EVALUATION OF INTERFERENCE BY INSULIN-LIKE GROWTH FACTOR-I (IGF-I) BINDING-PROTEINS IN A RADIOIMMUNOASSAY FOR IGF-I IN SERUM FROM DAIRY-COWS

Insulin-like growth factor I (IGF-I) circulates in serum bound to a number of different binding proteins (BPs). With antibodies currently available, BPs must be dissociated and inactivated or removed from serum prior to measurement of IGF-I by radioimmunoassay (RIA). Serum samples which spanned a 13-fold range in IGF-I concentration were obtained from lactating dairy cows and used to develop conditions for assay of IGF-I with minimal interference from BPs. Removal of BPs from serum by acid-ethanol extraction resulted in interference in the RIA. Therefore, serum was incubated with 0.1 M glycyl-glycine HCl to inactivate BPs as suggested by Underwood et al. (11). Time, temperature and pH were optimum when serum was incubated for 48 hr at 37 C, pH 3.7. Binding protein inactivation was evaluated by ability of glycyl-glycine incubated serum to reassociate with I-125-IGF-I. In addition, BPs isolated by gel filtration of glycyl-glycine incubated serum were tested for interference in the RIA. The concentration of IGF-I in serum where inactivated BPs were removed by acid gel filtration was compared to corresponding glycyl-glycine incubated serum. There was a 1:1 relationship which intersected at zero indicating that total IGF-I could be measured. Therefore, incubation of serum with glycyl-glycine is a reliable method for measuring total IGF-I in serum from dairy cows.