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| Product Name | Acetylcholinesterase Activity Assay Kit, Ellman's Method, Colorimetric |
| Catalog No. | ETR-HMM-0091 |
| Description | Colorimetric acetylcholinesterase (AChE) activity assay kit based on the classical Ellman's method for quantitative measurement of AChE and butyrylcholinesterase (BChE) activity in blood, serum, plasma, tissue homogenates, and cell lysates. Acetylthiocholine (ATCh) is hydrolyzed by AChE to thiocholine and acetate. The thiocholine product reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to produce 5-thio-2-nitrobenzoic acid (TNB), a yellow compound with maximum absorbance at 412 nm. The rate of TNB production (delta A412/min) is directly proportional to cholinesterase activity. The kit is suitable for enzyme characterization, inhibitor screening (organophosphates, carbamates, therapeutic cholinesterase inhibitors), pesticide exposure assessment in research, and Alzheimer's disease drug discovery targeting cholinesterases. |
| Intended Use | Quantitative measurement of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) activity in: erythrocyte and serum/plasma samples; brain and muscle tissue homogenates; cultured neuronal cells; recombinant AChE/BChE characterization; screening of cholinesterase inhibitors (Alzheimer's therapeutics, pesticides, nerve agent antidotes); environmental toxicology research. |
| Principle / Technology | Acetylthiocholine (ATCh) + H2O --(AChE)--> thiocholine + acetate. Thiocholine reacts stoichiometrically with DTNB (Ellman's reagent): thiocholine + DTNB --> TNB (yellow, A412) + mixed disulfide. The rate of increase in absorbance at 412 nm is measured kinetically. Molar extinction coefficient of TNB at 412 nm = 14,150 M^-1 cm^-1. The reaction is specific to cholinesterases; butyrylthiocholine (BTCh) can be used as alternative substrate for BChE-specific measurement. Use of selective inhibitors (BW284c51 for AChE, iso-OMPA for BChE) allows differentiation of AChE and BChE activity in mixed samples. |
| Detection Method | Add sample (serum, plasma, tissue homogenate, or purified enzyme) to assay buffer containing DTNB (0.3-0.5 mM) and ATCh substrate (0.5-1 mM); incubate at 25-37 C; monitor A412 kinetically every 15-30 s for 5-10 min; calculate activity: U/mL = (delta A412/min x reaction volume x dilution factor) / (14.15 x sample volume), where 14.15 is the millimolar extinction coefficient of TNB. |
| Sample Type | Whole blood (erythrocyte AChE), serum or plasma (BChE), brain homogenate (AChE), muscle homogenate (AChE), cultured neuronal cells, purified recombinant human AChE or BChE, tissue from toxicology studies. |
| Performance Range / Specifications | Detection range: 0.05-50 U/L cholinesterase in cuvette; linear range: up to 0.1 delta A412/min (approximately 0.7 umol substrate hydrolyzed/min); assay time: 5-10 min kinetic at 25-37 C; blood AChE typical activity: 5-12 U/mL whole blood; serum BChE typical activity: 4-12 U/mL. |
| Sensitivity / LOD | Detection limit: 0.05 U/L (approximately 0.05 nmol substrate/min/mL); corresponds to AChE activity in approximately 10 nL of normal human whole blood. |
| Specificity | Acetylthiocholine is hydrolyzed by both AChE and BChE (AChE approximately 2-3x greater activity than BChE). Butyrylthiocholine is specific for BChE (AChE shows <1% activity). Use selective inhibitors: BW284c51 (1 uM) selectively inhibits AChE; iso-OMPA (tetraisopropyl pyrophosphoramide, 10 uM) selectively inhibits BChE. Non-specific thiocholine production by other esterases is negligible under the assay conditions (pH 7.4-8.0). |
| Reaction Conditions / Protocol | Optimal pH 7.4-8.0 (phosphate or Tris buffer); DTNB concentration 0.3-0.5 mM; ATCh concentration 0.5-1.0 mM (near-saturating, Km ~0.1 mM); temperature 25-37 C; measure kinetically at 412 nm. |
| Components / Formulation | ATCh Substrate (acetylthiocholine iodide, lyophilized, 50 mg), BTCh Substrate (S-butyrylthiocholine iodide, lyophilized, 25 mg — for BChE-specific measurement), DTNB (Ellman's reagent, lyophilized, 25 mg, 2 vials), Assay Buffer (5x, 50 mL: 0.5 M potassium phosphate pH 7.4), Protocol. |
