound of formula (I) or a
15 composition containing it is used as a control, standard or adjuster in a prodefensin-A6
immunoassay.
Defensins are a family of antimicrobial peptides involved in host defense
against microbial attacks. In the mature form, they consist of 30 to 40 amino acids and
have the property of selectively disaggregating membranes. Like other eukaryotic
20 proteins, defensins can be present not only in mature protein form but also in precursor
form. The term “prodefensin” is then used. Prodefensin-A6 (Uniprot accession No.
Q01524) has been described as possibly being of use as a marker in the context of
cancer and in particular of colorectal cancer, in particular in patent application
WO 2010/112777 by the applicant.
25 Prodefensin-A6 protein epitopes and mimotopes are known and are described
for example in patent application WO 2010/112777. In one particular mode of the
invention, the peptide sequences E1 and E2 are chosen independently from the
following groups of sequences, given that, if E1 is chosen from one group, E2 is chosen
from another group:
30 Group 1:
- Sequence 4: NYVTPPWAIFRH
19

- Sequence 5: WTGVLSPTQEYR
- Sequence 6: SHLTPPWMDYRV
- Sequence 7: VMAVTCSTCDSR
- Sequence 8: LTPPTEDLRPPD 5 Group 2:
- Sequence 9: YGNHSCTHIGHC
- Sequence 10: GPSYTCLHFGHC
- Sequence 11: TEREVHNWFPFH
Group 3:
10 - Sequence 12: YPHPWSMHVIRA
- Sequence 13: TTTPHPWALFAV
- Sequence 14: TPHPWQRWVVYS
- Sequence 15: EDVLRWHPEWPG Group 4:
15 - Sequence 16: YHETWPPKSAQL
- Sequence 17: YHDNWPQPSRSW
- Sequence 18: QHNHQRHGAMGA
- Sequence 19: YHDMWPMSGRMA
- Sequence 20: YHDNWPPLNGAR 20 - Sequence 21: YHDMWPAIQLSP

- Sequence 22: YHEKFPGPVVLP Group 5:
- Sequence 23: QAEDDPLQAK
These controls, standards and/or adjusters are particularly suitable for use in
25 processes for detecting and/or quantifying an analyte by immunoassay in a test sample
that may contain said analyte.
Thus, another subject of the invention relates to a process for detecting an analyte by immunoassay in a test sample that may contain said analyte, comprising
i. an immunoassay test by bringing said test sample into contact with one
30 or more binding partners for the analyte,
20

ii. a test to verify the validity of the immunoassay test by bringing a
bi-epitope compound of formula I as previously defined or a composition as previously
defined, by way of positive control, into contact with said one or more binding partners
for the analyte,
5 iii. the reading of the immunoassay test if the validity verification test is
positive,
iv. the determination of 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.
10 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.
Among the samples of food origin, mention may be made, nonexhaustively, of a sample of milk products (yoghurts, cheeses, etc.), of meat, of fish, of eggs, of fruit, of
15 vegetables, of water, of beverages (milk, fruit juice, soda, etc.). Of course, these
samples of food origin may also come from sauces or more elaborate dishes or non-transformed or partially transformed raw materials. A food sample may also be derived from an animal feed, such as oil cakes or animal meals. All these samples, if they are not liquid, are pretreated so as to be in liquid form.
20 As previously indicated, the sample may be of environmental origin and may
consist, for example, of a surface sampling, a water sampling, etc.
The sample may also consist of a biological sample, of human or animal origin, which may correspond to samplings of biological fluid (urine, total blood or derivatives such as serum or plasma, saliva, pus, cerebrospinal fluid, etc.), of stools (for example
25 choleraic diarrhea), nose, throat, skin, wound, organ, tissue or isolated cell samplings,
or swab samples. This list is obviously not exhaustive.
Generally, the term “sample” refers to a portion or an amount, more particularly a small portion or a small amount, taken from one or more entities for analytical purposes. This sample can optionally have undergone a pretreatment, involving for
30 example mixing, diluting or else milling steps, in particular if the starting entity is in
the solid state.
21

