Contents of Kit
The DNA Damage ELISA kit contains the following components in sufficient quantities for 96 wells. These reagents are sufficient to assay one standard curve and 39 samples in duplicate or two standard curves and 30 samples in duplicate.
1. 8-OHdG Immunoassay Plat, 96 well plate 12 x 8 removable strips and plate frame. Pre-coated plate with 8-OHdG: BSA conjugate
2. 8-OHdG Standard, 25 μL, 10 μg/mL stock solution of 8-hydroxy-2'deoxyguanosine
3. Sample Diluent, 50 mL Buffer to dilute standards and samples
4. 20× Wash Buffer, 100 mL Concentrated solution of buffer and surfactant
5. Anti-8-OHdG, 25 μL Monoclonal antibody specific for 8-OHdG
6. Antibody Diluent, 6 mL Buffer for dilution of Anti-8-OHdG
7. Anti-Mouse IgG: HRP Conjugate, 25 μL Anti-Mouse IgG conjugated to horseradish peroxidase
8. HRP Conjugate Diluent, 11 mL Buffer for dilution of Anti-Mouse IgG: HRP Conjugate
9. TMB Substrate, 10 mL Stabilized tetramethylbenzidine substrate
10. Stop Solution 2, 10 mL Acid stop solution to stop color reaction
11. Plate Sealer Two adhesive plate sealers
Storage
All reagents are stable as supplied at 4°C, except the 8-OHdG Standard, which should be stored at -20°C. For optimum storage, the 8-OHdG Standard should be aliquotted into smaller portions and stored at -20°C. Avoid repeated freeze/thaw cycles.
Unused wells of the 8-OHdG Immunoassay Plate should be resealed with desiccant in the foil pouch provided and stored at 4°C until the kit's expiry date.
Precision
1. Intra-Assay Precision (Within Run Precision)
To determine 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 < 10%.
2. Inter-Assay Precision (Between Run Precision)
To determine 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 < 10%.
General Description
The DNA Damage ELISA (enzyme-linked immunosorbent assay) is a fast and sensitive competitive immunoassay for the detection and quantitation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in urine, serum, and saliva samples. 8-OHdG has become a frequently used biomarker of oxidative DNA damage and oxidative stress. Measurement of urinary 8-OHdG may be useful as an indicator of oxidative damage.
The DNA Damage ELISA uses an 8-OHdG monoclonal antibody to bind, in a competitive manner, 8-OHdG in the sample, standard or pre-bound to the wells of the 96-well immunoassay plate. Anti-8-OHdG bound to 8-OHdG in the sample or standard are washed away while those captured by the immobilized 8- OHdG are detected with a secondary antibody: HRP conjugate. The assay is developed with tetramethylbenzidine substrate and the absorbance is measured in a microplate reader at 450nm. The intensity of the yellow color is inversely proportional to the concentration of 8-OHdG.
Intracellular and extracellular free radical species can be potentially damaging to the living cell. Intracellular free radical species are produced as a result of normal metabolism and extracellular forms are produced as a result of ultraviolet radiation or ionizing radiation. Reactive oxygen species (ROS) are of particular interest in the research of oxidative damage and disease. The various ROS include the highly reactive hydroxy radical (●OH), superoxide radical (O2 ●- ), hypochlorite ion (OCl●- ) and non-radical hydrogen peroxide (H2O2). DNA, lipids, and proteins are cellular targets for oxidative damage by ROS and the order of preference for modification depends on location of ROS production, availability of metal ions, and the relative ability for the target to be oxidized1 . Cells have acquired a number of defense mechanisms to cope with oxidative damage by ROS and other free radicals. The simplest defense mechanisms involve Vitamin C and E intercepting free radicals, becoming radicals themselves and protecting cellular biomolecules from damage. Complex defense mechanisms involve enzymes such as superoxide dismutase, catalase and glutathione peroxidase that have evolved to reduce ROS levels. Low background levels of damage occur even in normal cells because ROS have a tendency to escape the defense mechanisms. However, when the defense mechanisms cannot prevent the accumulation of ROS, then there is an increase in cellular damage.
Modified lipids and proteins are removed via normal lipid-, protein- turnover mechanisms. However, modified DNA cannot be replaced and has to be repaired. Numerous DNA repair mechanisms have evolved in the cell and have become the focus of research in many disease states. Removal of DNA damage and restoration of the continuity of the DNA duplex response, activation of the DNA damage checkpoint, which stops cell cycle and prevents the transmission of damaged chromosomes, changes in the transcriptional response of the cell and apoptosis are some of the important DNA damage response reactions. 8-hydroxy-2'-deoxyguanosine (8-OHdG) is a modified nucleoside base, which is the most commonly studied and detected by-product of DNA damage that is excreted in the urine upon DNA repair. Urinary 8-OHdG and its analogs, 8- hydroxyguanosine and 8-hydroxyguanine, are linked to many degenerative diseases. The association of ROS and the use of 8-OHdG as a biomarker of oxidative stress have been investigated in many diseases, including bladder and prostate cancer cystic fibrosis, atopic dermatitis and rheumatoid arthritis. Parkinson's disease, Alzheimer's disease and Huntington's disease are neurodegenerative diseases that are thought to be caused by exposure to neurotoxins in people with a genetic predisposition for these diseases. Oxidative stress is associated with the pathogenesis of these diseases and elevated levels of DNA damage have been measured in a wide range of neurological conditions.
CRITICAL ASSAY PARAMETERS AND NOTES
1. The DNA Damage ELISA kit contains a pre-coated microtiter plate (8-OHdG Immunoassay Plate) with removable wells to allow assaying on separate occasions. Run both standards and samples in duplicate.
2. Include a standard curve each time the assay is performed.
3. The following kit components should be brought to room temperature prior to use: 8-OHdG Immunoassay Plate, Sample Diluent, Wash Buffer, Antibody Diluent, HRP Conjugate Diluent, TMB Substrate, Stop Solution 2.
4. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents are ready to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
5. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 15 minutes.
Mix all reagents and samples gently, yet thoroughly, prior to use. Avoid foaming of reagents.
6. To avoid cross contamination, change disposable pipette tips between the addition of each standard, sample, and reagent. Use separate reagent troughs/reservoirs for each reagent.
7. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated.
8. Consistent, thorough washing of each well is critical. If using an automatic washer, check washing head before use. If washing manually, ensure all wells are completely filled at each wash. Air bubbles should be avoided.
9. Exercise appropriate laboratory safety precautions when performing this assay.
10. In this protocol, room temperature refers to 20-28°C. The room temperature should remain within this range throughout the assay.
Citations
COA
Certificate of Analysis
