Stable chromogenic test reagent and its use in coagulation-diagnostic tests
View Patent ↗The present invention relates to a chromogenic test reagent which comprises a chromogenic peptide substrate and an inhibitor of fibrin polymerization, which is particularly suitable for being used in coagulation-diagnostic tests and which is distinguished by the fact that it exhibits a nigh degree of stability and/or a long shelf life in the liquid state.
1. A composition comprising:
a) a chromogenic peptide substrate, wherein the chromogenic peptide substrate comprises a substrate of thrombin, and wherein the chromogenic group is selected from para-nitroaniline (pNA), 5-amino-2-nitrobenzoic acid (ANBA), 7-amino-4-methoxycoumarin (AMC), quinonylamide (QUA), dimethyl 5-aminoisophthalate (DPA), and their derivatives; and
b) an oligopeptide inhibitor of fibrin polymerization, wherein the oligopeptide inhibitor of fibrin polymerization:
i. inhibits polymerization of fibrin monomers which are formed under the influence of thrombin; and
ii. comprises the peptide sequence GPRP-X—NH 2 (SEQ ID NO: 1), wherein G is the amino acid glycine, P is the amino acid L-proline, R is the amino acid L-arginine, and X is selected from the amino acids alanine and glycine; and
wherein the pH of the composition is between 3.0 and 6.0.
2. The composition as claimed in claim 1 , further comprising acetate ions.
3. The composition as claimed in claim 1 , wherein the chromogenic peptide substrate is a para-nitroaniline-coupled peptide substrate.
4. The composition as claimed in claim 1 , wherein the chromogenic peptide substrate is a 5-amino-2-nitrobenzoic acid-coupled peptide substrate.
5. The composition as claimed in claim 1 , wherein the chromogenic peptide substrate comprises the sequence Ala-Gly-Arg-R 1 , wherein R 1 is the chromogenic group.
6. The composition as claimed in claim 1 , wherein the chromogenic peptide substrate comprises the sequence Msc-VaI-Arg-R 1 , wherein Msc is methylsulfonylethyl-oxycarbonyl and R 1 is the chromogenic group.
7. The composition as claimed in claim 1 , further comprising one or more stabilizers.
8. A method for preparing the composition as claimed in claim 1 , comprising the steps of:
combining the chromogenic peptide substrate and the oligopeptide inhibitor of fibrin polymerization; and
adjusting the pH to between pH 3.0 and 6.0 by adding a proton donor.
9. The method as claimed in claim 8 , wherein the proton donor is acetic acid.
10. A method for determining a coagulation parameter in a whole blood or plasma sample, comprising:
(a) adding to the whole blood or plasma sample the composition as claimed in claim 1 ;
(b) determining the chromogenic signal of the chromogenic peptide substrate; and
(c) calculating the coagulation parameter based upon the chromogenic signal of the chromogenic peptide substrate.
11. The method of claim 10 , wherein the coagulation parameter is thrombin generation.
12. The method of claim 10 , wherein the coagulation parameter is the endogenous thrombin potential of the sample.
13. The method of claim 11 , wherein the coagulation parameter is the coagulation time of the sample.
14. The composition as claimed in claim 1 , wherein the pH of the composition is between 3.0 and 5.0.