Radioimmunoassay for the detection of active-site specific thrombin inhibitors in biological fluids. I. Assay characteristics and quantitation of recombinant hirudin.
A sensitive radioimmunoassay (RIA) for the quantitation of recombinant (r) hirudin in biological fluids is described. Taking advantage of the highly specific hirudin-thrombin interaction, a monoclonal antibody to human alpha-thrombin was used to capture hirudin-thrombin complexes in a competitive binding assay. Quantitation of r.hirudin in buffer, plasma or urine at concentrations ranging from 0.17 to 20 ng/ml (1.7 x 10(-3) to 2 x 10(-2) antithrombin units/ml) was achieved. In the absence of competing unlabelled r.hirudin the assay also measured alpha-thrombin (from 2 x 10(-4) to 1 x 10(-2) NIH units/ml) in citrated or defibrinated human plasma. A series of peptides corresponding to the carboxyl-terminal region of hirudin and with varying anticoagulant activities did not displace 125I-r.hirudin in the RIA described, confirming published data that these hirudin fragments bind to a site distant to the catalytic site of thrombin. The assay was used to test hirudin clearance after bolus i.v. injections of 0.1 mg r.hirudin [Val1-Val2] into human volunteers. The plasma concentrations and elimination kinetics of r.hirudin were in good agreement with published data and a close correlation between hirudin plasma concentration and prolonged clotting time was observed.
Lackmann
M
M
Heart Research Institute, Camperdown, NSW, Australia.
Hoad
R
R
Kakakios
A
A
Geczy
C L
CL
D000911:Antibodies, Monoclonal / Q000032:analysis; D001665:Binding Sites / Q000502:physiology; D001826:Body Fluids / Q000737:chemistry*; D006629:Hirudins / Q000032:analysis* / Q000097:blood / Q000652:urine; D006801:Humans; D007457:Iodine Radioisotopes; D010976:Platelet Count; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000032:analysis / Q000097:blood / Q000652:urine; D012015:Reference Standards; D012016:Reference Values; D012680:Sensitivity and Specificity; D013917:Thrombin / Q000032:analysis / Q000037:antagonists & inhibitors*; D013918:Thrombin Time
D000911:Antibodies, Monoclonal; D006629:Hirudins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; D013917:Thrombin