Bioluminescent enzyme immunoassay for captopril



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Captopril is an orally active antihypertension agent. A sensitive method for captopril quantitation in biological fluid is highly desired for clinical and diagnostic practices. The few assay methods available so far do not fully satisfy these needs. In this project, a bioluminescent enzyme immunoassay for captopril has been developed. Polyclonal anti-captopril antibody was induced with captopril-protein conjugate in rabbits. A captopril derivative bearing a thiopyridine was linked to an essential cysteinyl thiol of Vibrio harveyi luciferase through a mixed disulfide linkage. The luciferase is catalytically inactive in this conjugated form but can be released from the conjugate and activated by reduction with dithiothreitol. A fixed quantity of anti- captopril antibody was allowed to react with a constant level of the Captopril-Iuciferase conjugate and various amounts of captopril. An excess amount of immobilized goat anti-rabbit immunoglobulin was added subsequent to a period of incubation. After separating the immunoprecipitate from the soluble phase by centrifugation, the inactive luciferase in the precipitated Captopril-luciferase conjugate was reactivated by dithiothreitol and the in vitro bioluminescence activity was determined. The higher the amount of non-labeled captopril is in the original sample, the lower the luciferase activity that will be detected. [...]



Immunoenzyme technique, Captopril