Alcohol Assay Kit (Colorimetric)

1. Ethanol Standard : One 500 μL vial of pure ethanol (MW 46.07, 17.15N)
2. 10X Assay Buffer : Three 1.5 mL vials
3. 100X Enzyme Mixture : One 100 μL vial
4. 50X Colorimetric Probe : One 200 μL amber vial 2

1. 96-well microtiter plate
2. 10 μL to 1000 μL adjustable single channel micropipettes with disposable tips
3. 50 μL to 300 μL adjustable multichannel micropipette with disposable tips
4. Multichannel micropipette reservoir
5. Microplate reader capable of reading at 570 nm

• Simple assays that quantitatively measure the alcohol concentration (primary alcohols only) in various samples using a 96-well microtiter plate format and is not ethanol specific
• Each kit provides sufficient reagents to perform up to 100 assays, including blanks, standards and unknown samples

• 10X Assay Buffer should be thawed/maintained at 4 °C during assay preparation. Precipitation may be visible after thawing, mix well to dissolve the precipitate. The solution is stable for 1 week at 4 °C. For longer term storage, the solution should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.
• 1X Assay Buffer: Dilute the 10X Assay Buffer Concentrate with deionized water. Stir to homogeneity. .
• Ethanol Standard and 100X Enzyme Mixture should be thawed/maintained at 4 °C during assay preparation. All are stable for 1 week at 4 °C. For longer term storage, each should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.
• 50X Colorimetric Probe should be thawed/maintained at room temperature during assay preparation. Any unused material should be aliquoted and frozen at -80 °C to avoid multiple freeze/thaws.

• Plasma: Collect blood with an anticoagulant such as heparin, citrate or EDTA and mix by inversion. Centrifuge the blood at 1000 x g at 4 °C for 10 minutes. Collect plasma supernatant without disturbing the white buffy layer. Sample should be tested immediately or frozen at -80 °C for storage. Plasma must be diluted before assaying (1:20 to 1:100 in 1X Assay Buffer).
• Serum: Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant 4 without disturbing the white buffy layer. Samples should be tested immediately or frozen at -80 °C for storage. Serum must be diluted before assaying (1:20 to 1:100 in 1X Assay Buffer).
• Saliva: Samples should be tested immediately or frozen at -80 °C for storage. Saliva must be diluted before assaying (1:20 to 1:100 in 1X Assay Buffer).
• Urine: This kit is not recommended for urine samples.

Each ethanol standard and sample should be assayed in duplicate or triplicate. A freshly prepared standard curve should be used each time the assay is performed.

1. Add 10 μL of the diluted ethanol standards or samples to the 96-well microtiter plate.
2. Maintain all components/mixtures at 4 °C. According to Table 3 (below), prepare the desired volume of Reaction Mixture (based on the # of tests) in the following sequence: a. In a clean tube, add the appropriate volume of deionized water. b. To the water add the corresponding volume of 10X Assay Buffer. Mix well. c. Next, add the corresponding volume of 100X Enzyme Mixture. d. Finally, add the corresponding volume of 50X Colorimetric Probe. Mix well and immediately use. Note: Reaction Mixture will appear slightly pink in color. This is normal background and should be subtracted from all absorbance values. Deionized 10X Assay 100X Enzyme 50X Total Volume # of Tests in Water (mL) Buffer (mL) Mixture (μL) Colorimetric of Reaction 96-well Plate Probe (μL) Mixture (mL) (90 μL/test) 8.7 1 100 200 10 100 4.35 0.5 50 100 5 50 2.175 0.25 25 50 2.5 25 Table
3. Preparation of Reaction Mixture
4. Transfer 90 μL of the above Reaction Mixture to each well (already containing 10 μL of ethanol standard or sample).
5. Cover the plate wells to protect the reaction from light.
6. Incubate at 37 °C for 30 minutes.
7. Read absorbance in the 540-570 nm range on a microplate reader.
8. Calculate the concentration of ethanol within samples by comparing the sample absorbance to the standard curve. Negative controls (without ethanol) should be subtracted. 5