Indirect sandwich ELISA for human antithrombin III employing the interaction between D-biotin and streptavidin

Human antithrombin III (hATHI) is a natural inhibitor of many serine proteases involved in the intrinsic pathway of blood coagulation. The concentration ofhATIIIhas been determined by various immunoelectrophoretic methods and by assaying its biological activity. More sensitive methods are needed for the determination of hA Till in biological fluids which contain low amounts ofhATIH, for example urine and conditioned cell culture media, or which are not available in great amounts, such as cerebrospinal or follicular fluid. Here we report the use of an indirect capture sandwich ELJSAfor the determination of hA Till. In contrast to other methods, this assay is very sensitive and multiple samples can be easily processed simultaneously. The influence of varying the amounts and concentrations of reagents used in the ELJSA is discussed. Using the procedure described, hATIII was detected in cell culture media conditioned by recombinant CHO cells at concentrations as low as 30 ng mf1. The relative error of all determinations was below 10%.