Radioimmunoassay of cholecystokinin: production and evaluation of antibodies.

In order to produce antibodies for the radioimmunoassay of cholecystokinin the following animals were immunized: 10 rabbits and 3 guinea pigs with cholecystokinin 1—33 coupled to bovine serum albumin, 9 rabbits and 2 guinea pigs with uncoupled cholecystokinin 1-33, 5 rabbits with cholecystokinin 10-20 coupled to bovine serum albumin and 4 rabbits with sulphated cholecystokinin 26-33 coupled to bovine serum albumin. The titer, the binding energy and the specificity of the antibodies were evaluated. Cholecystokinin 1—33, coupled to [I]hydroxyphenylpropionic acid-succinimide ester (Bolton-Hunter reagent) to a specific activity of 8.1-14.7 MBq/^g (219—398 Ci/ g) (n = 8), was used äs label. After the fourth immunization 5 rabbits immunized with albumin coupled cholecystokinin 1—33 had antibody titers between 1:22500 and 1:80000, while one rabbit immunized with uncoupled cholecystokinin 1-33 had a titer of 1:52000. The affinity constant of these 6 antibodies ranged from K = 2.0 x 10 to 72.3 x 10 1/mol. One antibody was specific for all sulphated forms of cholecystokinin, while the other 5 antibodies were specific for the large molecular forms of cholecystokinin. In the guinea pigs immunized with cholecystokinin 1—33 and in the rabbits immunized with cholecystokinin fragments antibody titers were less than 1:500. Concentrations of cholecystokinin in an aqueous-acid extract of human and hog upper small intestine were 48.7 ± 6.7 pmol/g and 46.2 ± 3.4 pmol/g, when measured with antibody T204 specific for sulphated forms of cholecystokinin, and 49.7 ± 1.9 pmol/g and 39.2 ±1.6 pmol/g, when measured with antibody 1703 specific for large forms of cholecystokinin. After ingestion of 60 ml corn oil in 6 normal subjects, plasma cholecystokinin increased from 2.7 ± 0.5 pmol/1 to 6.8 ±1.7 pmol/1 (p < 0.05) when measured with antibody T204, and from 1.0 ± 0.3 pmol/1 to 5.7 ±1.0 pmol/1 (p < 0.05) when measured with antibody 1703.