Acetylcholinesterase linked to SP, antiserum, and samples were added to the wells and incubated for 24h at 4˚C. The plates were washed five times with phosphate-buffered saline (PBS) containing 0.05% Tween-20 (PBS-Tween 20), then Ellman’s Reagent (which contains the substrate for acetylcholinesterase) was added to each well, and the absorbance was measured at 405nm. With a microplate reader (Dade Behring BEP, Germany).