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.