A NEW IMMUNOASSAY FOR SOLUBLE FIBRIN ENABLES A MORE SENSITIVE DETECTION OF THE ACTIVATION STATE OF BLOOD-COAGULATION INVIVO

A novel sandwich immunoassay for measurement of soluble fibrin in plasma has been developed. For immunization we used the synthetic heptapeptide Gly-Pro-Arg-Val-Val-Glu-Arg representing the amino terminus of the alpha-chain of human fibrin. A monoclonal IgG1 antibody was obtained by conventional hybridoma technology. To increase convenience, the sandwich immunoassay was developed for the Enzymun-Test(TM) systems which are based on streptavidin pre-coated tubes. The new assay was designed with the same fibrin specific antibody both in biotinylated and in peroxidase-labelled form. Fibrin in native plasma samples could only be detected after pre-incubation of the plasma with chaotropic ions. Test results were calculated using a standard curve comprising six fibrin standards (0-50 mug/ml). Precison of the method was satisfactory; intra-assay CVs using plasma samples ranged between 5.0% (23.7 mug/ml) and 12.4% (0.2 mug/ml). CVs of interassay precision measurements using standards as samples range between 7.3% (25.0 mug/ml) and 11.4% (1.0 mug/ml). The lower detection limit was 0.12 mug/ml. Investigations of normal range in 70 age-matched healthy individuals resulted in a mean of 1.12 mug/ml. Linearity was excellent; recovery of high fibrin plasma after dilution with normal plasma was always between 100 and 107%. Fibrin specificity was due to the monoclonal antibody 2B5 used and no cross reactivity with fibrinogen, fibrinogen split products or fibrin D-dimer was observed. Fibrin fragment El (studied by Dempfle CE, et al Blood Coag Fibrinol 1993; 4: 79- 8 6) and fragments X and Y showed moderate cross reactivity in the assay and caused some overestimation of fibrin at high fibrin split product concentrations. Initial clinical studies revealed significantly lowered plasma fibrin levels in patients on long-term and stable oral anticoagulation (0.21 +/- 0.08 mug/ml) compared to a symptom-free control group with a history of venous thrombosis not receiving anticoagulant treatment (1.82 +/- 0.34 mug/ml). In conclusion the new assay allows a very sensitive detection of soluble fibrin in plasma.