MEASUREMENT OF AN ANALOG OF INSULIN-LIKE GROWTH-FACTOR-I IN BLOOD-PLASMA USING A NOVEL ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Long-Arg(3)-IGF-I (LR(3)IGF-I) is a synthetic analog of IGF-I that has much lower affinity for IGF-binding proteins than do native IGFs-I an d -II. Comparisons of the effects of LR(3)IGF-I with those of IGFs-I a nd -II in in vitro and in vivo studies have proved useful in defining the functions of IGF-binding proteins. We have developed a sensitive n oncompetitive nonisotopic assay of LR(3)IGF-I. Mouse IgG 1A7-F5-E5 bin ds an epitope that contains the substituted arginine(3) in LR(3)IGF-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 c ommon to IGF-I and LR(3)IGF-I. The ELISA system was able to detect as little as 50 pg LR(3)IGF-I in 100 mu l and the native peptides IGFs-I and -II have less than 0.01% activity. Blood plasma from animals treat ed with pharmacologically active doses of this growth factor analog co uld be diluted 33.3-fold before assay, at which concentrations plasma had no significant effect on the assay. The ELISA response to LR(3)IGF -I was unaffected by the presence of IGF-binding proteins. The intra-a ssay and interassay coefficients of variation are 2.8 and 7.3% respect ively. Recovery of LR(3)IGF-I added to blood plasma was similar to 90% . The ELISA was used to measure LR(3)IGF-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 establi shed LR(3)IGF-I RIA that requires size exclusion chromatography of pla sma under acidic conditions to eliminate IGF-binding protein artefacts from the RIA. There was a positive correlation between results obtain ed by the two assays. The LR(3)IGF-I ELISA permits discrimination betw een the exogenous synthetic IGF-I analog and the endogenous native IGF s-I and -II in animals treated with this growth factor without tile ne ed for radio-iodination of LR(3)IGF-I and elimination of the requireme nt for extraction of plasma before assay.