Description
Acetylcholinesterase Assay Kit (BA0009) (BA0009)
The Acetylcholinesterase Assay Kit (SKU: BA0009) provides a rapid colorimetric method for determining acetylcholinesterase (AChE) activity in blood, serum, plasma and other biological samples. AChE hydrolyses the neurotransmitter acetylcholine into choline and acetic acid, and cholinesterase levels are widely used to establish safety precautions relating to organophosphate insecticide exposure. The assay is based on an improved Ellman method in which thiocholine produced by AChE forms a yellow colour with 5,5'-dithiobis(2-nitrobenzoic acid), measured at 412 nm. With a detection range of 10 to 600 U/L, the procedure simply requires adding a single working reagent and reading the optical density at 2 and 10 minutes at room temperature. It can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
| Product Name: | Acetylcholinesterase Assay Kit (BA0009) |
| SKU: | BA0009 |
| Detection Method: | Colorimetric (OD 412 nm) |
| Detection Range: | 10 to 600 U/L in 96-well plate |
| Sample Type: | ['Blood', 'Serum', 'Plasma', 'Tissue lysate', 'Cell lysate'] |
| Species Reactivity: | All |
| Assay Time: | ~10 minutes (readings at 2 and 10 min) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Room temperature |
| Shelf Life: | 6 months after receipt |
| Shipping: | Room Temperature |
Acetylcholinesterase (EC 3.1.1.7, AChE), also known as RBC cholinesterase, is found primarily in the blood and neural synapses. Low serum cholinesterase activity may relate to exposure to insecticides or to one of a number of variant genotypes. AChE catalyses the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation. Cholinesterase levels of cells and plasma are used as a guide in establishing safety precautions relative to exposure and contact with organic phosphate insecticides. This assay is based on an improved Ellman method, in which thiocholine produced by the action of acetylcholinesterase forms a yellow colour with 5,5'-dithiobis(2-nitrobenzoic acid). The intensity of the product colour, measured at 412 nm, is proportional to the enzyme activity in the sample.
- Sensitive and accurate. Detection range 10 to 600 U/L AChE activity in 96-well plate assay.
- Convenient. The procedure involves adding a single working reagent and reading the optical density at 2 min and 10 min at room temperature.
- High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.
- Direct assays of acetylcholinesterase activity in blood, serum, plasma and other biological samples.
- Evaluation of acetylcholinesterase inhibitors.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Prepare samples: dilute blood samples 40-fold in Assay Buffer (e.g. 5 µL blood with 195 µL Assay Buffer). Prepare tissue or cell lysates by brief sonication or homogenisation in 0.1 M phosphate buffer (pH 7.5), followed by centrifugation at 14,000 rpm for 5 min, using the supernatant. Ideally assay samples fresh, or refrigerate and assay within 24 hours. |
| 2 | Prepare the Working Reagent freshly and use within 30 min. Each reaction well requires 2 mg reagent; weigh the required amount and add 200 µL Assay Buffer per 2 mg reagent, then vortex to dissolve. |
| 3 | Transfer 200 µL water and 200 µL Calibrator separately into wells of a clear-bottom 96-well plate, and add 10 µL sample per well in separate wells. |
| 4 | Transfer 190 µL freshly prepared Working Reagent to all sample wells and tap the plate briefly to mix. |
| 5 | Read OD412nm at 2 min and at 10 min in a plate reader. |
AChE Activity = [(OD10 - OD2) / (ODCAL - ODH2O)] x n x 200 (U/L), where OD10 and OD2 are the OD412nm values of the sample at 10 min and 2 min, ODCAL and ODH2O are the OD412nm values of the Calibrator and water at 10 min, n is the dilution factor (n = 40 for whole blood) and 200 is the equivalent activity of the calibrator. If the AChE activity without the dilution factor exceeds 600 U/L, dilute the sample further in Assay Buffer, repeat and multiply by n. Unit definition: one unit of enzyme catalyses the production of 1 µmole of thiocholine per minute under the assay conditions (pH 7.5, room temperature).
| Component | Quantity | Storage |
| Assay Buffer (pH 7.5) | 30 mL | Room temperature |
| Reagent | 240 mg | Room temperature |
| Calibrator (equivalent to 200 U/L) | 4 mL | Room temperature |
