A Cell-based Potency assay for Insulin-like Growth Factor-I

Abstract

The authors developed a cell-based bioassay for determining the potency of recombinant human insulin-like growth factor I (IGF-I) using HU-3 human megakaryoblastic cell line. Cell proliferation was measured using the alamarBlue™fluorescence method. The addition of IGF-I resulted in a dose-dependent growth response after 48 hours under serum-free conditions. The effective range was 0·1–25 ng/ml with half-maximal response at approximately 2 ng/ml IGF-I. The assay is simple, requiring just three steps, performed in 96-well microtitre plates and is able to detect changes in activity of truncated analogues of IGF-I (such as des–Gly–IGF-I, des–Gly–Pro–IGF-I and des–Gly–Pro–Glu–IGF-I) as well as IGF-I samples that had been subjected to proteolytic or disulfide reduction treatments. This assay is precise, with interassay variability of less than 10% and accurate, with percentage recoveries of nearly 100%. The relative efficacies of other insulin-related peptides in stimulating cell growth of the cell line were examined. IGF-II was 5-fold less potent than IGF-I and insulin had little or no proliferative activity. In addition, the growth-promoting activity correlated well with IGF-I stimulation of glucose consumption in this system. In conclusion, the HU-3 human megakaryoblastic cell line constitutes a simple system for measuring the biological activity of recombinant IGF-I in quality control set-up. The safety, convenience and precision of the assay make it an attractive alternative to radioactive and other colorimetric methods.