DEVELOPMENT OF A SENSITIVE AND SPECIFIC ASSAY SYSTEM FOR CHOLECYSTOKININ TETRAPEPTIDE

Cholecystokinin is a gastrointestinal and neuropeptide which has been implicated in a wide range of physiological and behavioral processes. We have developed a sensitive and specific assay system to measure the various forms of cholecystokinin (CCK) in human plasma. This 3-step s ystem involves i) extraction of CCK fragments from plasma using revers e phase chromatography; ii) separation of peptides by high performance liquid chromatography; and iii) detection and quantification of pepti des with a double-antibody radioimmunoassay, using an antibody raised against cholecystokinin tetrapeptide (CCK-4) coupled to thyroglobulin and I-125 Bolton-Hunter CCK-4 as tracer. The antibody detects CCK-4, s ulfated CCK-8 (CCK-8s) and nonsulfated CCK-8 (CCK-sns) with equal affi nity. The lower limit of detection is 2.7 fmol, with an ED50 of 10.6 /- 2.2 fmol. Mean CCK-like immunoreactivity (CCK-LI) in the plasma of 12 healthy subjects was determined to be 12.9 +/- 2.1 pM CCK-4 equival ents. Concentrations of each individual peptide in plasma were determi ned to be 1.0 +/- 0.2 pM, 3.4 +/- 0.8 pM and 1.9 +/- 0.4 pM for CCK-4, CCK-8s and CCK-8ns respectively. (C) 1997 Elsevier Science Inc.