Excess antibody immunoassay for rat glandular kallikrein. Measurement of kallikrein complexed with inhibitors and in plasma.
We have recently developed an immunoradiometric assay (IRMA) for specific measurement of immunoreactive kallikrein which allows a simultaneous determination of the enzymatic activity of kallikrein. This paper describes the application of this method for measurements of glandular kallikrein complexed with inhibitors. Interference by low molecular weight inhibitors such as benzamidine and Trasylol was easily overcome by increasing the amount of immobilized anti-kallikrein antibody added in the assay, and by prolonging the incubation time of the antigen-binding step. The recovery of kallikrein in complex with plasma inhibitors was complete only when the anti-kallikrein antibody was immunoadsorbed onto a solid-phase sheep anti-rabbit immunoglobulin. The dose-response curve of glandular kallikrein in plasma paralleled that of purified kallikrein in both the immunoradiometric and the immunoenzymometric assays. The concentration of immunoreactive glandular kallikrein in normal rat plasma was 12.8 +/- 4.3 nU/ml. The enzymatic activity of this immunoreactive kallikrein was 86% inhibited.
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000941:Antigens / Q000032:analysis / Q000276:immunology; D007611:Aprotinin / Q000097:blood / Q000378:metabolism*; D001550:Benzamidines / Q000378:metabolism; D002850:Chromatography, Gel; D004306:Dose-Response Relationship, Immunologic; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D007124:Immunoenzyme Techniques; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism*; D008297:Male; D051381:Rats; D013363:Submandibular Gland / Q000201:enzymology
D000906:Antibodies; D000941:Antigens; D001550:Benzamidines; D007611:Aprotinin; D007610:Kallikreins; C032157:benzamidine