Radioimmunoassay of Bradykinin

Abstract

IN the past few years highly sensitive and specific radioimmunological methods have been developed for the determination of many polypeptide hormones¹. Radioimmunoassays are usually based on the competitive reaction of a measured amount of polypeptide hormone labelled with iodine-125 and iodine-131 and varying amounts of unlabelled hormone with hormone antiserum. This report describes a radioimmunoassay technique for bradykinin, a nonapeptide of considerable physiological and pathological importance. Bradykinin differs from other polypeptide hormones, which have been assayed by this method, in its lack of immunogenicity and inaccessibility to iodination. Formation of antibody against this peptide was therefore induced by immunizing goats with a bradykinin–haemocyanin conjugate². An isotopic label was introduced into the molecule by acetylating its terminal amino-group with ¹⁴C-acetic anhydride^*. This approach makes it possible to use commercially available bradykinin and renders an isotopic synthesis of the peptide unnecessary. Antibody which binds ¹⁴C-acetyl bradykinin was demonstrated in antiserum by a gel nitration procedure which separates antibody-bound from unbound peptide. Partial to complete displacement of the labelled acetyl peptide from its antibody-complex was observed on addition of increasing amounts of unlabelled bradykinin or acetyl bradykinin to the incubation mixture of a fixed amount of ¹⁴C-acetyl bradykinin and antiserum. Unlabelled bradykinin was found to be about 10–20 times less effective in this reaction than unlabelled acetyl bradykinin.