Method for detecting the von Willebrand factor-cleaving protease activity of ADAMTS-13
View Patent ↗The invention relates to a diagnostic method for determining the von Willebrand factor (VWF) cleaving activity of ADAMTS-13 in a test medium during which the test medium is mixed with 0.5 to 5 U/ml of a von Willebrand factor (VWF) that does not contain ADAMTS-13, and after incubation, the ADAMTS-13 activity is determined based on the drop in the VWF-mediated aggregation of thrombocytes.
1. A diagnostic method for determining the von Willebrand factor (VWF)-cleaving activity of ADAMTS-13 in a test medium, the method comprising the following steps:
a) incubating art ADAMTS-13-free VWF with urea to form urea treated VWF,
b) providing a test medium comprising unquantified ADAMTS-13,
c) adding from 0.5 to 5 U per ml of said ADAMTS-13-free, urea treated von Willebrand factor to the test medium to form a reaction medium,
d) incubating the reaction medium,
e) adding platelets to the incubated reaction medium, and
f) quantifying the ADAMTS-13 activity based on the reduction in the VWF-mediated aggregation of the added platelets in the incubated reaction medium.
2. A diagnostic method for determining the VWF-cleaving activity of ADAMTS-13 in a test medium, the method comprising the following steps:
a) aggregating platelets by incubating the platelets with ADAMTS-13-free von Willebrand factor (VWF),
b) providing a test medium comprising an unquantified ADAMTS-13 activity,
c) adding the test medium to the aggregated platelets, and
d) quantifying the ADAMTS-13 activity base on the dissociation of the platelet aggregates.
3. The method as claimed in claim 1 , wherein the method is carried out in the presence of ristocetin.
4. The method as claimed in claim 1 , in which the reduction in the VWF-mediated aggregation of platelets is determined using a calibration curve, with normal human plasma which has been diluted with varying quantities of inactivated normal human plasma being used for constructing the calibration curve.
5. The method as claimed in claim 2 , in which the dissociation of the platelets is determined using a calibration curve, with normal human plasma which has been diluted with varying quantities of inactivated normal human plasma being used for constructing the calibration curve.
6. The method as claimed in claim 1 , wherein a serine protease inhibitor is used.
7. The method as claimed in claim 1 , wherein the test medium is blood plasma, blood serum, saliva, cerebrospinal fluid, cell culture supernatant or cell extract.
8. A diagnostic kit, containing an ADAMTS-13-free VWF and platelets, as well as urea for pretreating the ADAMTS-13-free VWF.
9. The diagnostic kit as claimed in claim 8 , wherein the ADAMTS-13-free VWF and the urea are present in one container.
10. The diagnostic kit as claimed in claim 8 , wherein said kit additionally contains ristocetin.
11. The method as claimed in claim 2 , wherein the method is carried out in the presence of ristocetin.
12. The method as claimed in claim 2 , wherein a serine protease inhibitor is used.
13. The method as claimed in claim 2 , wherein the test medium is blood plasma, blood serum, saliva, cerebrospinal fluid, cell culture supernatant or cell extract.