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Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine analysis by an improved ELISA: An inter-laboratory comparison study
Pavel Rossner1, Hilmi Orhan2, Gudrun Koppen3
1Department of Genetic Ecotoxicology, Institute of Experimental Medicine AS CR, Videnska 1083, 14220 Prague, Czech Republic.
Free Radical Biology & Medicine
|March 27, 2016
Summary
Standardizing ELISA methods for urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) improved lab agreement. However, interfering compounds in some samples mean ELISA is not yet a robust alternative to chromatography for oxidative stress assessment.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Biomarkers
Background:
- Urinary 8-oxo-7,8-dihydro-2 extquotesingle-deoxyguanosine (8-oxodG) is a key marker for whole-body oxidative stress.
- Enzyme-Linked Immunosorbent Assay (ELISA) is widely used for 8-oxodG detection but suffers from high inter-laboratory variability and poor agreement with chromatographic methods.
Purpose of the Study:
- To assess the inter-laboratory variability of ELISA for urinary 8-oxodG detection under standardized conditions.
- To compare ELISA results with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
- To identify potential interfering substances affecting ELISA accuracy.
Main Methods:
- An inter-laboratory comparison of ELISA for 8-oxodG was conducted using standardized sample pre-treatment (SPE), a single commercial kit, and strict temperature control.
- ELISA results were compared against HPLC-MS/MS.
- Gas chromatography-mass spectrometry (GC-MS) was used to tentatively identify interfering compounds in outlier samples.
Main Results:
- Standardization significantly improved inter-laboratory agreement for ELISA, with most results within 1SD of the mean.
- ELISA results showed moderate correlation with HPLC-MS/MS (R=0.679), improving to R=0.749 after excluding one laboratory's data.
- Excluding three outlier samples further enhanced agreement (R=0.869). Saccharides like d-glucose and d-galactose were identified as potential interferents in high-outlier samples.
Conclusions:
- Assay standardization enhances ELISA inter-laboratory agreement for 8-oxodG measurement, though some variability persists.
- Specific interfering compounds, particularly saccharides, can lead to overestimation of 8-oxodG by ELISA in certain urine samples.
- Despite improvements, ELISA is not yet a fully robust alternative to chromatographic techniques like HPLC-MS/MS for accurate 8-oxodG quantification.
