title: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine values determined by a modified ELISA improves agreement with HPLC-MS/MS.
abstract: ELISA is widely used for urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) analysis. It is the method of choice for laboratories that lack specialized chromatographic instrumentation. It allows fast, high-throughput sample analysis without a need for extensive samples processing. However, a lack of agreement between ELISA and chromatographic methods confines its application to the assessment of relative urinary 8-oxodG levels. We investigated various ELISA modifications, seeking optimal conditions that would yield a good agreement between ELISA and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS /MS). Purification of urine by solid phase extraction (SPE), then incubation with the anti-8-oxodG antibody at 4 °C overnight and subsequent normalization of 8-oxodG levels per urinary creatinine resulted in a near-perfect correlation and agreement in mean levels between ELISA and HPLC-MS /MS (r=0.917, p<0.001; and paired t-test p=0.803, respectively). Our data show that, after introduction of a simple modification, ELISA quantification urinary 8-oxodG substantially improves. Although more sample manipulation is required, the method retains its key advantages over chromatography (high-throughput analysis that does not require expensive instrumentation). This represents a significant advance for the ELISA, and encouraging its use in more studies adding to our knowledge of the role of this biomarker of oxidative stress in health and disease.
authors.affiliation: Department of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, Prague 142 20, Czech Republic. Electronic address: prossner@biomed.cas.cz.
authors.forename: Pavel
authors.initials: P
authors.lastname: Rossner
authors.forename: Vilas
authors.initials: V
authors.lastname: Mistry
authors.forename: Rajinder
authors.initials: R
authors.lastname: Singh
authors.forename: Radim J
authors.initials: RJ
authors.lastname: Sram
authors.forename: Marcus S
authors.initials: MS
authors.lastname: Cooke
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D015415:Biomarkers / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D006801:Humans; D008297:Male; D008875:Middle Aged; D018384:Oxidative Stress; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry / Q000592:standards
chemical_list: D015415:Biomarkers; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine
keywords: 8-OxodG; Discrepancy; ELISA; HPLC–MS /MS; Oxidative stress; Urine