An Enzyme Immunoreceptor Assay for the Quantitation of Insulin-like Growth Factor-I and Insulin Receptors in Bovine Muscle Tissue

Abstract

To investigate the regulation of insulin-like growth factor-I (IGF-I) and insulin receptor in muscle, a sensitive enzyme immunoreceptor assay (EIRA), which allows for the determination of both affinity and capacity of the receptors, was developed. After solubilization with Triton X-100, receptors were immobilized in microtiter plates using receptor specific monoclonal antibodies that recognize the intracellular β-domain of the respective receptors (clone 17A3 and clone 29B4). The immobilized receptors were labeled with either biotinylated IGF-I or insulin. The bound hormones were detected with a streptavidin-horseradish peroxidase technique. The assay had a detection limit of 1 fmol receptor/well. The intraassay variation was 9% (n = 22) for the IGF-I receptor concentration and 12% (n = 22) for the insulin receptor. The interassay variation was 5% (n = 4) for the IGF-I receptor and 10% (n = 4) for the insulin receptor. The coefficient of variation of the dissociation constants (Kd) was 26% (n = 7) for the IGF-I receptor and 24% (n = 7) for the insulin receptor. The assay system was used to study the effect of growth hormone treatment upon IGF-I and insulin receptors in bovine skeletal muscle. Three groups of 12 heifers (13 months old) were treated with either 320 or 640 mg recombinant bovine somatotropin (slow release preparation) every fortnight for 3 months. When samples of m. splenius were assayed for IGF-I and insulin receptors, there was no difference between groups for receptor concentration or affinity. The amount of receptor (average of all groups) was 49 ± 11 fmol/mg protein (insulin) and 97 ± 18 fmol/mg protein (IGF-I). The average Kd obtained was 0.76 ± 0.22 nM (insulin) and 0.66 ± 0.18 nM (IGF-I).