125I- [Tyr8] -Somatostatinを標識抗原とするSomatostatinのRadioimmunoassayの基礎的検討

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Somatostatin analogs, which have been used as tracers in various somatostatin (GIF) radioimmunoassay systems such as Nα-tyrosyl-, [Tyr1] - and [Tyr11] -GIF, contain tryptophan at position eight in the structure. These peptides tend to be subject to oxidative degradation during the radioiodination procedure and labile under storage conditions. In the present study, we therefore developed a radioimmunoassay for GIF using 125I- [Tyr8] - GIF as a labelled antigen which was a far more stable tracer. [Tyr8] -GIF was radioiodinated by the lactoperoxidase method. The 125I- [Tyr8] -GIF was purified on a CMC column chromatogram (1 × 8cm) by eluating either with the stepwise method of 0.002M (19ml) - 0.2M ammonium acetate, pH 4.6, or with the linear gradient method of 0.05M (50ml) -0.25M (50ml) ammonium acetate, pH 4.6. Each 2ml fraction was collected in polystyrene tubes. Immunoreactive 125I- [Tyr8 J -GIF existed in the] _last peak under both elution conditions. The fractions containing 125I- [Tyr8]-GIF in the ammonium acetate buffer were stored in the polystyrene tubes at -20°C until used. Under these conditions, 125I_ [Tyr8] -GIF was completely adsorbed on the surface of the polystyrene tube, while free 125I remained in the ammonium acetate buffer. Immediately before use, the stock solution was thawed, and the ammonium acetate buffer was discarded by eliminating free 125I which had been liberated from the tracer during storage. Then, 1.0ml of buffer (0.1% gelatin-0.2% BSA-0.025M EDTA-0.15M NaC1-0.01M phophate, pH 7.4 : GBPBS) was added to the tube, which was vigorously shaken for 5 min with a ThermoMixer. Eighty to 90% of 125I- [Tyr8] -GIF attached to the wall was recovered in GBPBS by this procedure. The repurification on the CMC column chromatogram of the recovered tracer in GBPBS after storage of one month revealed that 88.2% of applied radioactivity was eluated at the same position of 125I- [Tyr8] -GIF. An autoradiogram of enzymatic hydrolysates of 125I- [Tyr8] -GIF showed that the labelled amino acid was 3-iodotyrosine. To elucidate the biological activity of iodinated [Tyr8]-GIF, [Tyr8]-GIF was iodinated with KI by the lactoperoxidase method. On the CMC column chromatogram, the products were divided into three fractions designated as Fr.I, Fr.II and Frill. Fr.II was identified as [3-iodotyrosine8] -GIF by comparison with the elution position of 125I- [Tyr8] -GIF on the CMC column chromatogram. The in vivo potency of Fr.II to inhibit the release of TSH stimulated by TRH was 14.9% of GIF, indicating that the biological activity of iodinated [Tyr8] -GIF was markedly increased as compared with that of [Tyr8] -GIF (less than 0.5% of GIF). Specific antiserum R 501 for GIF at the final dilution of 1 : 2,500 bound 39.3% of 125I- [Tyr8] -GIF. The binding of the tracer to the antibody reached near the maximal level 19 hours after incubation at 4°C. For the separation of the free and bound tracers, the optimal incubation time with dextran coated charcoal was 15 minutes at 4°C. In the assay, glass tubes were better than polystyrene tubes because they lowered the non-specific binding of the free tracer. Based on the above results, we performed the assay as follows : 0.2ml GBPBS, 0.1ml standard or sample solution, 0.1ml 125I- [Tyr8] -GIF (7,000-10,000cpm) and 0.1ml antiserum R 501 (1 : 500) were combined in glass tubes. After the first incubation at 4°C for 48 hours, the free and bound tracers were separated by the dextran coated charcoal method or the double antibody method.
日本内分泌学会の論文

<SUP>125</SUP>I- [Tyr<SUP>8</SUP>] -Somatostatinを標識抗原とするSomatostatinのRadioimmunoassayの基礎的検討

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