Measurement of an analog of insulin-like growth factor-I in blood plasma using a novel enzyme-linked immunosorbent assay

Abstract

Long-Arg³-IGF-I (LR³IGF-I) is a synthetic analog of IGF-I that has much lower affinity for IGF-binding proteins than do native IGFs-I and -II. Comparisons of the effects of LR³IGF-I with those of IGFs-I and -II in in vitro and in vivo studies have proved useful in defining the functions of IGF-binding proteins. We have developed a sensitive noncompetitive nonisotopic assay of LR³IGF-I. Mouse IgG 1A7–F5–E5 binds an epitope that contains the substituted arginine³ in LR³IGF-I and was used as the solid phase antibody. The solution phase antibody was a rabbit immunoglobulin Nelson which binds to an epitope that is common to IGF-I and LR³IGF-I. The ELISA system was able to detect as little as 50 pg LR³IGF-I in 100 μl and the native peptides IGFs-I and -II have less than 0•01% activity. Blood plasma from animals treated with pharmacologically active doses of this growth factor analog could be diluted 33•3-fold before assay, at which concentrations plasma had no significant effect on the assay. The ELISA response to LR³IGF-I was unaffected by the presence of IGF-binding proteins. The intra-assay and interassay coefficients of variation are 2•8 and 7•3% respectively. Recovery of LR³IGF-I added to blood plasma was 90%. The ELISA was used to measure LR³IGF-I concentrations in plasma of cows treated with a pharmacologically active dose of this peptide and the results were compared with those obtained by a previously established LR3IGF-I RIA that requires size exclusion chromatography of plasma under acidic conditions to eliminate IGF-binding protein artefacts from the RIA. There was a positive correlation between results obtained by the two assays. The LR³IGF-I ELISA permits discrimination between the exogenous synthetic IGF-I analog and the endogenous native IGFs-I and -II in animals treated with this growth factor without the need for radioiodination of LR³IGF-I and elimination of the requirement for extraction of plasma before assay.