8-Hydroxy-2-Desoxyguanosine ELISA Kit

Catalog No. ABIN956126

Product Details

Target: 8-Hydroxy-2-Desoxyguanosine

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Competition ELISA

Application: ELISA

Analytical Method: Quantitative

Characteristics: 1. The 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 enzyme-labeled secondary antibody is removed by a wash step.
5. Addition of a chromatic substrate results in the development of color in proportion to the amount of antibody bound to the plate.
6. The color reaction is terminated and absorbance 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 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: 0.125, 0.25, 0.5, 1, 4, 10 ng/mL (1 mL each)
Plate Seal: 2 sheets

Dilute the washing solution 5 times (v/v) with distilled water for use.

Material not included: 50 µL pipettor and tips
8-channel micropipettor (50-200 µL) and pipette tips
Reagent trays for 8-channel micropipettor
Refrigerator
Microtiter plate reader (450 nm).

Application Details

Plate: Pre-coated

Reagent Preparation:

A. Test Sample
1. Avoid freezing and thawing of samples. In order to confirm the aptitude of assay methods on a new sample, implementing recovery test of 8-OHdG calibrator added into the new sample is recommended.
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. Allow complete dissolving.
   3. Add 50 µL of sample or calibrator per well. To insure accuracy, do not use outer most wells.
   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 4°C for overnight.
   5. Pour off contents of wells into sink. Pipette 250 µL 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.
   6. Reconstitute the secondary antibody with the secondary antibody solution. Allow complete dissolving.
   7. 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 in room temperature for 1 hour.
   8. At the end of the incubation period, repeat washing as in step 5.
   9. Reconstitute the chromatic solution (enzyme substrate solution) with 100 times volume of the diluting solution. Add 100 µL of the reconstituted enzyme substrate per well. Shake the plate from side to side and mix fully. Incubate at room temperature for 15 mins in the dark, i.e. shield the plate with aluminum foil.
   10. Add 100 µL of the stop solution. Shake the plate from side to side and mix fully.
   11. Measure the absorbance at 450 nm using the mircotiter 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. The kit is stable until the expiration date shown on the label.

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