A MICROTITER ASSAY FOR FACTOR-XIII USING FIBRINOGEN AND BIOTINYLCADAVERINE AS SUBSTRATES

Efforts to develop an improved assay for plasma and tissue transglutam inase have led us to a convenient, sensitive microtiter plate assay fo r coagulation factor XIII using human fibrinogen as an immobilized sub strate. Factor XIII was activated in the presence of calcium, thrombin , and immobilized fibrinogen and then assayed by adding biotinylcadave rine. The reaction was terminated by adding EDTA and the level of inco rporated biotin was measured with streptavidin-beta-galactosidase. In this assay, the analytical range for human platelet factor XIII was 0. 01-100 ng and 1-100 ng for guinea pig liver transglutaminase. Fibrinog en-coated plates gave more than 100-fold increase in sensitivity compa red with N,N-dimethylcasein-coated microtiter plates. The intraassay c oefficient of variation was less than 5% (n = 12) and interassay less than 6% (n = 4). The sensitivity of this assay reduced the volumes of plasma samples required and consequently eliminated the need to remove fibrinogen from such test samples. As expected, factor XIII activity could be inhibited by putrescine and antibodies against factor XIII as well as by a monoclonal antibody that bound to the carboxyl terminus of human fibrin gamma-chains. The assay provided a sensitive, simple, and rapid method for measuring factor XIII. (C) 1994 Academie Press, I nc.