Direct Radioimmunoassay of Rat Urinary Kallikrein: Its Application to the Determination of Active and Inactive Kallikrein Concentration after HPLC Analysis

Abstract

Rat urinary kallikrein (RUK) was purified to apparent homogeneity by a three-step procedure and antibodies were raised in rabbits. Renal kallikrein exists as an active and an inactive form. A specific radioimmunoassay (RIA) was developped to measure directly the total kallikrein. The antibody used in this radioimmunoassay recognized both forms. No cross reactivity was detected with trypsin, esterase A or human urine. When iodinated rat urinary kallikrein was used, the detection range was between 0.125 and 16 ng with 6.5% intraassay variation and 8.1% between assay variation. Intrarenal kallikrein was measured in renal tissue after homogeneisation and solubilisation. Correlations between this RIA and the kininogenase activity or the amidolytic activity were highly significant. Since kallikrein exists as an inactive precursor the direct measurement of the total immunoreactive protein differs from activities determinations. An HPLC ion exchange system has been developped to separate active and inactive forms directly from urine, with a recovery of 79 ± 11%. This procedure permits measurement of inactive forms. Rat urine contains as much inactive kallikrein as active kallikrein.