Description
Ethanol Test Kit (Colorimetric) (BA0085) (BA0085)
The Ethanol Test Kit (Colorimetric) (SKU: BA0085) provides a simple, direct and automation-ready procedure for measuring ethanol concentration. Quantitative determination of ethanol has applications in basic research, drug discovery, clinical studies and the alcoholic beverage industry. This ultrasensitive assay is based on alcohol dehydrogenase-catalysed oxidation of ethanol, in which the formed NADH is coupled to the formazan (MTT) chromogen. The intensity of the product colour, measured at 565 nm, is proportional to the ethanol concentration in the sample. The convenient procedure requires no 37C heater and can be readily automated for high-throughput analysis.
| Product Name: | Ethanol Test Kit (Colorimetric) (BA0085) |
| SKU: | BA0085 |
| Detection Method: | Colorimetric |
| Detection Range: | 0.0008 to 0.1 vol % |
| Sample Type: | Serum, plasma, urine and saliva |
| Species Reactivity: | All |
| Assay Time: | 30 minutes at room temperature |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
Ultrasensitive colorimetric determination of ethanol at 565 nm. Alcohol dehydrogenase oxidises ethanol, generating NADH that is coupled to an MTT formazan chromogen; the colour at 565 nm is proportional to ethanol concentration.
- Sensitive and accurate; detection limit 0.0008 vol % (140 uM or 8 ppm) and linearity up to 0.1%
- Convenient add-incubate-stop procedure with no 37C heater required
- High-throughput; readily automated for thousands of samples per day
- Product colour read at 565 nm
- Direct assays of ethanol in serum, plasma, urine and saliva samples
- Pharmacology: effects of drugs on alcohol metabolism
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 | Reagent preparation. Reconstitute Enzyme A by adding 120 uL Enzyme Buffer to the Enzyme A tube, ensuring it is fully dissolved by pipetting up and down. Store reconstituted Enzyme A at -20C and use within 1 month. |
| 2 | Calibration curve. Prepare 0.1% alcohol premix by mixing 25 uL of the 1% standard and 225 uL distilled water, then dilute as shown in the dilution table. Transfer 10 uL standards into wells of a clear flat-bottom 96-well plate. |
| 3 | Samples. Add 10 uL sample per well in separate wells. Saliva samples should be diluted 10-fold in PBS prior to assay. |
| 4 | Reaction. For each reaction well, prepare working reagent by mixing 80 uL Assay Buffer, 1 uL Enzyme A, 1 uL Enzyme B, 2.5 uL NAD and 14 uL MTT; fresh reconstitution is recommended. Add 90 uL working reagent per well quickly, tap to mix briefly and thoroughly and incubate 30 minutes at room temperature. Add 100 uL Stop Reagent per well and tap to mix. |
| 5 | Read optical density at 565 nm (520-600 nm). |
Subtract the blank (water, #4) from the OD values for the standard wells and plot the standard curve (dOD vs ethanol concentration) to determine the slope. [Ethanol] = (OD-sample - OD-blank) / slope x n (%), where n is the dilution factor (10 for saliva). Conversions: 1 vol % ethanol equals 170 mM or 785 mg/dL. If the sample concentration exceeds 0.1%, dilute in distilled water, repeat and multiply by the dilution factor.
| Component | Quantity | Storage |
| Assay Buffer | 10 mL | -20C |
| NAD Solution | 1 mL | -20C |
| MTT Solution | 1.5 mL | -20C |
| Standard (1% Ethanol) | 1.5 mL | -20C |
| Enzyme A | Lyophilised | -20C |
| Enzyme B | 120 uL | -20C |
| Enzyme Buffer | 150 uL | -20C |
| Stop Reagent | 12 mL | -20C |
