- Biochemical Assay (BCA)
- Sample Type
- Serum, Plasma, Urine, Saliva
Sensitive and accurate. Detection limit 0.0008 vol % (140 μM or 8 ppm), linearity up to 0.1 % ethanol in 96-well plate assay.
Convenient. The procedure involves adding working reagent, incuba- ting 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.
- Assay Buffer: 10 mL. NAD Solution: 1 mL. MTT Solution: 1.5 mL. Enzyme Mix: 120 µL. Stop Reagent: 12 mL. Standard: 1.5 mL 1% ethanol.
- Material not included
- Pipeting (multi-channel) devices. Clear-bottom 96-well plates (e.g. Corning Costar) and plate reader.
- Application Notes
Direct Assays: ethanol in serum, plasma, urine and saliva samples.
Pharmacology: effects of drugs on alcohol metabolism.
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%).
1. Calibration Curve. Prepare 0.1% alcohol Premix by mixing 25 µL 1% Standard and 225 µL distilled water. Transfer 10 µL standards into wells of a clear bottom 96-well plate. Samples: add 10 µL 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 80 µL Assay Buffer, 1 µL Enzyme Mix, 2.5 µL NAD and 14 µL MTT. Fresh reconstitution is recommended. Add 90 µL Working Reagent per well quickly. Tap plate to mix briefly and thoroughly. Incubate 30 min at room temperature. Add 100 µL Stop Reagent per well. Tap plate to mix.
3. Read optical density at 565 nm (520-600nm).
- For Research Use only
- -20 °C
Magne, Blanc, Legrand, Lucas, Barouki, Rouach, Garlatti: "ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes." in: Journal of hepatology, Vol. 54, Issue 4, pp. 729-37, 2011 (PubMed).
Echevarria, Toms, Jouandot: "Alcohol-induced behavior change in zebrafish models." in: Reviews in the neurosciences, Vol. 22, Issue 1, pp. 85-93, 2011 (PubMed).
Ou, Stockmeier, Meltzer, Overholser, Jurjus, Dieter, Chen, Lu, Johnson, Youdim, Austin, Luo, Sawa, May, Shih: "A novel role for glyceraldehyde-3-phosphate dehydrogenase and monoamine oxidase B cascade in ethanol-induced cellular damage." in: Biological psychiatry, Vol. 67, Issue 9, pp. 855-63, 2010 (PubMed).
Suwannarangsee, Oh, Seo, Kim, Rhee, Kang, Chulalaksananukul, Kwon: "Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha." in: Applied microbiology and biotechnology, Vol. 88, Issue 2, pp. 497-507, 2010 (PubMed).
Jeong, Osei-Hyiaman, Park, Liu, Bátkai, Mukhopadhyay, Horiguchi, Harvey-White, Marsicano, Lutz, Gao, Kunos: "Paracrine activation of hepatic CB1 receptors by stellate cell-derived endocannabinoids mediates alcoholic fatty liver." in: Cell metabolism, Vol. 7, Issue 3, pp. 227-35, 2008 (PubMed).
- Magne, Blanc, Legrand, Lucas, Barouki, Rouach, Garlatti: "ATF4 and the integrated stress response are induced by ethanol and cytochrome P450 2E1 in human hepatocytes." in: Journal of hepatology, Vol. 54, Issue 4, pp. 729-37, 2011 (PubMed).
Quantitative determination of ethanol by enzymatic colorimetric (565nm) method.
Procedure: 30 min.
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. This 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.