High molecular somatostatin. A possible interfering factor in radioimmunoassay

Cyclic Tyr1-somatostatin (Tyr1-somatotropin release inhibiting factor, Tyr1-SRIF) is radioiodinated by the lactoperoxidase method. Purification is achieved by Sephadex G-25 adsorption chromatography. Specific anti-SRIF serum (FA1) has been raised in rabbits. A dose-response curve is obtained in the range of 5-5000pg per tube using an antiserum dilution of 1:2000. There is little cross-reaction with linear somatostatin and none with oxytocin, (Lys-, Arg-) vasopressin, valinomycin, polymyxin, insulin, glucagon, hGH, LH-RH and TRH, each at a concentration of 10ng/ml. The percentage of non-specific binding is 17% for a radioimmunoassay (RIA) incubation time of 18h at 40C. Bound and free antigen are separated by the charcoal technique. With the radioimmunoassay described, no native cyclic SRIF could be measured in human plasma. For recovery tests, extraction procedures are necessary. Thin-layer chromatography (TLC) and polyacrylamide disc electrophoresis (disc-PAGE) are performed to identify the presumed high molecular 125I-Tyr1-SRIF associate. This high molecular associate may represent an interfering factor in the radioimmunoassay for cyclic SRIF. The following observations provide experimental evidence for this assumption: 1. The first elution peak ''V0'' (mol.wt.>5000) of the Sephadex G-25 adsorption chromatography increases with the storage time of the labelled Tyr1-SRIF. 2. In the radioimmunoassay the non-specific binding value increases from 17% to >30%(B/T) after a longer period of incubation. 3. TLC of ''V0'' and of the 125I-Tyr1-SRIF fractions from the ''retarded'' peak of the G-25 chromatography shows similar Rsub(f)-values in acidic elution systems. 4. Disc-PAGE of 125I-Tyr1-SRIF demonstrates radioactivity in gel fractions corresponding to an Rsub(f) value of 0.35. This radioactivity can be extracted with acidic solvents. (author)