A Direct Radioimmunoassay for Human Urinary Kallikrein

Abstract

A protein-binding radioimmunoassay (RIA) has been developed for human urinary kallikrein (HUK). HUK was purified to apparent homogeneity and rabbit anti-human urinary kallikrein serum was prepared. Radio-iodination of HUK was performed in 20 µg batches with 1 mCi of ¹²⁵I-Bolton-Hunter reagent in 0.1 M sodium borate buffer, pH 8.5, at 0°C. The 30 min reaction was terminated by adding 0.5 ml of 0.2 M glycine. Radiolabelled HUK was purified on a Sephacryl S-200 column (1 × 50 cm) in 0.05 M Tris HC1 plus 0.2% gelatin, pH 7.4 (RIA buffer). ¹²⁵I-HUK emerged as a single, uniform peak at V_e/V_o of 1.3. The RIA reaction mixture contained 15,000 cpm of ¹²⁵I-HUK, 0.1 ml of diluted anti-kallikrein serum, 0.1 ml unlabelled kallikrein (1–100 ng) or the unknown sample and RIA buffer to a total of 0.5 ml. The reaction mixture was incubated at 22°C for 18 hr, and 100 µl of goat-anti-rabbit IgG serum was added to each tube. The tubes were incubated 18 hr at 4°C, centrifuged 4,000 rpm for 30 min and, after decantation, the precipitate was counted for ¹²⁵I. The RIA reported here is sufficiently sensitive to detect 1 ng of kallikrein and should offer a useful technique to determine alterations of the kallikrein-kinin system in response to stimuli; both physiologic and pathologic.