Synthetic bioepitope compounds which can be used as standards in the biological assays of Troponin I

1. Biepitope compound consisting of formula I:

Ξ-E 1 -Z-E 2 -Ψ  (I)

in which:

E 1 and E2, which are identical or different, represent a peptide sequence comprising a minimum epitope of troponin I,

Z represents

a chain of formula II:

—NH—(CH 2 ) m —CO—  (II)

 in which m represents an integer from 1 to 10,

or a mixed construct of formulae III to V:

-[pep 1 -NH—(CH 2 ) m —CO] p -  (III)

-[pep 1 -NH—(CH 2 ) m —CO-pep 2 ] p -  (IV)

-[NH—(CH 2 ) m —CO-pep2] p -  (V)

 in which

m represents an integer from 1 to 10,

p represents an integer from 1 to 5, and

pep 1 and pep 2 which are identical or different, represent a peptide chain containing from 2 to 10 amino acids forming an extended epitope of troponin I with the sequences E 1 an E 2 , respectively,

Ξ represents

a hydrogen atom, an acetyl group, a peptide sequence of 1 to 10 amino acids, an N-α-acetylated peptide sequence of 1 to 10 amino acids, a cysteinyl, biotinyl or biocytinyl group, a peptide sequence of 1 to 10 amino acids carrying a cysteinyl, biotinyl or biocytinyl residue,

a linear or branched amino(C 1 -C 10 alkyl)carbonyl chain,

an N-α-acetylated linear or branched amino(C 1 -C 10 alkyl)carbonyl chain,

a mixed construct of at least one peptide sequence of 1 to 10 amino acids and at least one linear or branched amino(C 1 -C 10 alkyl)carbonyl chain, or

a mixed construct of at least one peptide sequence of 10 to 10 amino acids and at least one linear or branched amino(C 1 -C 10 alkyl)carbonyl chain carrying a cysteinyl, biotinyl, or biocytinyl residue,

Ψ represents

a hydroxyl radical, an amino radical, a peptide sequence of 1 to 10 amino acids, or a peptide sequence of 1 to 10 amino acids carrying an amino-terminal group,

a linear or branched amino(C 1 -C 10 alkyl)carbonyl chain,

a linear or branched amino(C 1 -C 10 alkyl)carbonyl chain carrying a hydroxyl radical or an amino radical,

a mixed construct of at least one peptide sequence of 1 to 10 amino acids and at least one linear or branched amino(C 1 -C 10 alkyl)carbonyl chain, or

a mixed construct of at least one peptide sequence of 1 to 10 amino acids and at least one linear or branched amino(C 1 -C 10 alkyl)carbonyl chain carrying a hydroxyl radical or an amino radical.

2. The biepitope compound according to claim 1 , in which E 1 and/or E 2 , which are identical or different, represent a peptide sequence selected from the group consisting of

SEQ ID NO: 1 -Thr Glu Pro His-,

SEQ ID NO: 2 -His Ala Lys Lys-,

SEQ ID NO: 3 -Pro Ala Pro IIe Arg Arg Arg-

SEQ ID NO: 4 -Leu Leu Gly Ala Arg, and

SEQ ID NO: 5 -Arg Lys Asn IIe-.

3. The biepitope compound according to claim 1 of formulae Ia or Ib:

Ξ-Thr Glu Pro His-Z-Leu Leu Gly Ala Arg-Ψ  (Ia)

Ξ-Pro Ala Pro IIe Arg Arg Arg-Z-Thr Glu Pro His-Ψ  (Ib)

in which

Z includes sequence

SEQ ID NO: 26 -Ala Lys Lys-NH—(CH 2 ) m —CO-Ala Met Met Gln Ala-,

wherein m represents an integer from 1 to 10,

Ξ represents an acetyl radical, a cysteinyl radical, or a biotinyl or biocytinyl radical optionally linked with a peptide sequence residue selected from the group consisting of:

SEQ ID NO: 57 -Gly A Tyr Arg Ala Tyr Ala-,

SEQ ID NO: 58 -Gly Gly Asn Tyr Arg Ala Tyr Ala-,

SEQ ID NO: 59 -Asn Tyr Arg Ala Tyr Ala-, and

SEQ ID NO: 60 -Arg Pro Ala- and

Ψ represents an amino radical or a peptide sequence selected from the group consisting of

SEQ ID NO: 61 -Ala Lys Glu and

SEQ ID NO: 62 -Ala Lys Lys Lys Ser Lys.

4. The biepitope compound according to claim 1 , selected from the group consisting of

Ac-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys-NH(CH 2 ) 5 —CO-ala Met

     1              5                   10                      15

Met Gin Ala Leu Leu Gly Ala Arg Ala Lys Glu-NH 2 (SEQ ID NO:28)

         20                  25

and

Cys-Gly Asn Tyr Arg Ala Tyr Ala Thr Glu Pro His Ala Lys Lys-NH(CH 2 )—CO-Ala Met

      1              5                   10                      15

Met Gin Ala Leu Gly Ala Arg Ala Lys Glu-NH 2 (SEQ ID NO:53).

         20              25

5. Composition containing the biepitope compound according to claim 1 in solution in water, in plasma, or in a buffer solution.

6. Composition according to claim 5 , wherein the buffer solution is selected from the group consisting of phosphate, succinate, and Tris-HCl buffer solutions.

7. Composition according to claim 5 , wherein the buffer solution is selected from the group consisting of

a succinate buffer solution pH=5-6 containing Kathon, Régilait, and EDTA,

a succinate buffer solution pH=5-6 containing Kathon, casein, and EDTA,

a phosphate buffer solution KH 2 PO 4 /K 2 HPO 4 0.1 M and pH=6.5-7.5 containing Kathon, Régilait and EDTA, and

a phosphate buffer solution KH 2 PO 4 /K 2 HOP 4 0.1 M pH=6.5-7.5 containing Kathon, casein, and EDTA.

8. A method for immunoassaying troponin I, wherein the biepitope compound according to claim 1 is used as a standard.

9. The method according to claim 8 , wherein the immunoassay is a sandwich immunoassay.

10. A kit for carrying out an immunoassay comprising (a) the biepitope compound according to claim 1 or (b) a composition containing the biepitope compound in solution in water, in plasma, or in a buffer solution.