Characterization of an Antiserum Used in a Radioimmunoassay for Arginine-Vasopressin: Implications for Reference Standards*

Abstract

The region of the arginine-vasopressin (AVP) molecule critical for binding to the effective antibodies in a RIA [radioimmunoassay] was localized to the vicinity of the Phe3 position by using the cross-reaction in the assay between AVP and a number of its structural analogs. Binding was almost independent of any direct contributions from components of the tripeptide tail of AVP. Using this RIA disequilibrium conditions of incubation produce a 5-fold increase in assay sensitivity over equilibrium conditions. Amino acid analysis revealed that 3 synthetic peptide preparations used as reference standards comprised only 70-80% of their weight as peptide. This finding indicates the need to correct such reference standards for their peptide content. The ratio of rat vasopressor activity to RIA activity of these 3 preparations as well as of a natural AVP preparation approximated unity. Results obtained comparing measurements of AVP in rat neural lobes by RIA and rat vasopressor assay show a correlation between RIA and bioassay of 0.9406, a slope of 1.086, and an intercept of 20 mU [units], suggesting good agreement for AVP determined by these 2 assay systems.