Development of cholecystokinin radioimmunoassay using synthetic CCK-10 as immunogen

Using an antiserum generated against synthetic CCK-10, we have developed a radioimmunoassay specific for the carboxyl-terminus of cholecystokinin (CCK). Three rabbits were immunized with synthetic sulfated carboxy-terminal CCK decapeptide (CCK-10) conjugated to keyhole limpet hemocyanin. Using 125I-CCK-39 prepared by the Iodogen method as a tracer, we found that all immunized rabbits produced antibodies against the conjugate. Antiserum R016 had the highest titer (1:225,000 after four immunizations) and was studied most extensively. R016 recognizes all molecular forms of CCK, including unsulfated and oxidized forms, but has negligible cross-reactivity with gastrin and other peptides. Using CCK-8 as a standard, the assay has a minimum detection limit of 0.5 pM and an ED50 of 11.5 pM. Serial dilutions of water/acid extracts of canine intestine were parallel to serial dilutions of sulfated CCK-8, CCK-33 and CCK-39. The assay was used to measure CCK concentrations in canine plasma after C18 Sep-Pak extraction; the concentration of immunoreactive CCK increased from a basal value of 7.8 +/- 1.0 to 9.5 +/- 1.2 and 11.1 +/- 1.2 pM 30 and 60 min postprandially (P less than 0.05 by paired analysis). This sensitive and uniquely specific CCK radioimmunoassay should be useful in characterizing several aspects of CCK physiology and the method for generating CCK antisera should be of value to other investigators.