The measurement of growth hormone bioactivity in patient serum using an eluted stain assay

The Journal of Clinical Endocrinology and Metabolism
M T DattaniN J Marshall

Abstract

We have adapted the MTT-ESTA bioassay for human GH (hGH) to measure the lactogenic bioactivity of the hormone in human serum. This highly quantitative in vitro colorimetric bioassay is based upon the reduction of a tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), to its formazan by lactogen-activated Nb2 cells. Relatively high concentrations of human serum (1-10%) modified responses to the hormone in a complex manner. As the serum effects varied between samples, it proved impossible to adapt the bioassay by the conventional approach of using a lactogen-depleted serum as a representative matrix. However, as the Nb2 cells were exceptionally sensitive to hGH, the serum effects could be diluted out. We adopted a dilution strategy by which all samples of human serum were included in the bioassay at a concentration of 0.625% or less. A valid assay was obtained, as judged by the criteria of parallelism between diluted samples and hGH standards, and recoveries of spiked samples that were close to 100%. Hormonal specificity was achieved with the use of a highly specific anti-PRL antiserum. A within-assay precision of between 2-5% over the dose range of 0.03-0.96 microgram hGH/L was attained. As only...Continue Reading

Citations

Feb 11, 1999·Journal of Endocrinological Investigation·M BozzolaP Chatelain

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