The sample analyzed may generally contain, or is suspected of containing, at least one analyte representative of the presence of microorganisms or of a disease to be detected, characterized or monitored.
The steps of this process for detecting an analyte by immunoassay are steps
5 widely known to those skilled in the art which have been previously described. In
particular, the first step consists in bringing the test sample into contact with one or
more binding partners for the analyte, preferably two binding partners for a sandwich
test. As previously described, one of the two partners can be coupled to a label so as to
form a conjugate or a tracer. The other binding partner can be captured on a solid
10 support as is known to those skilled in the art. The term “capture partner” is then used
for the latter and “detection partner” for the former.
The measured signal emitted by the conjugate is then proportional to the amount of analyte in the biological sample.
The binding partners for the analyte of interest are any molecule capable of
15 binding to the analyte. By way of example of binding partners for the analyte, mention
may be made of the binding partners of immunological nature or origin, such as
(monoclonal or polyclonal) antibodies and antibody fragments, well known to those
skilled in the art, and also binding partners which are not of immunological nature or
origin, such as nanofitins, receptors for the analyte if they exist, aptamers, DARPins or
20 any other molecule which is known to have an interaction with said analyte.
Nanofitins (tradename) are small proteins which, like antibodies, are capable of binding to a biological target, thus making it possible to detect it, to capture it or quite simply to target it within an organism.
Aptamers are oligonucleotides, generally RNA or DNA, identified in libraries
25 containing up 1015 different sequences, by a combinatorial method of in vitro selection
called SELEX for “Systematic Evolution of Ligands by Exponentiel Enrichment”
(Ellington AD and Szostak JW., 1990). Most aptamers are RNA compounds, owing to
the capacity of RNA to adopt varying complex structures, thereby making it possible to
create, at its surface, cavities of various geometries, making it possible to bind various
30 ligands. They are biochemical tools of interest that can be used in biotechnological,
22

diagnostic or therapeutic applications. Their selectivity and their ligand-binding properties are comparable to those of antibodies.
“DARPins” for Designed Ankyrin Repeat ProteINS (Boersma YL and
Plütckthun A, 2011) are another class of proteins which make it possible to mimic
5 antibodies and to be able to bind with high affinity and high selectivity to target
proteins. They derive from the family of ankyrin proteins which are adaptor proteins that make it possible to bind the membrane proteins integral to the spectrin/actin network which constitutes the “vertebral column” of the cell plasma membrane. The structure of ankyrins is based on the repetition of a unit of approximately 33 amino
10 acids and the same is true for DARPins. Each unit has a secondary structure of helix-
turn-helix type. DARPins contain at least three, preferably four to five repeated units and are obtained by screening of combinatorial libraries.
The term “label” is intended to mean, in particular, any molecule containing a group that is reactive with a group of the binding partner, directly without chemical
15 modification, or after chemical modification so as to include such a group, which
molecule is capable of directly or indirectly generating a detectable signal. A nonlimiting list of these direct detection labels consists of:
• enzymes which produce a signal detectable for example by colorimetry,
fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase,
20 beta-galactosidase or glucose-6-phosphate dehydrogenase,
• chromophores such as fluorescent, luminescent or dye compounds,
• radioactive molecules such as 32P, 35S or 125I,
• fluorescent molecules such as Alexas or phycocyanines, and
• electrochemiluminescent salts such as organometallic derivatives based 25 on acridinium or on ruthenium.
Indirect detection systems can also be used, for instance ligands capable of reacting with an anti-ligand. The ligand then corresponds to the label for constituting, with the binding partner, the conjugate.
Ligand/anti-ligand pairs are well known to those skilled in the art, which is the
30 case for example with the following pairs: biotin/streptavidin, hapten/antibody,
23

antigen/antibody, peptide/antibody, sugar/lectin, polynucleotide/polynucleotide
complementary thereto.
The anti-ligand may then be directly detectable by the direct detection labels
previously described or may itself be detectable by another ligand/anti-ligand pair, and
5 so on.
These indirect detection systems can result, under certain conditions, in an
amplification of the signal. This signal amplification technique is well known to those
skilled in the art, and reference may be made to the prior patent applications
FR 2781802 or WO 95/08000 by the applicant.
10 Depending on the type of labeling used, those skilled in the art will add reagents
which enable the visualization of the labeling or the emission of a signal detectable by any type of appropriate measuring device, for instance a spectrophotometer, a spectrofluorimeter, a densitometer or else a high-definition camera.
The step which is the test to verify the validity of the immunoassay is as
15 described previously.
The immunoassay test i) and the verification test ii) can be carried out in any order, simultaneously or successively, optionally on the same solid support.
The reading of the immunoassay test is also a step widely known to those
skilled in the art which depends on the test used.
20 Finally, the last step consists of the determination of 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 the detection, the compounds of the invention are also suitable for
25 the quantification of an analyte in a test sample. Thus, another subject of the invention
relates to a process for quantifying an analyte by immunoassay in a test sample that may contain said analyte, comprising
i. an immunoassay test by bringing said test sample into contact with one
or more binding partners for the analyte,
30 ii. a test to verify the validity of the immunoassay test by bringing a
positive control into contact with said one or more binding partners for the analyte,
24

