Acetylcholinesterase (ACHE) hydrolyzes acetylcholine, which is essential in regulating nerve impulses throughout the nervous system by turning off neural signals and removing the neurotransmitter from the synaptic cleft. ACHE is primarily located in the neuromuscular junctions, central nervous system, and red blood cell membranes. Inhibition of ACHE leads to an accumulation of acetylcholine leading to flaccid paralysis, bradycardia, cardiac arrest, and neurological effects like epilepsy. A depletion of cholinergic activity and a corresponding need to inhibit ACHE are linked to neurodegenerative conditions like Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), and Parkinson’s disease. Monitoring acetylcholinesterase is critical in the diagnosis and management of neurological conditions.

Acetylcholinesterase Activity Assay Kit provides a simple, reproducible, and sensitive tool of measuring ACHE activity in plasma, serum, cell lysates, saliva, and other biological liquid samples. In this assay, acetylcholinesterase hydrolysis acetylthiocholine into thiocholine which reacts with 5, 5’-dithiobis (2-nitrobenzoic) acid (DTNB) to form 5-thio-2-nitrobenzoic acid (TNB2-), which generates a strong yellow color at 412 nm. Since in crude biological samples other enzymes can also reduce DTNB, therefore, acetylcholinesterase specific inhibitor is utilized to determine ACHE specific activity. The difference between the two results is the DTNB reduction due to ACHE activity.

• Microplate reader capable of measuring absorbance at 412 nm
• Precision pipettes to deliver 2 µL to 1 mL volumes
• Tubes to prepare sample dilutions
• 5 mL tubes
• Multi-channel pipettes to deliver 20 µL to 200 µL volumes
• MilliQ water or another type of double distilled water (ddH₂O)

1. Add 45 µL of each sample, or positive control to the 96-well microtiter plate.
2. Each sample replicate requires two paired wells, in one sample well, add 5 µL of the inhibitor. In the other sample well, add 5 µL of assay buffer so the final volume in each well is 50 µL. Incubate the samples for 10 minutes at room temperature with shaking protected from light.
3. Add 100 µL of each standard to the 96-well microtiter plate.
4. Initiate the reaction by adding 50 µL Substrate Mix to each well. Carefully shake the plate for a few seconds to mix well.
5. For kinetic reading: Immediately start measuring absorbance at 412 nm continuously at room temperature and record data every 2 minutes for 30 minutes, protect from light. The standard curve can be read in end-point mode.