8-Hydroxy-2-Desoxyguanosine ELISA Kit

Catalog No. ABIN956125

Product Details

Target: 8-Hydroxy-2-Desoxyguanosine

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Analytical Method: Quantitative

Characteristics: 1. The anti-8-OHdG monoclonal antibody and the sample or calibrator are added to the microtiter plate which has been pre-coated with 8-OHdG. The 8-OHdG monoclonal antibody reacts competitively with the 8-OHdG bound on the plate and the 8-OHdG in sample solution. Therefore higher concentrations of 8-OHdG in the sample solution lead to a reduced binding of the antibody to the 8-OHdG on the plate.
2. The antibodies which are bound to the 8-OHdG in the sample are washed away from the antibodies that have bound to the 8-OHdG coated on the plate.
3. An enzyme-labeled secondary antibody, which is added to the plate, binds to the monoclonal antibody which is bound to the 8-OHdG coated on the plate.
4. Unbound HRP-conjugated secondary antibody is removed by washing.
5. Addition of the substrate solution results in the development of color in proportion to the amount of anti-8-OHdG antibody bound to the plate.
6. The reaction is terminated by the phosphoric acid, and absorbance at 450 nm is measured.

Components: Pre-coated 8-OHdG Microtiter Plate: 96-wells (split type)
Primary Antibody: Anti 8-OHdG monoclonal antibody
Primary Antibody solution: Phosphate buffered saline, 6 mL
Secondary Antibody: HRP-conjugated anti mouse antibody
Secondary Antibody solution: Phosphate buffered saline, 12 mL
Chromatic Solution: 3,3',5,5'-tetramethylbenzidine, 0.25 mL
Diluting Solution: Hydrogen peroxide/citrate-phosphate buffered saline, 12 mL
Washing Solution (5X): concentrated phosphate buffered saline, 2 x 26 mL
Stop Solution: 1M Phosphoric acid, 12 mL
8-OHdG Calibrator Solution: Purified 8-OHdG (0.5, 2, 8, 20, 80, 200 ng/mL)
Plate Seal: 2 sheets

Material not included: Distilled water
50 µL micropipettor and pipette tips
8-channel micropipettor (50-200 µL) and pipette tips
Reagent trays for 8-channel micropipettor
A 37°C incubator
Microtiter plate reader (measuring wavelength = 450 nm).

Application Details

Plate: Pre-coated

Reagent Preparation:

A. Test Sample
1. Avoid freezing and thawing of samples.
2. Urine: If it's clear, pre-treatment is not necessary. Otherwise, centrifugation at 2,000-5,000 g for 10-15 mins is recommended for opaque samples only.
3. Serum: Blood samples must be separated to serum immediately. To separate interfering substances, filtration of serum using an ultra filter (cut off molecular weight 10,000) is necessary. Pre-treat ultra filter following the maker's manual. In order to reduce deviation, diluting samples by more than 2 times, while paying attention to concentration range is recommended.
4. DNA in tissue: Extraction and digestion of DNA in samples beforehand is necessary.

Assay Procedure:

1. Bring all reagents and samples to room temperature (20-25°C) before use.
   2. Reconstitute the Primary Antibody with the Primary Antibody Solution.
   3. Add 50 µL of sample or calibrator per well.
   4. Add 50 µL of reconstituted primary antibody per well. Shake the plate from side to side and mix fully. Cover the plate with adhesive strip, making sure it is sealed tightly. Incubate at 37°C for 1 hour.
   5. Mix 1 volume of washing solution (5X) with 4 volumes of distilled water.
   6. Pour off contents of wells into sink. Pipette 250 µL of washing solution into each well. After washing thoroughly by shaking the plate from side to side, dispose of washing solution. Invert plate and blot against clean paper towel to remove any remaining washing buffer. Repeat wash 2 more times. (The use of washing machines or aspirators is not recommended)
   7. Reconstitute the Secondary Antibody with the Secondary Antibody Solution.
   8. Add 100 µL of constituted secondary antibody per well. Shake the plate from side to side and mix fully. Cover the plate with an adhesive strip. Incubate 37°C for 1 hour.
   9. At the end of the incubation period, repeat washing as in step
   6.
   10. Prepare substrate solution. Add 1 volume of the Chromatic Solution to 100 volumes of Diluting Solution just before use. Add 100 µL of substrate solution per well. Shake the plate from side to side and mix fully. Incubate at room temperature for 15 mins in the dark.
   11. Add 100 µL of the Reaction Terminating Solution. Shake the plate from side to side and mix fully.
   12. Measure the absorbance at 450 nm using the microtiter plate reader.

Calculation of Results:

1. Use a calibration curve to determine the amount of 8-OHdG present in test samples.
   2. Generate the calibration curve by plotting absorbance versus log (concentration of calibrators).
   3. Use the absorbance values obtained from the test samples to determine the concentrations.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Storage Comment: Store at 4° C until the expiration date. After opening, the kit should be used within 2 weeks.

Expiry Date: The expiry date is stated on the label.

Target Details

Target: 8-Hydroxy-2-Desoxyguanosine

Alternative Name: 8-Hydroxy-2'-Desoxyguanosine

Target Type: Chemical