iii. the reading of the immunoassay test if the validity verification test is
positive, and
iv. the determination of the amount of said analyte in the test sample by
comparison of the signal of the immunoassay test with a standard curve obtained
5 beforehand, using a bi-epitope compound of formula (I) as previously defined or a
composition as previously defined.
Said steps of the quantification process are as previously defined. In particular,
the immunoassay test i) and the verification test ii) can be carried out in any order,
simultaneously or successively, optionally on the same solid support.
10 In this quantification process, the positive control may be any compound that is
of use 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 bi-epitope compound or a composition as previously defined.
The standard curve is prepared with the compound or the composition of the
15 invention. Nevertheless, any other appropriate standard solution can be used. Thus,
another subject of the invention relates to a process for quantifying an analyte by immunoassay in a test sample that may contain said analyte, comprising
i. an immunoassay test by bringing said test sample into contact with one
or more binding partners for the analyte,
20 ii. a test to verify the validity of the immunoassay test by bringing a
bi-epitope compound of formula I as previously defined or a composition as previously defined, as a positive control, into contact with said one or more binding partners for the analyte,
iii. the reading of the immunoassay test if the validity verification test is
25 positive, and
iv. the determination of the amount of said analyte in the test sample by
comparison of the signal of the immunoassay test with a standard curve.
The same characteristics and preferences previously described, in particular
with regard to the choice of the particular compounds and of the analytes, to the various
30 steps of the immunoassay, also apply to the detection and quantification processes of
the invention.
25

In particular, in all these detection and quantification processes, the analyte may be cardiac troponin I or prodefensin-A6.
The immunoassay processes of the invention involve the use of diagnostic kits
comprising the compounds or compositions of the invention, this constituting another
5 subject of the invention.
In addition to the compounds or compositions of the invention as described
above, the kits according to the invention can also contain the compounds required for
the implementation of a process for detecting or quantifying by immunoassay the
presence of an analyte of interest, for example by sandwich-type immunoassay, such as
10 the binding partners and all the compounds required for demonstrating the reaction
between the binding partner(s) and the analyte of interest.
The invention will be understood more clearly by means of the following
examples which are given by way of nonlimiting illustration, and also by means of the
figures, in which:
15 - figure 1 is a graph giving the fluorescence signal RFV, determined by the
VIDAS® automated device, emitted by a bi-epitope compound according to the
prior art (REF Compound), a bi-epitope compound according to the invention
(Compound 1) and a bi-epitope peptide corresponding to the bi-epitope
compound 1 of the invention, but not coupled to a carrier molecule (noncoupled
20 Peptide 1), as a function of their concentration;
- figure 2 is a graph giving the fluorescence signal RFV, determined by the
VIDAS® automated device, emitted by the bi-epitope compound according to
the prior art (REF Compound) and bi-epitope compounds according to the
invention (Compounds 1 and 3), as a function of their concentration;
25 - figure 3 is a graph giving the fluorescence signal RFV, determined by the
VIDAS® automated device, emitted by the bi-epitope compound according to the prior art (REF Compound) and bi-epitope compounds according to the invention (Compounds 1 and 4), as a function of their concentration.
30 EXAMPLES
Example 1: Peptide synthesis
26

