Development and Validation of Two Enzyme-Linked Immunosorbent Assay (ELISA) Methods for Recombinant Hirudin

Abstract

Recombinant hirudin is currently being developed as a potential prophylactic and therapeutic antithrombotic drug in various clinical indications such as angina and deep venous thrombosis. In this report, we have discussed the production of specific polyclonal antibodies to recombinant hirudin (rH) and the development of two ELISA methods to measure rH concentrations in biological fluids: a sandwich and a competitive ELISA method. Intra- and inter-assay variations in the two methods are extremely low (3-7%). The competitive ELISA method is rapid, simple and highly reproducible. Saturation binding curves, selection of appropriate incubation times, recovery of different hirudin variants and reactivity in the presence of thrombin are discussed. The methods can be easily adapted to monitor hirudin concentrations in the clinical laboratory for diagnostic purposes as well as for performing pharmacokinetic studies.

Ecarin Chromogenic Assay

“…Modifikationen dieser Methodenw urdenh insichtlichi hrerE ignung zumM onitoring vonH irudin unda nderen direktenT hrombininhibitoren untersucht (4,21,28,29,32,39). Als spezielle Tests wurden immunologischeM ethoden, HPLC-Methodens owie chromogene Tests oderspezielle Gerinnungstests zurBestimmungv on direktenT hrombininhibitoren entwickelt (1, 3,5,7,8,10,13,18,23,25,[35][36][37]. EinedetaillierteDiskussion derBestimmungsmethodenw urde vonN owak (26) und Hafner et al (11) vorgenommen.…”

HIT/HITT and alternative anticoagulation: current concepts

“…The drawback is the prolonged assay time (>1.5 h) and its availability. 13 More sensitive assays include chromogenic substrate-based assay and ECT. The chromogenic assay has a good linear correlation over a wide range of plasma hirudin concentrations.…”

Methods for the Monitoring of Direct Thrombin Inhibitors

“…30 Immunologic hirudin assays have good reproducibility (intra-assay imprecision < 4%, interassay imprecision < 7%) and recovery (92 to 107%). [33][34][35][36][37] The sandwich assay covers a large measurement range (0.1 to 5.0 mg/L), whereas the competitive method is highly sensitive at low concentrations (0.02 to 1.0 mg/L). 37 The major disadvantages of these methods are long assay times (> 1.5 hours) and their limited around-the-clock availability.…”

“…[33][34][35][36][37] The sandwich assay covers a large measurement range (0.1 to 5.0 mg/L), whereas the competitive method is highly sensitive at low concentrations (0.02 to 1.0 mg/L). 37 The major disadvantages of these methods are long assay times (> 1.5 hours) and their limited around-the-clock availability. In contrast to assays with monoclonal antibodies, tests with polyclonal antibodies also detect hirudin-thrombin complexes.…”