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Biochem Biophys Res Commun. 2013 Nov 1;440(4):725-30. doi: 10.1016/j.bbrc.2013.09.133. Epub 2013 Oct 8.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine values determined by a modified ELISA improves agreement with HPLC-MS/MS.

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1
Department of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, Prague 142 20, Czech Republic. Electronic address: prossner@biomed.cas.cz.

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.

KEYWORDS:

8-OxodG; Discrepancy; ELISA; HPLC–MS /MS; Oxidative stress; Urine

PMID:
24120947
DOI:
10.1016/j.bbrc.2013.09.133
[Indexed for MEDLINE]
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