ソマトスタチンのradioimmunoassayに関する基礎的検討

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Fundamental study of a radioimmunoassay for somatostatin (growth hormone release inhibiting factor : GIF or GHRIF) is described in detail. The antiserum to somatostatin was generated by immunizing rabbits with somatostatin conjugated human serum globulin. Tyr1 -somatostatin was radioiodinated by the method of Hunter and Greenwood with the modification using bovine serum albumin (BSA), without sodium metabisulfite to avoid reduction of the disulfide bond and then the labelled hormone was separated from 125I-BSA and free 125I by passage through a column (0.8 X15 cm) packed with Sephadex G-25. The column was equilibrated and eluted with 0.1 N acetic acid, pH 3.0, containing 0.25% BSA. The elution pattern of radioactivity revealed three peaks. The first and the second peak contained 125I-BSA and free 125I respectively. Labelled tyr1 -somatostatin was absorbed to Sephadex G-25 and eluted broadly after free 125I (the third peak). An antiserm at final dilution of 1 : 4000 bound 35% of this labelled hormone. However, the labelled hormone was damaged progressively even when stored at -20°C in 0.1N acetic acid, pH 3.0. The separation of the free and the bound hormone was performed by double antibody method. Based on the results of studies of assay's conditions (buffer composition, antibody concentration, incubation time, incubation temperature), the assay was carried out as follows : To each reaction tube 0.5 ml diluent buffer (0.01 M phosphate buffer, 0.15 M NaCl, pH 7.6, containing 1% BSA), 0.1 ml standard somatostatin or samples to be tested, 0.1 ml 125I tyr1-somatostatin (approximately 10000 cpm), 0.1 ml anti-somatostatin serum (diluted 1 : 500) were mixed. After the first incubation at 4°C for 4 days, 0.1 ml anti-rabbit-γ-globulin goat serum (diluted 1 : 20) and 0.1 ml normal rabbit serum (1%) were added. After thd second incubation at 4°C for 1 or 2 days, the assay tubes were centrifuged at 3000 rpm for 30 minutes and the precipitates were counted. Various synthetic hypothalamic, highly purified hypophysiotropic and pancreatic hormones did not interfere with the radioimmunoassay for somatostatin. A dose-response curve was obtained in a range from 7.8 pg to 500 pg per tube of standard somatostatin in this assay system. A dilution curve of rat hypothalamic extract paralleled that of standard somatostatin. Intra-assay coefficient of variation was 4.92%. These results of radioimmunoassay for somatostatin indicate that the assay system is sensitive and fairly specific and useful to investigate the physiological roles of somatostatin on the regulatory mechanism of various hormones and enzymes.
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ソマトスタチンのradioimmunoassayに関する基礎的検討

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