Measurement of Plasma Renin Activity by Angiotensin I Radioimmunoassay: A Modification of Haber's Method

Abstract

Radioimmunoassay of angiotensin I and its appli-cation to the measurement of plasma renin activity (PRA) was studied. The assay method consists of the incubation of plasma at 37°C, pH 5.5 for 6 hours with disodium ethylenediamine tetraacetic acid (EDTA) and di-isopropyl fluorophosphate (DFP) and the radioimmunoassay of angiotensin I generated during the incubation. The values of PRA determined by the present method showed a good correlation with that of bioassay. We examined to find the ideal conditions in each step of this procedure and found its high specificity, sensitivity and reproducibility for routine clinical use. Materials and Methods: 1) Angiotensin I antibody Angiotensin I antiserum was supplied from Schwarz Mann which was produced by injection of copolymers of asparatyl-isoleucyl⁵-angiotensin I and succinylated poly-L-lysine in a rabbit according to Haber's method. 2) Preparation of ¹25I-angiotensin I Angiotensin I was iodinated according to the method of Hunter and Greenwood. Anion exchange resin was employed on the procedure of the purification of ¹25I-angiotensin I. 3) Angiotensin I standard solution Asparatyl-isoleucyl 5-angiotensin I supplied from the Institute for Protein Research of Osaka University was dissolved to the Concentration of 500 μg/ml with 0.1M acetic acid. This solution was used as a stock standard solution. 4) Blood sampling Blood was drawn in the morning under fasting and recumbent position. Plasma was separated by centrifugation and was stored at -20°C. 5) Incubation One milliliter of plasma was pipetted into a polyethylene tube and incubated with EDTA and DFP at 37°C, pH 5.5 for 6 hours. 6) Radioimmunoassay Tris acetate buffer 0.2M, pH 7.4 containing lysozyme 0.1%, EDTA 0.05% was used as diluent for all samples and reagents. All procedures were performed in polyethylene tube. Standard angiotensin I (10, 25, 50, 75, 100, 200, 300 and 500 pg) or 10μl of samples were mixed with 6, 000 cpm of ¹25I-angiotensin I and 50μl of dilute antiserum in 1ml of tris buffer. The mixtures were incubated at 4°C for 24 hours. After the incubation, dextran coated charcoal was added to each tube and then shaken for 20 minutes and centrifuged. The supernatant was discarded and the precipitate was counted in a well-type scintillation counter. The binding ratio of each standard solution was calculated and plotted on a semilogarithm paper (standard curve). The binding ratio was used as a quantitative indicator to estimate angiotensin I content in each sample.