8-hydroxy-2-deoxy guanosine is a key marker for oxidative stress; it is formed during oxidative DNA damage through the oxidation of guanosine bases in DNA. This can occur from normal cellular metabolism or exposure of DNA to moieties, such as reactive oxygen species. 8-hydroxy-2-deoxy guanosine can exist as free nucleosides or incorporated into DNA.
The 8-hydroxy-2-deoxy Guanosine EIA Kit from StressMarq Biosciences can measure this marker from urine, plasma, culture media, cell lysates, tissue samples and saliva. This kit is a competitive ELISA assay utilizing an anti-mouse IgG coated plate and an 8-OH-2-dG tracer (conjugated to acetylcholinesterase (AChE)) to compete with 8-OH-2-dG in samples for binding to an anti-8-OH-2-dG mouse monoclonal antibody. Because the level of 8-OH-2-dG tracer is constant in the wells, the amount of tracer that can bind the monoclonal antibody is inversely related to the concentration of 8-OH-2-dG in standards and samples. The assay is developed using Ellman’s reagent, which contains the substrate for AChE; the reaction generates a yellow color with a strong absorbance at 412nm. The use of an AChE conjugated tracer has a number of benefits over other enzymes used in ELISAs; for example, AChE does not ‘self-inactivate’ and is stable during the experiment and hence the assay can be re-developed if need be.
I have used this kit to measure the formation of 8-OH-2-dG in mammary carcinoma cells after treatment with ionising radiation (IR). IR treatment leads to an increase in reactive oxygen species within cells, which in turn can cause 8-OH-2-dG formation. The protocol for measuring 8-OH-2-dG from cell lysates is slightly more complicated than that of urine, plasma or cell culture medium as you will be measuring both free and DNA incorporated 8-OH-2-dG. This means that DNA needs to be extracted and enzymatically digested (with nuclease P1 and alkaline phosphatase) prior to analysis. This adds cost to performing the experiment since a kit for extracting genomic DNA and the enzymes may need to be purchased.
Once samples are prepared, setting up the assay is relatively straightforward. Buffer, tracer, antibody and standards need to be reconstituted and diluted. A handy feature of this kit is the inclusion of differently colored dyes to add to the tracer and antibody; this enables you to clearly see which wells the different components have been added to. Once the assay is set up, it is incubated at 4°C for 18 hours and then developed. The development time can vary substantially over the development period and so it pays to check the absorbance at a few time points beginning at 30 minutes. The manufacturers indicate that the plate should be read when the absorbance of the ‘maximum binding’ control wells are between 0.3-1.0A.U. Analysis of the data can be done on the software which comes with most new plate readers; however it can also be done in Excel. The kit comes with comprehensive instructions for setting up and analyzing the assay.
I have found this assay to be relatively user friendly; however, the reproducibility within samples was not that good. Although we did experience some variance, (in one sample we had a standard deviation that was 15% of the average value), this did not preclude our ability to detect significant differences between groups of samples