Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

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Abstract
A sensitive and specific enzyme‐linked immunosorbent assay (ELISA) for Cholecystokinin octapeptide sulfate (CCK‐8S) has been developed using N‐terminal specific antibody for CCK‐8S. In this assay CCK‐8S coupled with poly‐L‐Glu (CCK‐poly‐Glu), which is adsorbed on a solid phase, competes with CCK‐8S for the binding sites of rabbit anti‐CCK antibody, and the complex of the immobilized antibody and CCK‐poly‐Glu is measured using goat anti‐rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is <24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2‐6.2% and the interassay coefficient of variation is 5.9–8.5%. This assay is sensitive enough to detect 9 pg of CCK‐8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6‐hydroxy‐dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.