Measurement of 8-Hydroxy-2′-Deoxyguanosine by a Commercially Available ELISA Test: Comparison with HPLC/Electrochemical Detection in Calf Thymus DNA and Determination in Human Serum

Abstract

Abstract 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a commonly used biomarker to assess oxidative lesions to DNA. Commercially available ELISA tests could provide a fast and simple method to measure this adduct. However, overestimations attributed to a lack of specificity of antibodies have been observed. We therefore decided to evaluate a commercial kit in calf thymus DNA oxidized with various concentrations of hydrogen peroxide. The comparison of ELISA with HPLC associated with electrochemical detection (EC) showed a good correlation with r = 0.962 (p<0.001) and a linear regression slope of 0.893 (95% Confidence Interval (CI) 0.738–1.080). Determination of 8-OHdG in serum could allow an integrated measure of damage and repair, but studies in this biological fluid are very limited, also due to lack of validated analytical procedures. Results were less convincing than those obtained with calf thymus DNA when the commercial assay was evaluated for determinations in human serum samples (n = 15). Measurements with this ELISA indeed gave values a thousand fold higher compared with those obtained by other laboratories with carbon-column liquid chromatography and ELISA after monoclonal antibody column chromatography. Various pretreatment steps or cross-reactivity reactions could explain these discrepancies. Our data suggest that the commercially available ELISA kits perform better for measurement of 8-OHdG in vitro experiments with DNA than for determinations of free 8-OHdG in more complex biological media such as serum.