Factor Xa-based heparin assay using a heparin-modifying component
The present invention is concerned with the diagnosis of coagulation and relates to a method for determining the heparin activity in a sample, where the sample is initially incubated with a heparin-modifying component, and then the heparin-dependent FXa inactivation is measured.
1 . A method for determining the heparin activity in a liquid sample, wherein the heparin-dependent inactivation of added factor Xa is quantified, comprising:
(a) providing a liquid sample;
(b) adding a heparin-modifying component to the sample; and
(c) adding factor Xa to the sample after addition of the heparin-modifying component.
2 . The method of claim in claim 1 , wherein the heparin-modifying component comprises:
(a) a heparinase;
(b) a glucuronidase;
(c) a mixture of different heparinases;
(d) a mixture of different glucuronidases; or
(e) a mixture of at least one heparinase and at least one glucuronidase.
3 . The method of claim 2 , wherein the heparin-modifying component comprises:
(a) heparinase I;
(b) heparinase II;
(c) heparinase III; or
(d) a mixture of any of (a), (b), and (c).
4 . The method of claim 1 , wherein the heparin-modifying component is used in a concentration range from 0.05 to 5.0 U/ml.
5 . The method of claim 1 , wherein the sample is incubated with a heparin-modifying component for 10 to 900 seconds before addition of factor Xa.
6 . The method of claim 1 , wherein the heparin-dependent inactivation of added factor Xa is quantified in the presence of added antithrombin.
7 . The method of claim 6 , wherein the antithrombin is added to the sample at the same time as the heparin-modifying component.
8 . The method of claim 1 , wherein additionally dextran sulfate is added to the sample.
9 . The method of claim 8 , wherein the dextran sulfate is added at the same time as factor Xa.
10 . The method of claim 1 , wherein the sample is a body fluid sample.
11 . The method of claim 1 , wherein further the heparin activity in the sample is quantified irrespective of the nature of the heparin present in the sample by means of a universal calibration curve which is suitable for all heparin variants and which has been constructed using any heparin.
12 . A test kit which comprises:
(a) a reagent comprising a heparin-modifying component; and
(b) a reagent comprising factor Xa.
13 . The test kit of claim 12 , which further comprises a reagent comprising a factor Xa substrate.
14 . The test kit of claim 12 , which further comprises a reagent comprising antithrombin.
15 . The test kit of claim 12 , wherein the reagent (a) further comprises antithrombin.
16 . The test kit of claim 15 , wherein the reagent (b) further comprises dextran sulfate.
17 . The test kit of claim 14 , wherein the antithrombin-containing reagent further comprises dextran sulfate.
18 . The test kit of claim 12 , wherein one or more of the reagents are lyophilized.
19 . The method of claim 1 , wherein the method is carried out using a test kit.
20 . The method of claim 1 , wherein the heparin-modifying component is used in a concentration range from 0.25 to 1.25 U/ml.
21 . The method of claim 1 , wherein the sample is incubated with a heparin-modifying component for 15 to 90 seconds before addition of factor Xa.
22 . The method of claim 1 , wherein the sample is incubated with a heparin-modifying component for 20 to 60 seconds before addition of factor Xa.
23 . The method of claim 19 , wherein the test kit comprises:
(a) a reagent comprising a heparin-modifying component; and
(b) a reagent comprising factor Xa.
24 . The method of claim 10 , wherein the body fluid is blood, blood plasma, serum or urine.