Adaptation and Analytical Validation of a Radioimmunoassay for the Measurement of Human Cholecystokinin for Use in Dogs



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Cholecystokinin (CCK) is an important neuroendocrine peptide in the gastrointestinal tract, being the major stimulant for exocrine pancreatic secretion and gall bladder contraction. As such, cholecystokinin release may be altered in many gastrointestinal diseases. Assays for the measurement of cholecystokinin in humans have previously been developed and validated. However, to our knowledge, no assay for the measurement of CCK in dogs has been analytically validated. Thus, the objectives of this study were to adapt a radioimmunoassay used for the measurement of plasma CCK in humans for use in dogs, perform the assay without human reagents, and to analytically validate this modified immunoassay for use with canine serum.

A human cholecystokinin radioimmunoassay protocol and antiserum were generously provided to us by the laboratory of Jens Rehfeld, Copenhagen, Denmark. Assay runs were set up to replace all human reagents that are part of the original protocol, followed by analytical validation of the adapted assay using canine serum samples by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A reference interval for cholecystokinin in canine serum was established using 90 serum samples from clinically healthy, fasted dogs (12 hrs), using the bottom 97.5th percentile.

The sensitivity of the assay was calculated to be 0.5 pmol/L. The lower limit of the working range of the assay was taken as the sensitivity at 0.5 pmol/L. For dilutional parallelism, observed-to-expected ratios ranged from 101.9 % to 253.6 % for 3 different canine serum samples at dilutions of 1 in 2, 1 in 4, and, 1 in 8. For spiking recovery, observed to expected ratios ranged from 96.1 % to 68.2 % for 3 different canine serum samples at 4 different spiking concentrations. Coefficients of variation for intra-assay variability for 4 pooled serum samples were 3.8, 13.5, 7.9, and 3.9 %. Coefficients of variation for inter-assay variability of 4 pooled serum samples were 12.3, 11.6, 7.4, and 6.4 %. The reference interval for serum CCK concentration was established as 0.0 to 2.8 pmol/L.

All objectives outlined above were successfully met, analytically validating a radioimmunoassay for the measurement of cholecystokinin in canine serum. The radioimmunoassay for CCK described here is sufficiently accurate, precise, and reproducible, but has limited linearity in the lower end of the working range.