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Ethanol Colorimetric Assay Kit
SKU: E-BC-K891-M-500
Ethanol Colorimetric Assay Kit
| SKU # | E-BC-K891-M |
| Detection Instrument | Microplate reader (440-460 nm, optimum wavelength: 450 nm) |
|
Detection method |
Colorimetric method |
Product Details
Properties
| Sample type | Serum, plasma, wine |
| Sensitivity | 0.27 μmol/mL |
| Detection range | 0.27–10.0 μmol/mL |
| Detection method | Colorimetric method |
| Assay type | Quantitative |
| Assay time | 30 min |
| Precision | Average inter-assay CV: 5% | Average intra-assay CV: 3.500% |
| Other instruments required | Micropipettor, Vortex mixer, Centrifuge, Incubator (37℃) |
| Storage | -20℃ |
| Valid period | 12 months |
Dilution of Sample
It is recommended to take 2~3 samples with expected large difference to do pre-experiment before formal experiment and dilute the sample according to the result of the pre-experiment and the detection range (0.27–17.0 μmol/mL).
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| Beer (3.6% alcohol) | 40-80 |
| White wine (12% alcohol) | 250-300 |
Detection Principle
Ethanol dehydrogenase can catalyze oxidative dehydrogenation of ethanol to acetaldehyde, and NAD+ is reduced to produce NADH. NADH, under the action of 1-mPMS, transfer electrons to WST-8 to produce the yellow product, which has a characteristic absorption peak at 450 nm. Therefore, ethanol content can be quantified by measure the OD value at 450 nm.
Kit Components & Storage
| Item | Component | Size 1(48 T) | Size 2(96 T) | Storage |
| Reagent 1 | Buffer Solution A | 10 mL × 1 vial | 20 mL × 1 vial | -20℃, 12 months |
| Reagent 2 | Enzyme Reagent | Powder × 1 vial | Powder × 2 vials | -20℃, 12 months, shading light |
| Reagent 3 | Buffer Solution B | 7 mL × 1 vial | 14 mL × 1 vial | -20℃, 12 months |
| Reagent 4 | Substrate | Powder × 1 vial | Powder × 2 vials | -20℃, 12 months, shading light |
| Reagent 5 | Chromogenic Agent | 1.5 mL × 1 vial | 1.5 mL × 2 vials | -20℃, 12 months, shading light |
| Reagent 6 | 10 μmol/mL Standard Solution | 1.8 mL × 1 vial | 1.8 mL × 2 vials | -20℃, 12 months |
| Microplate | 96 wells | No requirement | ||
| Plate Sealer | 2 pieces | |||
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.
Technical Data:
Parameter:
Intra-assay Precision
Three beer samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (μmol/mL) | 0.80 | 2.60 | 6.80 |
| %CV | 3.8 | 3.5 | 3.2 |
Inter-assay Precision
Three beer samples were assayed 20 times in duplicate by three operators to determine precision between assays
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (μmol/mL) | 0.80 | 2.60 | 6.80 |
| %CV | 4.8 | 5.1 | 5.1 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 96%.
| Standard 1 | Standard 2 | Standard 3 | |
| Expected Conc. (μmol/mL) | 2.7 | 5 | 7.5 |
| Observed Conc. (μmol/mL) | 2.6 | 4.7 | 7.2 |
| Recovery rate(%) | 98 | 94 | 96 |
Sensitivity
The analytical sensitivity of the assay is 0.27 μmol/mL. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.
Standard Curve
As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:
| Concentration (μmol/mL) | 0 | 2 | 3 | 4 | 6 | 8 | 9 | 10 |
| Average OD | 0.016 | 0.147 | 0.212 | 0.280 | 0.416 | 0.550 | 0.601 | 0.669 |
| Absoluted OD | 0.000 | 0.131 | 0.196 | 0.264 | 0.400 | 0.534 | 0.585 | 0.653 |