The peptide syntheses were carried out using either the ABI 433A synthesizer
from Applied Biosystems (Foster City, CA, United States), or the Liberty synthesizer
from CEM Corporation (Matthews, NC, United States). The Rink Amide MBHA resin
(Cat. No. 855003, Novabiochem®, Merck Millipore, Molsheim, France) was used as
5 polymeric solid support.
At the end of the 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 approximately 2 hours. After elimination of the polymer by filtration, the peptides were isolated by precipitation
10 from 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™ 120Å C18, 10 µm column (Mandel Scientific Company Inc., Guelph, Ontario, Canada). Each peptide was eluted with a stepwise gradient of acetonitrile
15 (from 0 to 95%) in aqueous solution containing 0.1% of trifluoroacetic acid, the percentage of acetonitrile of the steps 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 carried out in order to verify and characterize the peptides obtained.
20 For each peptide, an analytical HPLC profile was generated on a Chromolith®
High Resolution RP-18 encapped reverse-phase column (Merck Millipore, Molsheim, France). The elution was carried out by means of a linear gradient of acetonitrile (from 0 to 100%) in aqueous solution containing 0.1% of trifluoroacetic acid and monitored by measuring the absorbance at 214 nm. This analysis makes it possible to determine
25 the level of purity of the peptide.
Each peptide was also analyzed by liquid chromatography-mass spectrometry (LC/MS) on a Zorbax Eclipse Plus C18 RRHD 2.1 × 50 mm column, particle size 1.8 µm (Agilent Technologies, Santa Clara, CA, United States) coupled to an Accurate-Mass Q-TOF LC/MS 6540UHD mass spectrometer (Agilent Technologies). This
30 analysis makes it possible to determine the molar mass of the peptide.
27

The sequences of the peptides synthesized and also the characterization results are presented in Table 1.
Table 1. Sequences and characteristics of the peptides synthesized.

Identifier Analyte Sequence Amount
obtained
(mg) Purity Molar
mass
measured
(Daltons)
Peptide 1 TnI ATEPHAKKK-Ado2-C-Ado2-AGLGFAELQDL-NH2 78.8 97% 2806.47
Peptide 2 TnI ATEPHAKKKC-NH2 48.0 97% 1110.60
Peptide 3 TnI AGLGFAELQDLC-NH2 50.0 95% 1234.60
Peptide 4 TnI KISASRKLQLKT-Ado2-C-Ado2-AGLGFAELQDL-NH2 59.7 99% 3169.75
Peptide 5 TnI ATEPHAKKKGGGSCSGGG
AGLGFAELQDL-NH2 10.0 89% 2741.36
Peptide 6 PDEF-A6 QAEDDPLQAK-Ado2-C-Ado2-WTGVLSPTQEYR-NH2 32.0 99% 3215.50
5 The abbreviation TnI corresponds to cardiac troponin I and PDEF-A6 to
prodefensin-A6. The abbreviation Ado corresponds to 8-amino-3,6-dioxaoctanoic acid (CAS No.: 134978-97-5).
Table 2. Summary of the bi-epitope compounds according to the invention obtained
10 (formula I) and tested in terms of immunoreactivity

Identifier Analyte Epitope E1 Epitope E2 Arm X Arm Y Carrier molecule
Compound 1 TnI ATEPHAKKK AGLGFAELQDL (Ado)2 (Ado)2 BSA
Compound 2 TnI KISASRKLQLKT AGLGFAELQDL (Ado)2 (Ado)2 BSA
Compound 3 TnI ATEPHAKKK AGLGFAELQDL (Ado)2 (Ado)2 IgG
Compound 4 TnI ATEPHAKKK AGLGFAELQDL GGGS SGGG BSA
Compound 5 PDEF-A6 QAEDDPLQAK WTGVLSPTQEYR (Ado)2 (Ado)2 BSA
28

The abbreviation TnI 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 bovine serum albumin. The IgG is rabbit immunoglobulin G. 5
Example 2: Preparation of the bi-epitope compounds
The bi-epitope compounds were obtained by carrying out covalent couplings between, on the one hand, the peptides obtained in Example 1 and, on the other hand, carrier molecules. Table 2 presents in detail the various bi-epitope compounds
10 according to the invention that were prepared. All these compounds correspond to
formula I. Table 3, for its part, summarizes all of the couplings performed, while specifying the peptide and carrier-molecular pairs.
The procedure for the couplings is the following:
Firstly, the protein chosen as carrier molecule was activated in the presence of
15 an excess of Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-
carboxylate, CAS No.: 92921-24-9, Cat. No. 22322, Pierce, Thermo Scientific, Villebon sur Yvette, France). For the bovine serum albumin (BSA, Proliant Health & Biologicals, Ankeny, IA, United States), a 1/20 BSA/SMCC molar ratio was chosen. Thus, the BSA was diluted to 10 mg/ml in PBS (phosphate buffered saline), pH 7.2,
20 and 53 µl of a solution of sulfo-SMCC at 25 mg/ml in water, prepared
extemporaneously, was added dropwise. After incubation for 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 containing 150 mM NaCl, pH 6.8, in dialysis tubing having a cut-off threshold of 12 to 14 kDa. The dialysis was performed
25 at ambient temperature and the dialysis bath was changed every hour, 3 times. After the
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 50 mM phosphate buffer containing 150 mM NaCl, pH 6.8. This step makes it possible to modify the surface of the carrier molecule which from then on bears several reactive
30 groups of maleimide type.
29

The peptide to be coupled was dissolved at 5 mg/ml in 50 mM phosphate buffer containing 150 mM NaCl and 5 mM EDTA, pH 6.8, taking into account the purity. A 1/10 BSA/peptide molar ratio was chosen. Thus, 2.35 mg of BSA-SMCC at a concentration of 5 mg/ml (0.47 ml) were added to 1 mg of peptide at a concentration of 5 5 mg/ml (200 μl). This mixture was incubated for a minimum of 16 hours at 2/8°C on a wheel. The reaction was then blocked by adding 0.1 M of 2-mercaptoethylamine (CAS No.: 60-23-1, cysteamine) in 50 mM phosphate buffer containing 150 mM NaCl, pH 6.8, prepared extemporaneously. After incubation for 20 ± 5 minutes on a wheel at the laboratory temperature, the peptide-BSA conjugate was dialyzed against a PBS buffer,
10 pH 7.2, in dialysis tubing having a cut-off threshold of 12 to 14 kDa. The dialysis was continued for a minimum of 16 hours at 2/8°C. After the dialysis, the protein concentration was adjusted to a theoretical concentration of 2 mg/ml of BSA in PBS buffer, pH 7.2. The concentration of the peptide-BSA conjugate was then determined by measuring the absorbance at 280 nm. This step allows the reaction between the
15 maleimide groups and the sulfhydril groups (-SH) of the peptide, at the level of the terminal or median cysteine depending on the peptide sequence to be coupled, in order to form thioether bonds.
The compounds 1, 2, 4 and 5 were all obtained by applying the procedure described above. For the REF compound, which corresponds to the bi-epitope
20 compound as described in patent US 6 114 180, the same procedure was also applied, except that 2 peptides (peptide 2 and peptide 3) were placed in the presence of BSA-SMCC simultaneously and each peptide was coupled at a theoretical BSA/peptide molar ratio of 1/10. For the compound 3, peptide 1 was coupled to the rabbit polyclonal immunoglobulin G (bioMérieux). The procedure was identical, except that the BSA
25 was replaced with another carrier molecule. The theoretical carrier molecule/peptide molar ratio was 1/10.
30

Table 3. Summary of the peptide-carrier molecule couplings

Identifier Peptide Carrier molecule
Bi-epitope compounds
according to the
invention Compound 1 Peptide 1 BSA (bovine serum albumin)

Compound 2 Peptide 4 BSA

Compound 3 Peptide 1 Rabbit immunoglobulin G

Compound 4 Peptide 5 BSA

Compound 5 Peptide 6 BSA
Bi-epitope compound according to the prior art (US 6 114 180) REF compound Peptide 2 and Peptide 3 BSA
Example 3: Study of the immunoreactivity of the bi-epitope compound 1 according to the invention
5 The study of the bi-epitope nature of the compounds was carried out by means
of a cardiac troponin I immunoassay using the VIDAS® immunoanalysis automated device (bioMérieux). The single-use tip serves both as solid phase for the reaction and as pipetting system. The cartridge is composed of 10 wells (X0 to X9 covered with a sealed and labeled sheet of aluminum. The first well (X0) comprises a precut part so as
10 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 various reagents required for the analysis are contained in the intermediate wells. All the steps of the test are carried out automatically by the instrument. They consist of a succession of cycles of suctioning/blowing back of the reaction medium. The cardiac troponin I immunoassay
15 was carried out by means of a single-step sandwich test.
a) Sensitization and passivation of the tips The characteristics and the suppliers of the antibodies used are presented in Table 4. The tips were sensitized with 300 µl of a solution of the 19C7 and B90 monoclonal antibodies each diluted to 2.5 µg/ml in a PBS buffer, pH 6.2. After
20 approximately 20 h of incubation at +18/25°C with the sensitizing solution, the tips
were emptied. 300 µl of this same solution containing 10 g/l of bovine serum albumin are then added. The passivation continues at +18/25°C overnight. The tips are emptied, dried, and then stored at +4°C until use, in a moisture-free environment.
31

