Development and validation of two enzyme-linked immunosorbent assay (ELISA) methods for recombinant hirudin.
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.
Iyer
L
L
Department of Pharmacology, Loyola University Medical Center, Maywood, IL 60153, USA.
Adam
M
M
Amiral
J
J
Fareed
J
J
Bermes
E
E
D000918:Antibody Specificity; D001826:Body Fluids / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006629:Hirudins / Q000032:analysis*; D006801:Humans; D008297:Male; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results
D006629:Hirudins; D011994:Recombinant Proteins