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Measurement of growth hormone bioactivity in human serum using a lactogenic Eluted Stain Bioassay (ESTA)

Dattani, Mehul Tulsidas; (1995) Measurement of growth hormone bioactivity in human serum using a lactogenic Eluted Stain Bioassay (ESTA). Doctoral thesis (M.D), UCL (University College London). Green open access

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Abstract

In 1988, Ealey et al. described an in vitro Eluted Stain Assay (ESTA) for the measurement of the lactogenic bioactivity of human growth hormone (hGH) and human prolactin (hPRL). The assay is based upon the use of Nb2 rat lymphoma cells which express lactogenic receptors responding to both hGH and hPRL. It utilises the colorimetric reduction of a yellow tetrazolium salt, MTT, to its purple formazan by activated Nb2 cells. The assay is highly sensitive, quantitative and precise. Using this bioassay, I have investigated the in vitro effects of 1) a recombinant human growth hormone binding protein (rhGHBP) and 2) a metallic ion (zinc; Zn2+) on the bioactivity of hGH. I have also adapted the assay for the measurement of the lactogenic bioactivity of hGH in human serum and subsequently used the bioassay to assess hGH bioactivity in a number of different clinical settings. Recombinant hGHBP, which has a similar affinity and binding capacity to the naturally-occurring high affinity hGHBP, inhibited the in vitro bioactivity of 22kDa hGH in a dose-dependent manner. This inhibition was observed with physiological concentrations of the hGHBP and was competitive and specific. My data suggests that simple Michaelis-Menton kinetics do not apply to the interaction between hGH and its receptor, as represented by hGHBP which is identical to the extracellular domain of the somatogenic GH receptor. This is therefore consistent with the recent model of hGH interacting with two hGH receptors in order to achieve its biological effects. The 20kDa alternative splice variant of hGH exhibited approximately 10% of the potency of 22kDa hGH in the ESTA bioassay. Its bioactivity was inhibited by hGHBP in a similar fashion to that of hGH. The concentrations of hGHBP required to inhibit hGH bioactivity by 50% (IC50) were similar for both 20 and 22 kDa hGH, suggesting that the binding affinity of 20kDa for somatogenic GH receptors may be comparable to that of 22kDa hGH. This challenged previous findings suggesting that hGHBP does not interact with 20kDa hGH, and hence the extracellular domain of the somatogenic receptor. Additionally, zinc (Zn2+) led to a modest potentiation of 22kDa hGH bioactivity. This was at variance with previous binding studies which had suggested an 8000-fold increase in the binding affinity of hGH for the lactogenic receptor. However 50μM Zn2+ led to a much greater potentiation of 20kDa hGH bioactivity. This marked potentiation was also observed at physiological concentrations of the metallic ion. This discrimination had not been previously reported. In order to adapt the assay for the measurement of serum GH bioactivity, (i) nonspecific "blank" effects of serum were overcome by a 160-fold dilution of sera and, (ii) hormonal specificity achieved by using a monoclonal anti-serum to hPRL. The bioassay generally agreed well with GH determinations made with the Hybritech immunoradiometric assay (IRMA) when applied in a variety of clinical situations eg. acromegalic patients, Laron- type dwarfism and constitutional delay of growth and puberty. However, differences were observed between the bioassay and the IRMA during provocative tests of GH secretion. These discrepancies were particularly pronounced at the peaks of GH secretion. The additional bioactivity was labile on freezing and storage, when the discrepancy between the two assays disappeared. Hence this additional bioactivity may be due to labile isoforms of hGH which may also be bioactive, but not detected in the specific Hybritech IRMA.

Type: Thesis (Doctoral)
Qualification: M.D
Title: Measurement of growth hormone bioactivity in human serum using a lactogenic Eluted Stain Bioassay (ESTA)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Bioactivity; Eluted Stain Bioassay; Hormone; Human serum; Lactogenic
URI: https://discovery.ucl.ac.uk/id/eprint/10099758
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