Patent ID: 6140062
Filing Date: 2000-10-31
Classification: C12Q,G01N,Y10S

Abstract:
Method of determining the heparin content, which method comprises the following steps:(a) adding a known amount of thrombin (FIIa) or activated coagulation factor X (FXa) to a mixture which comprises a known concentration of chromogenic substrate (S), a known concentration of antithrombin (AT) and a sample having an unknown heparin concentration, the amount of FIIa or FXa being chosen such that at most 20%, of the S present is reacted during the period which the AT needs to deactivate all the FIIa or FXa present,(b) allowing the reactions which occur to proceed and determining the final concentration of the reacted chromogenic substrate p-nitroanilide ([pNA].sub.final) after completion of the reactions,(c) using the [pNA].sub.final found to determine the rate constant (k.sub.dec ) of the reaction of FIIa or FXa with AT using the relationship: ##EQU4## where: *=a multiplication operationKm=Michaelis constant=(k.sub.cat +k.sub.-1)/k.sub.1, (2)k.sub.cat =rate constant for the formation of the product pNA from the intermediate complex FIIa-S or FXa-S,k.sub.1 =rate constant for the formation of an intermediate complex FIIa-S or FXa-S from FIIa or FXa and S,k.sub.-1 =rate constant for the decomposition of the intermediate complex FXa-S or FIIa-S into FXa or FIIa and S,[S]=initial concentration of the substrate,[AT]=initial concentration of AT, and[E]=initial concentration of FXa or FIIa, and(d) determining the heparin concentration in the sample using the value of k.sub.dec thus found from a predetermined calibration curve of k.sub.dec against heparin concentration, the temperature being constant during the entire method.