Preparation of a homogeneous extract of human growth hormone, isohormone B, and its 125I-labelling for utilization in radioligand assays

Non-destructive polyacrylamide gel electrophoretic (PAGE) tecnique, with direct UV-densitometry, was set up to permit both qualitative and quantitative studies of human growth hormone (hGH) isohormone purification is presented. This tecnique was used on a preparative scale to obtain milligram amounts of the fundamental form of hGH, isohormone B(Ih-B). Reversed electrophoresis was employed to elute the protein band form the gel. Retention of bio-and immunoactivity was demonstrated via two separate experiments. An 'in vivo' bioassay, based on the weight increase of hypophysectomized rats with a 2 x 2 factorial assay design, was used to compare the true somatotrophic activity of an hGH preparation submitted to the purification process with that of a single control preparation. Retention of immunoactivity was confirmed by studying the antibody binding properties of purified and radioiodinated Ih-B and by determination of its absolute immunopotency against a calibrated secondary standard. Radioimmuno assay curves, determined using Ih-B, as a standard and labelled preparation, showed its applicability in setting up assays based on more homogeneous reagents. (Author)