The quantitation of human growth hormone by a radioreceptor assay using an established human cell line.
Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The assay should fulfil established pharmacopoeial requirements for quantitation of hormones. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 X 10(7) cells ml-1 were incubated with 5-20 X 10(-12) M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations.
D000818:Animals; D001835:Body Weight / Q000187:drug effects; D002460:Cell Line; D005260:Female; D013006:Growth Hormone / Q000032:analysis / Q000378:metabolism / Q000592:standards*; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D011485:Protein Binding; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature; D013997:Time Factors
D013006:Growth Hormone