A cell-based potency assay for insulin-like growth factor-I.
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.
Gazzano-Santoro
H
H
Genentech Inc., South San Francisco, CA 94080, USA.
Chen
A
A
Mukku
V
V
D001681:Biological Assay / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008533:Megakaryocytes / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013678:Technology, Pharmaceutical
D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D005947:Glucose