Využitie metódy ELISA na štúdium enzýmovej hydrolýzy butyrylcholínesterázy v biologickom materiáli

Abstrakt

The aim of this project was to design ELISA assay using new antibodies against human butyrylcholinesterase, which enable one to work with “pure” enzyme. The amount of primary antibodies, human plasma and substrate were determined from the saturation curves. For the detection, 1 mM butyrylthiocholine substrate in presence of Ellman's reagent (0.5 mM DTNB) and 5 mM HEPES, pH 7.4 or 1 mM indoxylacetate in 0.1 mM phosphate buffer saline, pH 7.4 were used. This method makes it possible to study both steady-state and pre-steady-state phases in butyrylcholinesterase-catalyzed hydrolyzes. In conclusion, we propose a simple and reliable method to study butyrylcholinesterase activity in a small amount of biological samples, while captured “pure” enzyme can be used for studying the enzyme kinetics and activity changes in selected pathological models.