Radioimmunoassay of cholecystokinin

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Abstract
A highly sensitive and specific radioimmunoassay for cholecystokinin (CCK) has been developed. Fully immunoreactive [125I]CCK33 was prepared by chloramine T-catalyzed iodination followed by purification by gel filtration and ion exchange chromatography. A high titer of antiserum was obtained by multiple immunizations of rabbits with 15% pure porcine CCK without conjugation. The antiserum was highly specific for CCK33 and CCK39, with 40% of the binding sites recognizing CCK8 at high affinity, but reacted weakly with gastrin. Plasma interference was eliminated by an XAD-2 resin column extraction technique with high recovery of CCK. The overall assay sensitivity was 3.3 pM with intra-and interassay coefficients of variation determined with a plasma of 11.2 pM at 9.6 and 20.8%, respectively. The assay was capable of detecting linear increments of both CCK33 and CCK8 added into plasma and intravenous infusion of CCK8 as low as 0.03 μg/kg/hr in dogs. The assay was validated by its ability to monitor increase of plasma CCK immunoreactivity after ingestion of a meat meal in both humans and dogs, as well as following intragastric infusion of liver extract meal and intraduodenal infusion of phenylalanine in dogs. When the CCK8 and gastrin binding sites of the antiserum were removed by immunoadsorption, the treated antiserum remained capable of measuring a postprandial change in plasma CCK concentration, indicating that CCK33-like immunoreactivity was present in the plasma.