A novel metholdoogy has been developed to assay clotting factors, using fibrinogen as a substrate. Two forms of fibrinogen are prepared, one having fibrinogen non-covalently associated with a solid-phase (microtiter plates), and the other having fibrinogen covalently associated with an enzyme in solution phase. When the two forms are converted to fibrin, the enzyme label associates with the solid phase. This technique has been named enzyme- linked coagulation assay or ELCA. Thrombin and any enzyme or cofactor which affects the generation of thrombin from plasma can be assayed using this methodology, at a sensitivity much higher than any other available technique, and at very low reagent cost. This project aims at converting this potential into a useful system for measurement of clotting factors and effectors. Reagent mixtures will be developed for all clotting facors of clinical interest, which will consist of peroxidase-fibrinogen mixed with purified factors or substrate plasmas specific for a factor of interest. Samples will be diluted and reagents will be added using a robotic system designed to be used in microtiter plate assays. Results will be read using a new rapid microplate reader which permits kinetic analysis rather than endpoint readings. The combination of high assay sensitivity by using the ELCA methodology and convenience of sample handling and data manipulation by using automated systems will considerably improve the quality of analysis of clotting factors and effectors.
| Triscott, M X; Bottoms, J D; Beard, G A et al. (1990) Enzyme linked fibrinolytic assay (ELFA). A new method for the measurement of t-PA in plasma using enzyme labelled fibrin. Thromb Res 59:723-33 |
| Doellgast, G J; Triscott, M X; Buss, D H et al. (1988) Extrinsic-pathway enzyme-linked coagulation assay (EP-ELCA). A clot-based alternative to prothrombin time for measurement of extrinsic pathway factors in plasma. Clin Chem 34:294-9 |
