A CELL-BASED POTENCY ASSAY FOR INSULIN-LIKE GROWTH-FACTOR-I

The authors developed a cell-based bioassay for determining the potenc y of recombinant human insulin-like growth factor I (IGF-I) using HU-3 human megakaryoblastic cell line. Cell proliferation was measured usi ng the alamarBlue(TM) fluorescence method. The addition of IGF-I resul ted in a dose-dependent growth response after 48 hours under serum-fre e conditions. The effective range was 0.1-25 ng/ml with half-maximal r esponse 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-IG F-I and des-Gly-Pro-Glu-IGF-I) as well as IGF-I samples that had been subjected to proteolytic or disulfide r eduction treatments. This assay is precise, with interassay variabilit y of less than 10% and accurate, with percentage recoveries of nearly 100%. The relative efficacies of other insulin-related peptides in sti mulating 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 act ivity. In addition, the growth-promoting activity correlated well with IGF-I stimulation of glucose consumption in this system. In conclusio n, the HU-3 human megakaryoblastic cell line constitutes a simple syst em for measuring the biological activity of recombinant IGF-I in quali ty control set-up. The safety, convenience and precision oi the assay make ii an attractive alternative to radioactive and other colorimetri c methods. (C) 1998 The International Association of Biological Standa rdization.