Measurement of urinary kallikrein activity by kinin radioimmunoassay

OA Carretero, NB Oza, A Piwonska, T Ocholik… - Biochemical …, 1976 - Elsevier
OA Carretero, NB Oza, A Piwonska, T Ocholik, AG Scicli
Biochemical Pharmacology, 1976Elsevier
A method for measuring urinary kallikrein activity by kinin radioimmunoassay (RIA) is
described. Kinins were generated by incubating urine with partially purified dog kininogen in
the presence of peptidase inhibitors. Antibodies against kallidin were induced in rabbits by
injecting kallidin coupled to ovalbumin. One of the antisera generated was used at a final
dilution of 1: 18,000 to obtain a 40 per cent binding of approximately 3000 cpm (10 pg) of
bradykinin (S-tyrosin)-[125 I] triacetate ([125 I] bradykinin). Synthetic kallidin (10–500 pg) …
Abstract
A method for measuring urinary kallikrein activity by kinin radioimmunoassay (RIA) is described. Kinins were generated by incubating urine with partially purified dog kininogen in the presence of peptidase inhibitors. Antibodies against kallidin were induced in rabbits by injecting kallidin coupled to ovalbumin. One of the antisera generated was used at a final dilution of 1:18,000 to obtain a 40 per cent binding of approximately 3000 cpm (10 pg) of bradykinin (S-tyrosin)-[125I]triacetate ([125I]bradykinin). Synthetic kallidin (10–500 pg) was used to construct standard curves. When kinins generated by urinary kallikrein were also used, the two displacement curves for [125I]bradykinin were similar. The RIA was sensitive to 10 pg kinins. The antiserum cross-reacted with bradykinin, methionyl- kallidin, and kininogen, but not with angiotensin, oxytocin or SQ 20,881. To increase the specificity of the RIA, the kininogen was removed by ethanol precipitation followed by QAE-Sephadex-A50 chromatography. Kallikrein activity in 81 human urine samples and 8 samples obtained from a dog undergoing stop-flow procedure was measured by RIA and bioassay. Correlations of r = 0.81 and 0.94 were found. This RIA is useful for measuring kallikrein activity in rat, dog and human urine.
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