| Storage Conditions | Lyophilized substrates and DTNB at -20 C desiccated (protect from light and moisture); Assay Buffer at 2-8 C; reconstituted substrates and DTNB: prepare fresh or aliquot and store at -20 C for 2 weeks. |
| Shelf Life | 12 months from date of manufacture; lyophilized reagents stable at -20 C for 24 months. |
| Package Specifications | 1 kit (sufficient for approximately 200 tests in 200 uL assay volume). |
| Product Form | Lyophilized powders (ATCh, BTCh, DTNB); liquid assay buffer concentrate. |
| Quality Control | Each lot tested for: DTNB purity >=99% by HPLC; ATCh purity >=98% by TLC; substrate blank (non-enzymatic hydrolysis) <0.002 A412/min at 37 C; linearity with purified electric eel AChE: R2 >0.99 over 0-1 U/mL; human serum BChE recovery within +/-10% of reference value. |
| Key Features | Classical Ellman's method; ATCh and BTCh substrates included; DTNB (Ellman's reagent) included; allows AChE/BChE differentiation; kinetic assay for accuracy; suitable for inhibitor screening; 96-well plate compatible. |
| Purity | DTNB: >=99% (single spot by TLC, single peak by HPLC); ATCh: >=98%; BTCh: >=98%; all components: free of cholinesterase inhibitors. |
| Concentration | ATCh working: 0.5-1.0 mM (Km ~0.1 mM for AChE); DTNB working: 0.3-0.5 mM (sufficient for TNB detection up to 1.0 A412); BTCh working: 1-5 mM (Km ~0.3 mM for BChE). |
| Activity / Unit Definition | 1 AChE unit (U) = 1 umol ATCh hydrolyzed per minute at 25 C, pH 7.4; specific activity of human erythrocyte AChE approximately 150-200 U/mg; human serum BChE approximately 30-50 U/mg. |
| Molecular Weight | Acetylthiocholine iodide: 289.18 g/mol; S-butyrylthiocholine iodide: 317.23 g/mol; DTNB: 396.35 g/mol; TNB: 227.3 g/mol. |
| Source / Origin | Synthetic substrates and DTNB; buffer salts: analytical grade; no animal-derived components. |
| pH Range / Optimal pH | Optimal pH 7.4-8.0 (potassium phosphate); activity decreases sharply below pH 6.5 and above pH 9.0; DTNB reactivity with thiols is optimal at pH 7.0-8.5. |
| Shipping Conditions | Ambient temperature for assay buffer; lyophilized substrates and DTNB on cold pack recommended. |
| Expiration Date / Stability | 12 months at recommended conditions; reconstituted DTNB and substrates: 2 weeks at -20 C. DTNB solutions are light-sensitive: store in amber vial or wrap in foil. |
| Regulatory / Compliance | For research use only; not for diagnostic or therapeutic use. |
| Compatibility | Samples must be free of cholinesterase inhibitors (organophosphates, carbamates). Hemolysis does not significantly affect erythrocyte AChE measurement but serum BChE measurement is affected if hemolysis >0.5% (release of erythrocyte AChE). Anticoagulants: heparin and citrate are compatible; EDTA partially inhibits AChE (chelates Ca2+). Avoid reducing agents (DTT, beta-mercaptoethanol) that reduce DTNB. For tissue homogenates, deproteinization is not required for kinetic measurement (initial rates unaffected by turbidity). |
| Recommended Buffer System | 5x Assay Buffer: 0.5 M potassium phosphate pH 7.4; prepare 1x working buffer fresh; DTNB reconstitution: 10 mM in 0.1 M potassium phosphate pH 7.4, 15 mg NaHCO3 per 25 mg DTNB. |
| Application Notes / Precautions | DTNB stock: dissolve in phosphate buffer with sodium bicarbonate (15 mg NaHCO3 per 25 mg DTNB) to enhance solubility. DTNB solution should be light amber color; dark yellow-brown indicates degradation. For AChE-specific measurement in whole blood, use BW284c51 (1-5 uM) to inhibit AChE or iso-OMPA (10-100 uM) to inhibit BChE before measuring remaining activity. The sum of AChE + BChE inhibitory activity should account for >95% of total activity. Pre-incubate samples with inhibitors for 10-15 min at RT before adding substrate. For inhibitor screening (e.g., donepezil, rivastigmine), pre-incubate enzyme with inhibitor for 15-30 min. Store blood samples on ice and assay within 4 hours of collection; AChE is stable at -80 C. |
| Batch-to-Batch Consistency | DTNB purity >=99%; ATCh blank hydrolysis <0.002 A412/min; human serum BChE recovery within +/-10%; AChE standard linearity R2 >0.99. |
For research use only, not for clinical use.
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