Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers.
BACKGROUND
8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer.


METHODS
We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep-anti-mouse antibody.


RESULTS
The concentration range of the calibration curve was 0-60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5+/-38.2 ng/mg creatinine) and prostate cancer (58.8+/-43.4 ng/mg creatinine) as compared to the healthy control (36.1+/-24.5 ng/mg creatinine).


CONCLUSIONS
Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.
Chiou
Chiuan Chian
CC
School of Medical Technology, Chang Gung University, Taoyuan 333, Taiwan.
Chang
Pi Yueh
PY
Chan
Err Cheng
EC
Wu
Tsu Lan
TL
Tsao
Kuo Chien
KC
Wu
James T
JT
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D015415:Biomarkers; D002138:Calibration; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D008297:Male; D018384:Oxidative Stress / Q000502:physiology*; D011471:Prostatic Neoplasms / Q000652:urine*; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D001749:Urinary Bladder Neoplasms / Q000652:urine*
D015415:Biomarkers; D011933:Reagent Kits, Diagnostic; D006151:Guanosine; C046215:8-hydroxyguanosine; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine