Ellman's Assay for Acetylcholinesterase and Butyrylcholinesterase Inhibition

The infusions were evaluated by the Ellman’s method, with some modifications (Ellman et al., 1961 (link)). Sample solution (50 µl, 2 mg/ml) was mixed with 120 µl DTNB (0.3 mM, 5,5-dithio-bis(2-nitrobenzoic) acid) and AChE (0.026 U/ml, AChE from Electric eel), or BChE (0.026 U/ml, BChE from horse serum) solution (25 µl) in Tris-HCl buffer 50 mM (pH 8.0) in a 96-well microplate and incubated for 20 min at 37°C. The reaction was then initiated with the addition of 20 µl of ATCI (1.5 mM, acetylthiocholine iodide) or BTCl (1.5 mM, butyrylthiocholine chloride). A blank was prepared for all reaction reagents without enzymes solution. The absorbances were recorded at 405 nm for 30 min at 37^oC and the results were expressed as IC₅₀ values (concentration range from 0.05 to 25 µg/ml).