Determination of thrombin-hirudin complex in plasma with an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for the determination of the thrombin-hirudin complex (TH) in plasma. The test is based on the sandwich principle and uses appropriate antibodies which selectively bind the corresponding moieties of the complex. The assay was calibrated by adding performed TH to normal human citrated plasma. The detection limit of the assay was 1.2 ng/ml. Mean coefficients of variation of 6.0% (intra-assay) and 6.4% (inter-assay) were found. The presence of TH was demonstrated in normal human plasma that was spiked with hirudin and subsequently activated in vitro by calcium-thromboplastin to generate thrombin. This complex was also found in plasma samples from pigs which had been treated with hirudin during experimentally induced septicaemia.