Cholinesterase assay by an efficient fixed time endpoint method

• An end-point method for cholinesterase determinations is performed.
• This method is based on stopping the reaction after a fixed reaction time.
• A large number of samples can be processed for complex kinetic assays.
• This assay can also be applied for manual, or with, automated workstations.
• This procedure allows to avoid undesired reactions by DTNB and TNB.

Many cholinesterase assays are performed to study the inhibition of cholinesterase (ChE) activity. Frequently a large number of samples are processed and Ellman's method [1] is the most commonly used [2] and [3]. Activity is estimated from the increment in absorbance between two reaction times when the reaction is not stopped. Bellino et al. [4] described a method based on Ellman's method whereby the reaction was stopped with SDS and then the absorbance was measured. In these methods, the chromogen reagent 5,5′dithiobis nitro benzoic acid (DTNB) is added with the substrate and colour is monitored. Some authors pointed that the chromogen can alter cholinesterase activity [5].
• A modification of Bellino's method is proposed for acetylcholine-hydrolyzing activity determinations that is based on stopping the reaction after a fixed substrate reaction time using a mixture of detergent SDS and DTNB.
• The method may be adapted to the user needs by modifying the enzyme concentration and applied for simultaneously testing many samples in parallel; i.e. for complex experiments of kinetics assays with organophosphate inhibitors in different tissues.