The use of nerve agents, organophosphorus compounds which irreversibly inhibit acetylcholinesterase, in conflicts and terrorism has gained renewed geopolitical status in recent years. As no “universal antidote” has ever been produced, the development of antidotes to reactivate enzyme is clearly warranted. Given the fact that nerve agents are restricted substances, the use of surrogates allows non-qualified laboratories to develop research on novel antidotes. We herein present an alternative and efficient synthesis of NEMP, O-(4-nitrophenyl) O-ethyl methylphosphonate, a venomous agent X (VX) surrogate, and Ellman’s test procedures for inhibition and reactivation of Electrophorus eel acetylcholinesterase involving the NEMP-generated adduct.

Straightforward, microscale, Ellman's cholinesterase test procedures are presented, useful for screening of novel compounds aiming at neurodegenerative diseases and antidotes towards nerve agents and pesticides. Herein, we detail the different procedure features and set forth guidelines on experimental choices researchers may make. The detailed procedure lowers the costs for Ellman´s test, what is highly desirable for low-budget groups in science-lagging and developing countries.