Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine.
While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 +/- 0.53 pmol/mumol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 +/- 0.39 pmol/mumol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.
Cooke
Marcus S
MS
Radiation and Oxidative Stress Group, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester, LE2 7LX, UK. msc5@le.ac.uk
Singh
Rajinder
R
Hall
Georgina K
GK
Mistry
Vilas
V
Duarte
Tiago L
TL
Farmer
Peter B
PB
Evans
Mark D
MD
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D002853:Chromatography, Liquid / Q000379:methods*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D012463:Saliva / Q000737:chemistry*
D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine