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Published on: May 4, 2012
Poor agreement between commercial ELISAs for plasma fetuin-A: An effect of protein glycosylation?
Edward R Smith1, Martin L Ford, Laurie A Tomlinson
1Department of Clinical Biochemistry & Immunology, Brighton & Sussex University Hospitals NHS Trust, Eastern Road, Brighton, BN2 5BE, UK. Edward.smith@bsuh.nhs.uk
Insights
Conflicting results on fetuin-A
Area of Science:
- Biochemistry
- Clinical Chemistry
- Nephrology
Background:
- Fetuin-A inhibits ectopic calcification.
- Associations between low fetuin-A and adverse outcomes in Chronic Kidney Disease (CKD) are inconsistent.
- Methodological differences may explain conflicting findings.
Purpose of the Study:
- To compare two commercial ELISA kits for measuring plasma fetuin-A.
- To investigate the impact of fetuin-A glycosylation on assay specificity.
Main Methods:
- Comparison of fetuin-A measurements using Biovendor and Epitope Diagnostics ELISA kits.
- Analysis of samples from patients with and without CKD.
- Evaluation of assay specificity concerning fetuin-A glycosylation.
Main Results:
- Poor agreement between the two ELISA kits was observed (R²=0.694).
- The Epitope Diagnostics kit showed a positive bias.
- The Epitope Diagnostics kit exhibited greater specificity for deglycosylated fetuin-A.
Conclusions:
- Discrepancies in reported associations of fetuin-A with biological variables may stem from differences in ELISA method specificity.
- Assay specificity for glycosylated versus deglycosylated fetuin-A is a critical factor.
Background:
Fetuin-A is a circulating inhibitor of ectopic calcification. Low plasma levels have been associated in some studies with increased vascular calcification, aortic stiffness and mortality in patients with Chronic Kidney Disease (CKD). However, there are other studies examining the association of fetuin-A with vascular parameters and mortality, which do not show these associations. These conflicting data may be explained by methodological differences.
Methods:
We compared plasma fetuin-A measurements made with two widely-used commercial fetuin-A ELISA kits (Biovendor, Modrice, Czech Republic; Epitope Diagnostics Inc., San Diego, US) in samples from patients with and without CKD. We evaluated the effect of differences in fetuin-A glycosylation status on assay specificity.
Results:
Deming regression analysis showed poor agreement between methods (for CKD cohort: y=-0.05+2.52x, S(y|x)=0.099g/L, R(2)=0.694). The Epitope Diagnostics kit demonstrated significant positive bias and greater specificity for deglycosylated fetuin-A relative to the Biovendor assay.
Conclusion:
The apparently contradictory nature of reports of the association of fetuin-A with biological variables may reflect differences in the specificity of different ELISA methods for glycosylated plasma fetuin-A.
