Table 1.

Summary of method validation

Validation parameter	Testing method	Acceptance criteria	Results
Specificity: competition	Positive samples with competing antigens (homologous and heterologous) at multiple concentrations ≤ 20 µg/mL	Heterologous competitors: ≤ 25% competition Homologous competitors ≥ 75% competition	All antigens and all samples ≤ 25% (heterologous competitors) and ≥ 75% (homologous competitors) at concentrations ≤ 20.0 µg/mL
Specificity: matrix effects	Spike recovery of assay reference standard	Recovery in the range of 100% ± 20% for ≥ 80% of samples for each matrix	100% of samples for each matrix had recovery 83–119%
Accuracy	Spike recovery using a sample of known concentration, spiked into negative/low sera	Recovery of 100% ± 20% (samples > 3× LLOQ) and 100% ± 25% (samples ≤ 3× LLOQ)	80–114% (samples > 3× LLOQ) and 84–124% (samples ≤ 3 × LLOQ)
Precision	Multiple determinations (≥ 5) of a sample panel (≥ 58 samples) for each antigen using at least two instruments for this experiment	Upper 95% CI of overall %GCV ≤ 25% and ≥ 80% samples with %GCV ≤ 20%	Upper 95% CI %GCV of ≤ 13.1% (repeatability) and ≤ 17.0% (intermediate precision) and ≥ 93% (repeatability) and > 88% (intermediate precision) samples had %GCV ≤ 20%
LLOQ verification	Clinical study samples (0.5× to 4× LLOQ) and assay reference standard	Overall %GCV ≤ 25% Spiked samples ≤ 3× LLOQ within ± 25% %RE	Overall %GCV ≤ 13.1% Spiked samples ≤ 3× LLOQ –16% to 24% %RE
Linearity/dilutability	Multiple determinations at 3–6 dilutions	Coefficient of determination > 0.95 and regression line slope 0.80–1.25	Coefficient of determination ≥ 0.9743 and regression line slopes 0.8010–1.1258

GCV geometric coefficient of variation, LLOQ lower limit of quantification, %RE percent relative error