Establishment of a radioimmunoassay of somatostatin in human plasma and in rat hypothalamus.

ABSTRACT

Synthetic cyclic somatostatin-14 (SRIF-14) was conjugated with carbodiimide onto hemocyanin to immunize rabbits, and anti-sera with high titers (15,000-100,000) and good sensitivity and specificity were obtained. 125I-Tyr1-SRIF was made and purified on CM-52 cation exchange column. The immunoreactive specific activity (SP.AC.) of the tracer was around 1013 mCi/mg, which was much higher than the commercial products and other reports. The sensitivity of the assay was around 16 pg/ml, and the detectable range 20-2000 pg/ml. Human plasma was freshly prepared in the presence of aprotinin. Rat hypothalamus was homogenized and extracted with HC1 followed by heating at 95 degrees C then centrifuged, and the supernatant assayed. The standard curves of the acetate buffer (ph 5.2), plasma doses, plasma recovery doses, hypothalamic extract doses, and hypothalamic extract recovery doses were all in good parallelism with each other. The recovery of SRIF from human plasma and from rat hypothalamus were 98.8 +/- 6.3% and 86.9 +/- 6.8%, respectively. The specificity of the antisera were very high, and they only cross-react with H2-SRIF and Tyr1-SRIF at 15% and 170%, respectively, of the immunoreactivity to cyclic SRIF-14. The Scatchard plot of the binding data showed a straight line with a Kd of 3.52 X 10(-12)M and binding capacity of 4.06 X 10(-10)M. The intra- and inter-assay coefficient of variation were 4.5% and 12%, respectively. When the synthetic cyclic SRIF (Stilamin) was infused intravenously into normal volunteers, there were reproducible plasma time-dose curves of SRIF-LI (SRIF Like Immunoreactivity), which revealed its plasma half life around 1.5 minutes, and metabolic clearance rate about 50 ml/kg/min.