Abstract
The establishment of radioimmunoassay for somatostatin is important for the assessment of its role in terms of humoral regulation, especially in the hypothalamo-pituitary relationship. The anti-somatostatin was prepared by the immunization of rabbits with somatostatin-HSG (α) conjugate. The antibody had no cross-reactivity with TRH, LHRH, pituitary hormones, insulin, C-peptide, or glucagon, but was cross-reactive with Des (Ala1, Gly2)-somatostatin, somatostatinamide, or Tyr1-somatostatin. 125I-Tyr1-somatostatin was prepared by the lactoperoxidase method, and purified through a carboxymethylcellulose column chromatography. The assay procedure was performed by incubation in 0.1 M phosphate buffer with 0.14 M NaCl, 0.025 M EDTA-2Na, 0.5% BSA (pH 7.4) at 4°C. The free and bound form were separated with polyethylene glycol. The double antibody technique and the dextran-coated charcoal technique were also employed for the separation of free and bound forms, but we prefer the former technique because of easy handling and cheap costs. The concentrations of immunoreactive somatostatin detected by this assay were 0.6-50 ng/ml.
Immunoreactive somatostatin in the tissue was extracted by aceton. The dilution curve of immunoreactive somatostatin extracted from the rat brain was paralleled to the standard curve gained by synthetic somatostatin. The recovery rate was approximately more than 50%. The extracts from the rat brain were identified by synthetic somatostatin on Sephadex G-25 column chromatography.
These data indicated this assay system might be useful for research applications concerning immunoreactive somatostatin in the tissues.
