8-hydroxy-2-deoxyguanosine (8-OHdG) is produced by the oxidative damage of DNA by reactive oxygen and nitrogen species and serves as an established marker od 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-OHdG are associated with the aging process as well a number of pathological conditions including cancer, diabetes, and hypertension.
In complex samples such as plasma, cell lysates, and tissues, 8-OHdG can exist either as the free nucleoside or incorporated in DNA. Once the blood enters the kidney, free 8-OHdG 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-OHdG than plasma. Urinary levels of 8-OHdG range between 2.7-13 ng/mg creatine, while plasma levels of free 8-OHdG have been reported to be between 4-21 pg/mL as determined by LC/MS.
ICT’s DNA Damage (8-OHdG) ELISA 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-deoxyguanosine-coated plate and an HRP-conjugated 8-OHdG specific detection antibody. This produces an assay with a useful range of 0.94 - 60 ng/mL, and a sensitivity of 0.59 ng/mL. Other highlights of this kit include a quick incubation time (60 minutes), stable reagents, and a user-friendly 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.
- Prepare the standard and samples in the Sample and Standard Diluent.
- Add 50 µL of prepared standards and samples in triplicate to appropriate wells.
- Add 50 µL of the diluted antibody preparation to the appropriate wells.
- Cover plate with Plate Cover and incubate at room temperature (20-25°C) for 1 hour.
- Dilute 10X Wash Buffer 1:10 with diH2O.
- Wash plate 4 times with 1X Wash Buffer.
- Add 100 µL TMB Substrate to each well.
- Cover plate with Plate Cover and incubate at room temperature in the dark for 30 minutes to develop the signal.
- Add 100 µL Stop Solution to each well.
- Measure absorbance using a plate reader at 450 nm.
- Plot the standard curve and calculate sample concentrations.