| Application | Enzyme-linked immunosorbent assay |
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| ELISA Kit Linearity | Linearity in urine was determined by taking two samples, one with a low diluted 8-hydroxy-2'-deoxyguanosine level of 793.4 pg/mL and one with a higher diluted level of 5,238 pg/mL, and mixing them in the ratios given below. The measured concentrations were compared to the expected values based on the ratios used. Linearity in serum and EDTA plasma were determined in a similar manner. Urine | High Sample | Low Sample | Expected Conc. (pg/mL) | Observed Conc. (pg/mL) | % Recovery |
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| 80% | 20% | 4,350 | 4,351 | 100.0 | | 60% | 40% | 3,460 | 3,375 | 97.5 | | 40% | 60% | 2,571 | 2,647 | 102.9 | | 20% | 80% | 1,682 | 1,596 | 94.8 | | | | Mean Urine Recovery | 98.8% | Serum | High Sample | Low Sample | Expected Conc. (pg/mL) | Observed Conc. (pg/mL) | % Recovery |
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| 80% | 20% | 1,311 | 1,338 | 102.0 | | 60% | 40% | 1,128 | 1,223 | 108.4 | | 40% | 60% | 945.8 | 968.4 | 102.4 | | 20% | 80% | 763.2 | 736.7 | 96.5 | | | | Mean Serum Recovery 102.3% | EDTA Plasma | High Sample | Low Sample | Expected Conc. (pg/mL) | Observed Conc. (pg/mL) | % Recovery |
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| 80% | 20% | 2,076 | 2,065 | 99.5 | | 60% | 40% | 1,772 | 1,709 | 96.4 | | 40% | 60% | 1,468 | 1,414 | 96.3 | | 20% | 80% | 1,164 | 1,148 | 98.6 | | | | Mean Plasma Recovery 97.7% | |
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| ELISA Kit Principle | The DetectX® DNA Damage Immunoassay Kit is designed to quantitatively measure DNA and RNA oxidized guanosine species. The assay detects all three oxidized guanine species; 8-hydroxy-2’-deoxyguanosine from DNA, 8-hydroxyguanosine from RNA and 8-hydroxyguanine, from digested DNA from DNA or RNA. These species may be present in serum, plasma, saliva, urine, dried fecal samples, and tissue culture media samples. Please read the complete kit insert before performing this assay. An 8-hydroxy-2’-deoxyguanosine (8-OHdG) stock solution is provided to generate a standard curve for the assay and all samples should be read off the standard curve. Standards or diluted samples are pipetted into a clear microtiter plate coated with an antibody to capture rabbit antibodies. An 8-hydroxyguanosine conjugate is added to the standards and samples in the wells. The binding reaction is initiated by the addition of a peroxidase-labeled mouse monoclonal antibody to 8-hydroxy-2’-deoxyguanosine to each well. After a 2 hour incubation, the plate is washed and substrate added. The substrate reacts with the peroxidaselabeled antibody that has reacted with the bound conjugate. After the reaction is stopped, the intensity of the generated color is detected in a microtiter plate reader capable of measuring 450 nm wavelength. The concentration of the 8-hydroxy-2’-deoxyguanosine in the sample is calculated, after making suitable correction for the dilution of the sample, using software available with most plate readers. |
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| ELISA Kit Reproducibility | Intra Assay Precision Three human samples were diluted with Assay Buffer and run in replicates of 20 in an assay. The mean and precision of the calculated 8-hydroxy-2'-deoxyguanosine concentrations were: | Sample | 8-Hydroxy-2'-deoxyguanosine Conc. (pg/mL) | %CV |
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| 1 | 423.6 | 11.7 | | 2 | 995.7 | 8.2 | | 3 | 1,187 | 7.1 | Inter Assay Precision Three human samples were diluted with Assay Buffer and run in duplicates in nineteen assays run over multiple days by five operators. The mean and precision of the calculated 8-hydroxy-2'-deoxyguanosine concentrations were: | Sample | 8-Hydroxy-2'-deoxyguanosine Conc. (pg/mL) | %CV |
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| 1 | 404.9 | 13.4 | | 2 | 887.8 | 8.3 | | 3 | 1,121 | 8.1 | |
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| ELISA Kit Component | | Component | Quantity |
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| Coated Clear 96 Well Plate | 1 or 5 Each | | 8-Hydroxy-2'-deoxyguanosine Standard | 70 uL or 350 uL | | DetectX® 8-Hydroxy-2'-deoxyguanosine Antibody | 3 mL or 13 mL | | DetectX® 8-Hydroxyguanosine EIA Conjugate | 3 mL or 13 mL | | Assay Buffer Concentrate | 28 mL or 55 mL | | Wash Buffer Concentrate | 30 mL or 125 mL | | TMB Substrate | 11 mL or 55 mL | | Stop Solution | 5 mL or 25 mL | | Plate Sealer | 1 or 5 Each | |
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| Additional Information | Background: Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check only by the existence of multiple antioxidant and repair systems as well as the replacement of damaged nucleic acids, proteins and lipids. Intracellular free radical species (ROS) are produced as a result of normal metabolism and extracellular forms are produced as a result of ultraviolet radiation or ionizing radiation. Cellular function may be interrupted or stopped if DNA damage corrupts the integrity of essential information contained in the genome. When individual bases are damaged, nonspecific DNA repair enzymes excise DNA lesions to release deoxynucleotides, and base specific repair glycosylases excise the corresponding base. Deoxynucleotides are enzymatically hydrolyzed to stable deoxynucleosides, and these repair products are transported through the blood and excreted in the urine. Damage to RNA is reflected in nucleoside adducts. It is widely thought that continuous oxidative damage to DNA is a significant contributor to the age-related development of the major cancers, such as those of the colon, breast, rectum, and prostate. Among numerous types of oxidative DNA damage, the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a ubiquitous marker of oxidative stress. 8-OHdG is physiologically formed and enhanced by chemical carcinogens. During the repair of damaged DNA in vivo by exonucleases, the resulting 8-OHdG is excreted without further metabolism into urine. |
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| :: | Detection Limit: 82.2 pg/mL |
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| :: | Cross Reactivity: The following cross reactants were tested in the assay and calculated at the 50% binding point. | Steroid | Cross Reactivity (%) |
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| 8-Hydroxy-2'-deoxyguanosine | 100% | | 8-Hydroxyguanosine | 27.32 | | 8-Hydroxyguanine | 9.50 | |
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| Reconstitution and Storage | 2°C to 8°C |
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| Sample Type | Serum, EDTA and Heparin Plasma, Saliva, Urine, Digested DNA, Fecal Extracts and Tissue Culture Media |
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| Sensitivity | 50.9 pg/mL |
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