HIGHLY SENSITIVE NONISOTOPIC IMMUNOASSAYS FOR CHOLECYSTOKININ USING VARIOUS DETECTION METHODS

We have developed sensitive, non-isotopic immunoassays for cholecystok inin (CCK) by using colorimetric, time-resolved fluorometric and chemi luminometric detection. The assay is based on competition between a fr ee CCK and biotinylated CCK-33 as tracer for rabbit anti-CCK-8 antibod ies on a goat anti-rabbit IgG antibody coated microtiter plate. After separation of bound and free fractions, a solution of streptavidin-hor seradish peroxidase conjugate (SA-HRP) or europium (III) ion chelate l abeled SA (Eu-SA) was added to the biotinylated CCK-33 bound on the mi crotiter plate. The microtiter plate was washed and then the SA-HRP ac tivity was measured by colorimetry or chemiluminometry and the Eu-SA a ctivity was measured by time-resolved fluorometry, respectively. Detec tion limits of CCK-8 were 1000 pg/mL by colorimetry, 125 pg/mL by time -resolved fluorometry, sad 1.95 pg/mL by chemiluminometry, respectivel y. Further, the sensitivity of the concentration yielding 50% inhibiti on value obtained by using a signal amplification method, catalyzed re porter deposition (CARD), was 135.7-fold by colorimetry, 18.5-fold by time-resolved fluorometry and 1.5-fold by chemiluminometry, better tha n that obtained without CARD.