Table 4. Antibodies used for the cardiac troponin I immunoassay

Antibody name Target Sequence of E1 or E2 Supplier (Cat. No.)
19C7 TnI KISASRKLQLKT Hytest (4T21-19C7)
B90 TnI ATEPHAKKK SDIX (B9085MA06-MA)
3D5F7 TnI AGLGFAELQDL bioMérieux (noncommercial)
7B9 TnC NA Hytest (4T27-7B9)
NA: not available. The abbreviation TnI corresponds to cardiac troponin I and the abbreviation TnC corresponds to cardiac troponin C. The abbreviation Cat. No. corresponds to the catalog reference of the supplier.
5
b) Immunoassay procedure
The test compounds were diluted in a PBS-BSA buffer at various concentrations and assayed as sample.
As soon as the VIDAS® tip is in contact with the sample, the immunological
10 reaction begins because the capture antibodies are immobilized on this tip. The
automated device mixes the test sample (135 µl) with 270 µl of the solution of
conjugate. 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 a 100 mM phosphate buffer, pH 6.4, also containing
15 150 mM of NaCl and filler proteins.
The incubation lasts 6.8 minutes at 37°C and enables the specific binding of the
cardiac troponin I, or of the cardiac TnI bi-epitope compounds, or the cardiac TnI
peptides, on the one hand to the antibodies adsorbed onto the tip and, on the other hand,
to the conjugates. The unbound components are then removed by 3 washes with a
20 200 mM Tris buffer, pH 7.8, containing 300 mM NaCl and 0.2% Triton X-100. During
the final revealing step, the 4-methylumbelliferyl phosphate substrate is suctioned up and then blown back in the tip; the enzyme of the conjugate catalyzes the reaction for hydrolysis of this substrate to 4-methylumbelliferone, the emitted fluorescence of which is measured at 450 nm. The value of the fluorescence signal (RFV=relative
32

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® automated device when the immunoreactivity of the REF
5 bi-epitope compound (prior art), of the bi-epitope compound 1 according to the
invention and of the noncoupled peptide 1 (Example 1) is compared. Figure 1 represents these same data in graph form. As a reminder, peptide 1 comprises 2 cardiac TnI epitopes, one recognized by the B90 capture antibody of the immunoassay previously described, and the other by the 3D5F7 detection antibody. In compound 1
10 according to the invention, peptide 1 is coupled via the median cysteine on BSA in
order to ensure better antigen presentation thereof and improved stability. The REF compound has the same two TnI epitopes also coupled to 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 level of the terminal cysteine. Compound 1, the REF
15 compound, which corresponds to a bi-epitope compound as described in patent
US 6 114 180, and peptide 1, which corresponds to a synthetic bi-epitope compound as described in patent application WO 98/24816, are both reactive in the cardiac TnI immunoassay, but their levels of reactivity are very different. Thus, in order to obtain a signal of approximately 1000 RFV, 21 µM of compound 1 are necessary compared
20 with 1118 µM of the REF compound, that is to say approximately 50 times less.
Compound 1 thus exhibits much better immunoreactivity than the REF compound. Moreover, the good dynamics of the VIDAS® signal obtained with Compound 1 is not reproduced with the REF compound, even when testing much higher concentrations of this compound. The noncoupled peptide 1 is much less well recognized than the two
25 bi-epitope compounds coupled to BSA.
Table 5. Immunoreactivity of the bi-epitope compounds 1, 3, 4 and REF and of cardiac TnI peptide 1
33

[c] peptide
in ng/mL REF compound
(89 kDa) Compound 1
(94 kDa)
Peptide 1 – BSA Noncoupled peptide 1
(28 kDa) Comp 3
(188
Peptid Ig ound
kDa)
e 1 – G Compound 4
(93 kDa)
Peptide 5 – BSA

