Human urinary kallikrein (HUK): large-scale purification and direct solid-phase radioimmunoassay (RIA)

HUK was purified from 1,000 liters of fresh urine by the following procedures: silica gel adsorption, gel filtration on Sephadex G-75, DEAE-Sephadex chromatography, bentonite treatment, affinity chromatography on aprotinin-Sepharose 4B, and rapid gel filtration on a TSK Gel G-3000 SWG column. Seventeen mg of HUK being found to be pure by means of various analyses was obtained. The pI values of the heterogeneous components of HUK were 3.5, 3.8, and 4.1, while the corresponding molecular weights of these components were 5.4 X 10(4), 4.9 X 10(4), and 4.4 X 10(4), respectively. The antigens (125I- and non-labeled HUK) were incubated for 4 hrs at 37 degrees C in polystyrene test tubes to which anti-HUK rabbit IgG had been immobilized. The quantitative range of the standard curve was 1-128 ng. This Ria principally recognized active form of HUK. Therefore, total and inactive HUK also could be determined by the combination of the RIA with trypsin treatment of the urine sample. The RIA correlated closely with both S-2266 amidolytic assay and kininogenase assay.