Determination of Human Prothrombin Activation Fragment 1+2 in Plasma with an Antibody against a Synthetic Peptide

Abstract

The present investigation describes a novel approach to prepare a specific antibody against prothrombin activation fragment 1 + 2 (F 1 + 2). The antibody discriminates between native prothrombin and F 1 + 2 in plasma. A synthetic peptide from the negatively charged region of F 1 + 2, which becomes the carboxy-terminal sequence after cleavage of prothrombin by factor Xa, was used for immunization of rabbits. Obtained antiserum was immunopurified and an enzyme-linked immunosorbent assay (ELISA) was constructed for determination of F 1 + 2. The test system follows the sandwich principle and uses two different antibodies directed against F 1 + 2 and prothrombin, respectively. The ELISA was calibrated with purified F 1 + 2 added to F 1 + 2-poor plasma. The lower limit of sensitivity of the assay was 0.02 nmol/l. Coefficients of variation of 6.9 to 10.4% (intraassay) and 6.7 to 11% (interassay) were found for F 1 + 2 concentrations between 0.08 and 4.9 nmol/l. A reference range from 0.32 to 1.2 nmol/l was calculated from 95 healthy donors (mean value +/- SD: 0.67 +/- 0.19 nmol/l). In patients with deep vein thrombosis (n = 7) confirmed by phlebography and in patients with pulmonary embolism (n = 8) confirmed by lung scan, F 1 + 2 levels were found up to 1.5 to 9.5 nmol/l. In plasma samples of patients under oral anticoagulant therapy in the stable state F 1 + 2 concentrations were found to be in the range of 0.08 to 0.5 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)