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Boster's ELISA Kit is a competitive assay that can be used for the quantification of 8-OHdG in urine, cell culture, plasma, and other sample matrices. The ELISA utilizes an 8-hydroxy-2-deoxy Guanosine-coated plate and an HRP-conjugated antibody for detection which allows for an assay range of 0.94 - 60 ng/mL, with a sensitivity of 0.59 ng/mL. The other highlights of this kit are a quick incubation time of 60 minutes, stable reagents, and an easy to use protocol. It is important to note that the 8-OHdG antibody used in this assay recognizes both free 8-OHdG and DNA-incorporated 8-OHdG. Since complex samples such as plasma, cell lysates, and tissues are comprised of mixtures of DNA fragments and free 8-OHdG, concentrations of 8-OHdG reported by ELISA methodology will not coincide with those reported by LC-MS where the single nucleoside is typically measured. This should be kept in mind when analyzing and interpreting experimental results.
| Product Name | DNA Damage (8-OHdG) ELISA Kit |
|---|---|
| SKU/Catalog Number | EK7114 |
| Description | Colorimetric detection of DNA Damage (8-OHdG). 96wells/kit, with removable strips. |
| Cite This Product | DNA Damage (8-OHdG) ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK7114) |
| Validated Species | All Animals |
| Application | ELISA *Our Boster Guarantee covers the use of this product in the above tested applications. |
| Cross Reactivity | 8-Hydroxy-2-deoxy Guanosine (8-OHdG): 100%. 8-Hydroxy Guanosine (8-OHG): 23%. 8-Hydroxy Guanine (8-oxoG): 23%. Guanosine: <0.01%. |
| Pack Size | 96wells/kit, with removable strips. |
| Sensitivity | 0.59 ng/mL *Sensitivity, or Lower Limit of Detection (LLD), is the minimum level of target protein the ELISA assay can detect. We measure 20 blank wells and if the O.D. value is 2 standard deviations higher than the blanks' average O.D. the sample can be deemed positive. |
|---|---|
| Assay Range | 0.94 - 60 ng/mL *This assay range is determined using common samples. For samples with low target protein concentrations, users can adjust the standard curve to extend the lower limit of assay range. |
| Sample Type | Cell lysates, Plasma, Sample matrices, Urine *The above listed samples are the ones valided with the assay. If you do not see your sample of interest listed, as long as there is enough level of target protein present in the sample, this Picokine? ELISA kit should detect it. **For protocol and tips regarding preparing your sample of interest, please check our ELISA sample preparation guide. |
| Storage | Store at 4°C and -20°C. (All reagents are stable as supplied at 4°C, except the standard, which should be stored at -20°C. ) |
| Description | Quantity |
| 8-hydroxy-2-deoxy Guanosine : BSA Coated Plate | 1 Plate |
| 8-hydroxy-2-deoxy Guanosine Standard | 1 vial/ 100uL |
| 8-hydroxy-2-deoxy Guanosine HRP Conjugated Monoclonal Antibody | 1 vial/75uL |
| Sample and Standard Diluent | 1 vial/50mL |
| 8-hydroxy-2-deoxy Guanosine Antibody Diluent | 1 vial/13mL |
| Wash Buffer Concentrate | 1 vial/50mL |
| TMB Substrate | 1 vial/ 13mL |
| Stop Solution | 1 vial/ 13mL |
| Plate Cover | 2 covers |
1. A plate reader capable of measuring absorbance at 450 nm.
2. Adjustable pipettes and a repeat pipettor.
3. Deionized or distilled water.
4. Materials used for Sample Preparation.
8-hydroxy-2-deoxy Guanosine (8-OH-dG) is produced by the oxidative damage of DNA by reactive oxygen and nitrogen species and serves as an established marker of oxidative stress. Hydroxylation of guanosine occurs in response to both normal metabolic processes and a variety of environmental factors (i.e., anything that increases reactive oxygen and nitrogen species). Increased levels of 8-OH-dG are associated with the aging process as well as with a number of pathological conditions including cancer, diabetes, and hypertension. In complex samples such as plasma, cell lysates, and tissues, 8-OH-dG can exist as either the free nucleoside or incorporated in DNA. Once the blood enters the kidney, free 8-OH-dG is readily filtered into the urine, while larger DNA fragments remain in the bloodstream. Because of the complexity of plasma samples, urine is a more suitable matrix for the measurement of free 8-OH-dG than plasma. Urinary levels of 8-OH-dG range between 2.7-13 ng/mg creatine, while plasma levels of free 8-OH-dG have been reported to be between 4-21 pg/ml as determined by LC-MS.
Click the images to enlarge.
Typical Standard Curve for the DNA Damage (8-OHdG) ELISA Kit (Enzyme-Linked Immunosorbent Assay)–EK7114 Assay Type: Competitive ELISA. Detection Method: Colorimetric Assay. Assay Range: 0.94 – 60 ng/ml.
Chemical Equation of the Oxidation of Guanosine
Diagram of the 8-OHdG Competitive ELISA
Urine Spike Assay
Diagram of the Preparation of the 8-OHdG Standards
Diagram of the Triplicate Sample Plate Format
Preview of the Calculations Worksheet
Intra-Assay Precision: Three samples of known concentration were assayed thirty times on one plate; the intra-assay coefficient of variation of the DNA Damage ELISA has been determined to be <5%.
Inter-Assay Precision: Three samples of known concentration were assayed thirty times in three individual assays; the inter-assay coefficient of variation of the DNA Damage ELISA has been determined to be <5%.
1. Prepare standard and samples in the Sample and Standard Diluent.
2. Add 50 ?L of prepared standards and samples in triplicate to appropriate wells.
3. Add 50 ?L of the diluted antibody preparation to the appropriate wells.
4. Cover plate with Plate Cover and incubate at room temperature (20-25°C) for 1 hour.
5. Wash plate 4 times with 1X Wash Buffer.
6. Add 100 ?L of TMB Substrate to each well.
7. Cover plate and develop the plate in the dark at room temperature for 30 minutes.
8. Add 100 ?L of Stop Solution to each well. <
9. Measure absorbance on a plate reader at 450 nm.
10. Plot the standard curve and calculate sample concentrations.
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