Reduction of dilution error in ELISAs using an internal standard

Dilution bias is a major cause of immunoassay variability due to the lack of an internal standard to determine the true versus the expected dilution value.

We used an internal control to measure dilution bias in an ELISA. Acridine-orange was added at the first dilution step and monitored throughout dilutions. Assay results were corrected using the fluorescent signal ratio between samples and reference. Acridine dilution correlated with analyte-specific assay measurements (R2 = 0.987). Correction of assay results with the measured dilution factor improved both accuracy and precision resulting in a reduction of >50% %CV reduction.

Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.