An Improved Radioreceptor Assay for Human Growth-hormone Using An Hplc-purified I-125-hgh

Human growth hormone (hGH) was labelled with 125Iodine to a specific activity of 50-80 μCi/μg by a stoichiometric modification of the chloramine-T method. After labelling, the crude reaction mixture containing 125I-hGH, unreacted iodide, chloramine-T, salts, etc. was purified by reverse-phase high performance liquid chromatography (HPLC) using a Synchropak RP-P column (Altech, USA) and a linear gradient. Biological activity of the purified material was measured by binding to IM-9 lymphocyte hGH receptors. The results were compared with those obtained after purification by gel filtration using a sephadex G-100 column. HPLC gave a much better separation than sephadex chromatography. The improved resolution was the result of an increase both in selectivity and efficiency. The analysis time was considerably reduced by HPLC (30 min vs 5 h). The recovery was comparable in both methods (95%). Specific binding of 125I-hGH to lymphocyte membrane receptors was higher by 20 % with the preparation purified by HPLC than with the sephadex material. The maximum bindability of the hormone by an excess of lymphocyte receptors was 86 % for the HPLC-purified 125I-hGH and 71 % for the sephadex-purified 125I-hGH. Both preparations behaved similarly in kinetic experiments. It is concluded that HPLC is a fast, convenient and reproducible method for purifying 125I-hGH for receptor studies.