Polyethylene glycol (PEG) coupled r-hirudin mutein is determined by biological methods-the coagulation system. In the present study, a competitive enzyme-linked immunosorbent assay (ELISA) is described that permits the measurement of PEG r-hirudin. The ELISA system adopts a rabbit IgG antibody to quantitatively detect PEG-hirudin in human plasma. A PEG-hirudin calibration curve ranged from 50 to 7000 ng/mL. The limit of detection was 87 ng/mL. The intraassay coefficients of variation (CV) ranged between 16 and 21%, and interassay CV between 8 and 22% for low and high PEG-hirudin concentrations, respectively. The recovery of the compound in plasma was between 96 and 111.5%. The interindividual differences between 100 and 5000 ng/mL PEG-hirudin were between 12 and 22%. The correlation of the concentration of PEG-hirudin determined with the ELISA and the ecarin clotting time was r = 0.902. No interactions between unfractionated heparin, low molecular-weight heparin, or phenprocoumon and PEG-hirudin were observed in the ELISA. Deficiencies of thrombin or antithrombin as well as low, normal, and high fibrinogen levels did not interfere with the assay. It is concluded that the ELISA determines the concentration of PEG-hirudin and is not influenced by other major anticoagulants or by plasma levels of some coagulation proteins.
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