Highly Sensitive Non-Isotopic Immunoassays for Cholecystokinin Using Various Detection Methods

We have developed sensitive, non-isotopic immunoassays for cholecystokinin (CCK) by using colorimetric, time-resolved fluorometric and chemiluminometric detection. The assay is based on competition between a free CCK and biotinylated CCK-33 as tracer for rabbit anti-CCK-8 antibodies on a goat anti-rabbit IgG antibody coated microtiter plate. After separation of bound and free fractions, a solution of streptavidin-horseradish peroxidase conjugate (SA-HRP) or europium (III) ion chelate labeled SA (Eu-SA) was added to the biotinylated CCK-33 bound on the microtiter plate. The microtiter plate was washed and then the SA-HRP activity was measured by colorimetry or chemiluminometry and the Eu-SA activity was measured by time-resolved fluorometry, respectively. Detection limits of CCK-8 were 1000 pg/mL by colorimetry, 125 pg/mL by time-resolved fluorometry, and 1.95 pg/mL by chemiluminometry, respectively. Further, the sensitivity of the concentration yielding 50% inhibition value obtained by using a signal amplification method, catalyzed reporter deposition (CARD), was 135.7-fold by colorimetry, 18.5-fold by time-resolved fluorometry and 1.5-fold by chemiluminometry, better than that obtained without CARD.