Alcoholic drinks are among the daily consumed beverages. Studies have shown heavy alcohol consumption may lead to various forms of liver diseases and to increased mortality rates. Quantitative determination of alcohol (ethanol, C2H5OH) has Applications in basic research, drug discovery, clinic studies and in the alcoholic industry.||Simple, direct and automation-ready procedures for measuring ethanol concentration are very desirable. Ethanol assay kit is based on alcohol dehydrogenase catalyzed oxidation of ethanol, in which the formed NADH is coupled to the formazan (MTT) chromogen. The intensity of the product color, measured at 565 nm, is proportionate to the ethanol concentration in the sample.||Applications:|Direct Assays: ethanol in serum, plasma, urine and saliva samples.|Pharmacology: effects of drugs on alcohol metabolism.||Key Features:|Sensitive and accurate. Detection limit 0.0008 vol % (140uM or 8ppm), linearity up to 0.1% ethanol in 96-well plate assay.|Convenient. The procedure involves adding working reagent, incubating for 30 min and stropping reaction. No 37°C heater is needed.|High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.||Kit Contents: (100 tests in 96-well plates)|Assay Buffer: 10ml NAD Solution: 1ml|MTT Solution: 1.5ml Enzyme Mix: 120ul|Stop Reagent: 12ml Standard: 1.5ml 1% ethanol|Storage conditions. Store Stop Reagent at room temperature and all other reagents at -20°C. Shelf life: 6 months after receipt.|Precautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.||Procedures:|1. Calibration Curve. Prepare 0.1% alcohol Premix by mixing 25ul 1% Standard and 225ul distilled water. Dilute standard as follows.|Transfer 10ul standards into wells of a clear bottom 96-well plate.|No Premix+H2O Vol (ul) Ethanol (%)|1 100ul+0ul 100 0.10|2 60ul+40ul 100 0.06|3 30ul+70ul 100 0.03|4 0ul+100ul 100 0|Samples: add 10ul sample per well in separate wells.|IMPORTANT: saliva samples should be diluted 10-fold in PBS prior to assay.|2. Reaction. Spin the Enzyme Mix tube briefly before pipetting. For each well of reaction, prepare Working Reagent by mixing 80ul Assay Buffer, 1ul Enzyme Mix, 2.5ul NAD and 14ul MTT. Fresh reconstitution is recommended. Add 90ul Working Reagent per well quickly. Tap plate to mix briefly and thoroughly. Incubate 30 min at room temperature. Add 100ul Stop Reagent per well. Tap plate to mix.|3. Read optical density at 565 nm (520-600nm).|4. Calculation:. Subtract blank (water, #4) from OD values for the standard wells. Plot Standard Curve (DOD vs Standard ethanol concentrations) to determine the slope. Sample ethanol concentration is calculated,|[Ethanol] =|ODSAMPLE–ODBLANK|Slope|× n (%)|where ODSAMPLE and ODBLANK are the OD565nm values of the sample and blank (water, #4). n is the dilution factor (n=10 for saliva). Note: if the sample ethanol concentrations are higher than 0.1%, dilute sample in distill water and repeat this assay. Multiply the results by the dilution factor.|Conversions: 1 vol % ethanol equals 170mM or 785 mg/dL.||Materials Required, But Not Provided:|Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.||General Considerations:|1. This assay is based on an enzyme-catalyzed kinetic reaction. Addition of Working Reagent should be quick and mixing should be brief but thorough. Use of multi-channel pipettor is recommended.|2. The following substances interfere and should be avoided in sample preparation: ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%).