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 hormone (hGH) are described. These characteristics are exploited for the development of an MTT-ESTA bioassay for purified preparations of lactogens such as growth hormone. The resulting in vitro bioassay is sensitive 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 hGH/l (114 ng/l). When utilizing quiescent Nb2 cells for bioassays, large magnitudes of response are observed. The major component of the response 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 that the presence of the hormone is required for the entire 96 h of the recommended bioassay incubation period to obtain the maximum response. At high doses, the dose-response relationship reaches a prolonged plateau which covers 4 orders of magnitude of incremental hormone concentrations. 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 recent 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.