Use of stabilised synthetic compounds in immunoassay
The invention concerns the use of at least a synthetic compound comprising: a carrier molecule having at least a hydrocarbon chain comprising at least 10, advantageously at least 12, preferably at least 14 and not more than 300, advantageously not more than 100, preferably not more than 50, selected'among C, N, O, S, P and Si, and comprising at least two reactive functions, or capable of reacting, for grafting lateral arms on said chain, and at least two different epitopes carried by said lateral arms grafted on said carrier molecule, as standard or control for immunoassay of said biological molecules, in particular of polypeptides or proteins.
1. A synthetic compound consisting of:
a) a cyclic or noncyclic polypeptide carrier molecule backbone, wherein the cyclic polypeptide carrier molecule backbone consists of 8 to 50 amino acid residues and the noncyclic polypeptide carrier molecule backbone consists of 7 to 50 amino acid residues, containing the sequence:
X 1 -A 1 -A 2 -A 3 -A 4 -A 5 -X 2 -A 6
wherein
X 1 and X 2 are lysine residues,
A 1 and A 2 represent an amino acid residue selected from the group consisting of proline, phenylalanine, glycine, alanine, valine, leucine, and isoleucine, with the proviso that one of A 1 and A 2 is selected from the group consisting of proline and phenylalanine,
A 3 represents an amino acid residue selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine,
A 4 represents an amino acid residue selected from the group consisting of arginine and histidine,
A 5 represents an amino acid residue selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine, and
A 6 represents an amino acid residue selected from the group consisting of glycine, alanine, valine, leucine, and isoleucine; and
b) a side arm grafted to the side chain of each of the two X 1 and X 2 lysine residues, the side arm carrying at least two different epitopes of human cardiac troponin I.
2. The synthetic compound according to claim 1 , wherein one of the amino acid residues A1 and A2 is proline.
3. The synthetic compound according to claim 1 , wherein each of the at least two side arms contains more than two different epitopes of human cardiac troponin I.
4. The synthetic compound according to claim 1 , wherein A 3 represents glycine.
5. The synthetic compound according to claim 1 , wherein A 4 represents arginine.
6. The synthetic compound according to claim 1 , wherein A 5 represents alanine.
7. The synthetic compound according to claim 1 , wherein A 6 represents glycine.
8. The synthetic compound according to claim 1 , wherein the polypeptide comprises a sequence selected from the group consisting of:
KGPGRAKG—(SEQ ID NO: 1),
KGFGRAKG—(SEQ ID NO: 2),
KPGGRAKG—(SEQ ID NO: 3), and
KFGGRAKG—(SEQ ID NO: 4).
9. The synthetic compound according to claim 1 , wherein each epitope comprises at least a sequence selected from the group consisting of:
TEPH (amino acid residues 10-13 of SEQ ID NO: 6)
ALLGAR, (SEQ ID NO: 8), and
FAEL (amino acid residues 26-29 of SEQ ID NO: 6).
10. The synthetic compound according to claim 1 , wherein the carrier molecule backbone is
Ac—KGPGRAKGPGSA—NH 2 (SEQ ID NO: 5) or
Ac—KGFGRAKGSGSA—NH 2 (SEQ ID NO: 7).
11. A composition containing at least one compound according to claim 1 in solution in water, in plasma, or in a buffer solution.
12. The composition according to claim 11 , wherein the buffer solution is selected from the group consisting of phosphate, succinate, and Tris HCl buffer solutions.
13. The composition according to claim 11 , wherein the buffer solution is selected from the group consisting of:
succinate buffer solution (pH=5-6) containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, skimmed milk powder and EDTA,
succinate buffer solution (pH=5-6) containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, casein and EDTA,
succinate buffer solution (pH=5-6) containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one and BSA,
phosphate buffer solution (KH 2 PO 4 /K 2 HPO 4 0.1 M, pH=6.5-7.5) containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, skimmed milk powder and EDTA,
phosphate buffer solution (KH 2 PO 4 /K 2 HPO 4 0.1 M, pH=6.5-7.5) containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, casein and EDTA, and
phosphate buffer solution (KH 2 PO 4 /K 2 HPO 4 0.1 M, pH=6.5-7.5) containing 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one and BSA.
14. Immunoassay kit comprising:
at least one synthetic compound as defined in claim 1 and
at least one reagent for carrying out an immunoassay.
15. Immunoassay kit comprising:
at least one composition according to claim 11 and
at least one reagent for carrying out an immunoassay.
16. The synthetic compound according to claim 1 , wherein:
the noncyclic polypeptide carrier molecule backbone consists of 7 to 30 amino acid residues.
17. The synthetic compound according to claim 1 , wherein:
the cyclic polypeptide carrier molecule backbone consists of 8 to 20 amino acid residues; and
the noncyclic polypeptide carrier molecule backbone consists of 7 to 20 amino acid residues.