THE DEVELOPMENT OF AN ELUTED STAIN BIOASSAY (ESTA) FOR HUMAN GROWTH-HORMONE

The basic characteristics of MTT-formazan production by both quiescent Nb2 cells and those activated by fetal calf serum or human growth hor mone (hGH) are described. These characteristics are exploited for the development of an MTT-ESTA bioassay for purified preparations of lacto gens such as growth hormone. The resulting in vitro bioassay is sensit ive and precise, with a detection limit of about 0.05 mU hGH/l (19 ng/ l) and a within-assay imprecision of 2.5% in the presence of 0.3 mU hG H/l (114 ng/l). When utilizing quiescent Nb2 cells for bioassays, larg e magnitudes of response are observed. The major component of the resp onse is clearly derived from metabolic activation of the cells, rather than increased cell proliferation. The response was abolished by anti -human growth hormone. Delayed addition of the latter demonstrated tha t the presence of the hormone is required for the entire 96 h of the r ecommended bioassay incubation period to obtain the maximum response. At high doses, the dose-response relationship reaches a prolonged plat eau which covers 4 orders of magnitude of incremental hormone concentr ations. A decline in response is observed at the highest dose tested, 10(6) mU hGH/l (385 mg/l). This auto-inhibition is consistent with rec ent reports of a reduction in response due to stoichiometric blockade of sequential receptor dimerisation which is crucial for activation of both somatogenic and lactogenic receptors by hGH.