Choline Acetyltransferase (ChAT)?is the enzyme responsible for synthesizing acetylcholine (ACh) from acetyl-CoA and choline in presynaptic neurons. It is crucial for cholinergic neurotransmission, influencing cognitive functions, muscle activation, and autonomic processes. ChAT is primarily localized in nerve terminals and is a biomarker for cholinergic neurons.ChAT synthesizes acetylcholine, while AChE breaks it down. They work in tandem to maintain the appropriate level of acetylcholine in the synaptic cleft for proper neural signaling.Thus a functional ELISA assay was conducted to detect the interaction of recombinant mouse ChAT and recombinant bovine ACHE. Briefly, ChAT was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to ACHE-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-ChAT pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant mouse ChAT and recombinant bovine ACHE was shown in Figure 1, the EC50?for this effect is 0.115ug/mL.