[c] S [c] S [c] S [c] S [c] S
0.5 – – 5.3 270 – – – – – –
1 – – 11 451 – – 5,3 175 11 60
2 – – 21 935 – – 11 360 21 118
5 56 62 53 1903 – – 27 882 54 295
10 112 120 106 3606 – – 53 1688 107 545
20 224 223 213 5911 713 2 106 3276 214 1100
50 559 617 – – 1781 21 266 6209 535 2569
100 1118 997 – – 3562 69 – – – –
200 2236 1484 – – 7125 356 – – – –
500 5589 2102 – – 17811 1825 – – – –
1000 11179 2155 – – 35623 3798 – – – –
2000 – – – – 71245 6140 – – – –
4000 – – – – 142491 7542 – – – –
The abbreviation [c] corresponds to the concentration in µM of the compound. The abbreviation S corresponds to the signal in RFV.
5 Example 4: Comparison of the immunoreactivity of the bi-epitope compounds
according to the invention using various carrier molecules
In this example, peptide 1 which comprises 2 different epitopes of cardiac TnI was coupled to 2 different carrier molecules: BSA (compound 1) and rabbit immunoglobulin G (compound 3). The obtaining of these bi-epitope compounds is
10 described in Example 2. The comparison of the immunoreactivity of these compounds
in the cardiac TnI immunoassay was carried out as described in Example 3 and the results are presented in Tableau 5 above and figure 2, which represents a graph giving the RFV fluorescence signals emitted by the various compounds, bi-epitope compound according to the prior art (REF Compound) and bi-epitope compounds according to the
15 invention (Compounds 1 and 3), as a function of their concentration. The results show
34

that compounds 1 and 3 are both reactive in the cardiac TnI immunoassay and exhibit a comparable reactivity which is much greater than that observed for the REF compound.
Example 5: Comparison of the immunoreactivity of the bi-epitope compounds
5 according to the invention using various spacer arms
In this example, the bi-epitope compounds compared differ only in terms of the spacer arm. In the case of compound 1, the two spacer arms are identical, it is a dimer of the Ado artificial amino acid. Compound 4, for its part, comprises the GGGS sequence as arm X and the SGGG sequence as arm Y. As a reminder, the two
10 compounds have 2 cardiac TnI epitopes and the carrier molecule is BSA. The obtaining
of these bi-epitope compounds is described in Example 2. The comparison of the immunoreactivity of these compounds in the cardiac TnI immunoassay was carried out as described in Example 3 and the results are presented in Table 5 above and figure 3, which represents a graph giving the RFV fluorescence signals emitted by the various
15 compounds, bi-epitope compound according to the prior art (REF Compound) and
bi-epitope compounds according to the invention (Compounds 1 and 4), as a function of their concentration. The results show that compound 4 comprising GGGS and SGGG arms is less well recognized than compound 1 comprising Ado arms, but is much greater than the REF compound.
20
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 carried out on D0, the day on which the solutions were prepared. The values obtained served as a reference for monitoring the stabilities. The
25 diluted solutions of the bi-epitope compounds were stored at +2/8°C and assayed on D7
(7th day after preparation). Table 6 below presents the variation in the RFV signal of the immunoassay between D0 and D7 (Signal D7 / Signal D0 × 100). Compounds 1 and 2 are stable and their antigenic properties are preserved when they are stored at +2/8°C for 1 week.
30
Table 6. Stability of compounds 1 and 2 at +2/8°C for 7 days
35

In a second step, a stability study of longer duration was carried out for compound 1 only, diluted in PBS, pH 6.2, containing 50 g/1 of BSA. When stored at +2/8°C, compound 1 is stable in dilute solution: 97% of the signal of the immunoassay is found at 1 month storage, 94% at 3 months and 90% at 6 months. When stored at +18/25°C, compound 1 is stable in dilute solution for approximately 1 month (88% of the signal is found). It is also important to note that compound 1 is capable of withstanding at least three freezing/thawing cycles at -20°C without any degradation of its antigenic properties. A higher number of freezing/thawing cycles was not tested.
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 bi-epitope compound 5 according to the invention comprising a mimotope
The bi-epitope compound 5 was designed so as to operate as a control and/or standard and/or adjuster during a prodefensin A6 immunoassay. Compound 5 combines a linear epitope (QAEDDPLQAKL) and a mimotope (WTGVLSPTQEYR). The prodefensin A6 immunoassay was carried out using the VIDAS® automated immunoanalysis device (bioMerieux), according to the protocol described in application WO 2010/112777, namely using, as capture antibody, the 12H4E1 clone (bioMerieux), which recognizes the linear minimal epitope of sequence EDDPLQ, and, as detection antibody, the 1H8C9 clone (bioMerieux), the epitope of which 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® automated device when various concentrations of

compound 5 (molecular molar mass: 98 155 Daltons) are tested. Compound 5 is indeed reactive in the prodefensin A6 immunoassay.
Table 7. Immunoreactivity of the bi-epitope compound 5

