Method for the quantification of activated coagulation factor VII (FVIIa)

• US 5,968,759 A
• Filed: 10/16/1996
• Issued: 10/19/1999
• Est. Priority Date: 10/18/1995
• Status: Expired due to Fees

• Current Assignee: AVENTIS BEHRING GMBH (CSL Limited)
• Original Assignee: Centeon Pharma GmbH
• Inventors: Romisch, Jurgen, Stohr, Hans-Arnold
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US08/731,580
• Time in Patent Office: 1,098 Days
• Field of Search: 435/13, 435/2, 435/23, 435/4, 435/183, 435/69, 514/56, 514/1, 424/530, 424/531, 424/529, 530/380, 530/381, 536/21, 436/69
• US Class Current: 435/13
• CPC Class Codes: G01N 2333/8128 Antithrombin III
• CPC Class Codes: G01N 2333/96447 Factor VII (3.4.21.21)
• CPC Class Codes: G01N 2333/96463 Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: G01N 2400/40 Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

The invention relates to a method for the quantification of coagulation factor VIIa (F VIIa) in FVIIa- or FVII/FVIIa-containing solutions by means of selective binding to immobilized soluble thromboplastin.

Claims

1. A method for the detection of activated factor VII (FVIIa) in protein solutions without promoting autoactivation of FVII, comprising

a) binding the FVIIa to immobilized soluble thromboplastin (sTF)

b) detecting the amount of bound or eluted FVIIa.

2. The method as claimed in claim 1, wherein, before the binding to immobilized soluble thromboplastin, the starting material is incubated with a combination of antithrombin and heparin and subsequently the heparin is neutralized.

3. The method as claimed in claim 1, wherein a protein A column is used for the sTF-Fc binding.

4. The method as claimed in claim 1, wherein the FVIIa activity is determined by amidolysis.

5. The method as claimed in claim 1, wherein the amount of FVIIa is determined via measuring the generation of FXa or FIXa.

6. The method as claimed in claim 1, wherein the amount of FVIIa is quantified by coagulometry with the aid of a dissolved soluble thromboplastin.

7. The method as claimed in claim 1, wherein the amount of FVIIa is determined by measuring the clotting activity/total activity ratio.

8. The method as claimed in claim 5, wherein the amount of FVIIa is determined via measuring the generation of FXa and FIXa.

9. The method as claimed in claims 1 or 2, wherein the immobilized soluble thromboplastin is prepared by:

a) making sTF-Fc by a recombinant method, and

b) immobilizing said sTF-Fc to a column which binds Fc.