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 degrees 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. (C) 2013 Elsevier Inc. All rights reserved.