Literature references
- Boersma YL and Plutckthun 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, Chemistry of Protein Conjugation and Cross-linking », CRC Press Inc., Boca Raton, Florida, United States

We Claim:
1. A bi-epitope compound of formula (I):
E1 -X-Z-Y-E2
Carrier molecule (I)
wherein:
El and E2, which may be identical or different, each independently represent a peptide sequence comprising at least one epitope of an analyte; and
X and Y, which may be identical or different, each independently represent a linker arm,
the carrier molecule is soluble and
Z represents an amino acid derivative bearing a thiol function before the bonding thereof with the carrier molecule.
2. The bi-epitope compound of formula (I) as claimed in claim 1, wherein the linker arms X and Y are amino acid derivatives each forming two peptide bonds -CO-NH-, one with El or E2 and the other with Z.
3. The bi-epitope compound of formula (I) as claimed in claim 2, wherein the linker arms X and Y, which may be identical or different, each independently comprise one or more amino acid derivatives.
4. The bi-epitope compound of formula (I) as claimed in any one of the preceding claims, wherein Z is chosen from a cysteine derivative, a homocysteine derivative and a penicillamine derivative.
5. The bi-epitope compound of formula (I) as claimed in any one of the preceding claims, wherein the carrier molecule is a protein of which the molecular weight is between 20 kDa and 700 kDa, preferably between 60 kDa and 250 kDa.

6. The bi-epitope compound of formula (I) as claimed in claim 5, wherein the carrier molecule is bovine serum albumin.
7. The bi-epitope compound of formula (I) as claimed in any one of the preceding claims, wherein El and E2 are peptide sequences comprising at least one cardiac troponin I epitope or prodefensin-A6 epitope.
8. A composition containing a bi-epitope 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. The use of a compound as defined in any one of claims 1 to 7 or of a composition as defined in claim 8, as a control or standard in an immunoassay.
10. The use of a compound as defined in any one of claims 1 to 7 or of a composition as defined in claim 8, as a control or standard in a cardiac troponin I immunoassay or prodefensin-A6 immunoassay.
11. A process for detecting an analyte by immunoassay in a test sample that may contain said analyte, comprising
i. an immunoassay test by bringing said test sample into contact with one
or more binding partners for the analyte,
ii. a test to verify the validity of the immunoassay test by bringing a
bi-epitope compound of formula (I) defined in any one of claims 1 to 7 or a composition as defined in claim 8, as a positive control, into contact with said one or more binding partners for the analyte,
iii. the reading of the immunoassay test if the validity verification test is positive, iv. the determination of 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.

12. The process for detecting an analyte by immunoassay as claimed in claim 11, wherein the analyte is cardiac troponin I or prodefensin-A6.
13. A process for quantifying an analyte by immunoassay in a test sample that may contain said analyte, comprising
i. an immunoassay test by bringing said test sample into contact with one or more binding partners for the analyte,
ii. a test to verify the validity of the immunoassay by bringing a positive control into contact with said one or more binding partners for the analyte,
iii. the reading of the immunoassay test if the validity verification test is positive, and
iv. the determination of the amount of said analyte in the test sample by comparison of the signal of the immunoassay test with a standard curve obtained beforehand using a bi-epitope compound of formula (I) as defined in any one of claims 1 to 7 or a composition as defined in claim 8.
14. The process for quantifying by immunoassay as claimed in claim 13, wherein the positive control is a bi-epitope compound of formula I as defined in any one of claims 1 to 7 or a composition as defined in claim 8.
15. A process for quantifying an analyte by immunoassay in a test sample that may contain said analyte, comprising
i. an immunoassay test by bringing said test sample into contact with one or
more binding partners for the analyte, ii. a test to verify the validity of the immunoassay test by bringing a bi-epitope 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, into contact with said one or more binding partners for the analyte, iii. the reading of the immunoassay test if the validity verification test is positive, and

iv. the determination of the amount of said analyte in the test sample by comparison of the immunoassay test with a standard curve.
16. The process for quantifying by immunoassay as claimed in any one of claims 13 to 15, wherein the analyte is cardiac troponin I or prodefensin-A6.
17. A kit for implementing an immunoassay, comprising a bi-epitope compound of formula I, as defined in any one of claims 1 to 7, or a composition as defined in claim 8.