Determination of components active in proteolysis
AssigneeChromogenix AB
InventorsLeif Aurell, Petter Friberger
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Abstract
The invention relates to a freeze-dried reagent combination containing all reagents required for determination of components active in a proteolysis such as proteases and especially proenzymes, cofactors, inhibitors and activators for proteases which reagent combination is enclosed in a container such as a cuvette. The invention also relates to a new process for the preparation of such a reagent combination in which process the conditions are controlled so that all reagents can exist in one single solution which is freeze-dried without the reagent being deactivated; and the use of the reagent combination in the fields of coagulation and fibrinolysis.
Claims11
A dry reagent combination for the determination of a blood coagulation factor or a fibrinolysis factor, said combination comprising, in a substantially unreacted form and in an amount sufficient for a single test, a chromogenic substrate for a blood coagulation protease colyophilized with an enzymatically active component reactive with said substrate, and a buffer at pH, such that the enzymatically active component is inactive but not denatured;
- wherein the component colyophilized with the substrate is a protease, and the factor to be determined is an inhibitor for the protease.
A process for the preparation of a reagent combination for the determination of an inhibitor for a protease, said process comprising the steps:
- (i) introducing into a container a sufficient amount for a single test of an aqueous solution of a chromogenic substrate and an enzymatically active component reactive with said substrate, said solution containing a buffer at a pH that provides non-denaturing conditions where said substrate and said active component are unreactive with each other, wherein said active component is a protease, and
- (ii) lyophilizing said solution while maintaining said unreactive conditions and said non-denaturing conditions.
In an assay method for the determination of a blood coagulation factor or a fibrinolysis factor, said method comprising:
- (i) incubating in a single step a biological plasma sample with a chromogenic substrate for a protease, and an enzymatically active component reactive with said substrate, the incubation being performed in an aqueous solution providing the appropriate pH for the enzymatic degradation of the substrate, and
- (ii) measuring of the substrate degradation which is a measure of the amount of the coagulation or the fibrinolysis factor in the sample,
- the improvement being adding the sample and an aqueous solvent that provides the appropriate pH-condition for the enzymatic substrate degradation to a colyophilized mixture comprising the substrate and the active component reactive with said substrate, and wherein the active component colyophilized with the substrate is a protease, and the factor to be determined is an inhibitor for the protease, and wherein a buffer is present at pH such that the enzymatically active component is inactive but not denatured.
Dependencies are inferred from the claim text.