RADIOIMMUNOASSAY OF SECRETIN IN ACIDIFIED PLASMA

Abstract

Antibody was readily produced in a rabbit against synthetic porcine secretin coupled to BSA [bovine serum albumin]. The final dilution of the antiserum to bind 50% of 1 f[femto]mol 125I-labeled secretin was 1:150,000. The effective equilibrium constant (Keff) according to Scatchard was 3.4 .times. 1011 l/mol, the average equilibrium constant (Ko) according to Sips 3.5 .times. 1011 l/mol, and the index of heterogeneity (.alpha.) according to Sips 1.00. No cross-reactivity was found for gastrin, glucagon and insulin. 125I-labeled synthetic porcine secretin was prepared by the Chloramine-T-method and the label purified on a Sephadex G-15 column. This was followed by a SP Sephadex C-25 column with a specific radioactivity of 1.150 .mu.Ci/nmol. This radioimmunoassy method has a detection limit of 1.6 pmol/ with 95% confidence limit, a within-assay precision of 9.6% and a between-assay precision of 12.8%. It allows detection of fasting plasma secretin in the low pmol/ range and the rather sharp rise ahd fall in plasma secretin subsequent to a brief period of duodenal acidification. The problems involved in measuring plasma secretin have been overcome by acidification of the plasma and by subtracting the apparent secretin concentration in corresponding secretin-free plasma prepared by incubation at 37.degree. C for 96 h for each subject.