Reduction of dilution error in ELISAs using an internal standard.
BACKGROUND
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.


METHODS
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.


CONCLUSIONS
Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.
Merck & Co., Inc., 770 Sumneytown Pike, PO Box 4, West Point, PA 19486, USA.
Thorsten
T
Verch
Merck & Co., Inc., 770 Sumneytown Pike, PO Box 4, West Point, PA 19486, USA.
Christopher
C
Roselle
Merck & Co., Inc., 770 Sumneytown Pike, PO Box 4, West Point, PA 19486, USA.
Mary
M
Shank-Retzlaff
D000165:Acridine Orange / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D007201:Indicator Dilution Techniques
D005456:Fluorescent Dyes; D000165:Acridine Orange
ELISA; dilution bias; internal standard