TABLE 1 

Results from assay robustness DoEsa

SerotypeRSD (%)
DayPrimary Ab
time
Primary Ab
temp
Secondary Ab
time
Secondary Ab
temp
ResidualTotal
10.000.000.000.002.8013.2513.95
31.515.5212.970.001.6110.8719.07
40.624.400.000.003.0714.1715.72
50.006.420.001.161.5414.0116.32
6A0.526.7311.690.002.2813.8721.01
6B0.000.003.290.942.0612.1313.35
7F0.619.298.201.130.8613.6119.97
9V1.951.510.000.291.7911.3112.28
140.000.000.000.003.0212.7913.65
18C0.500.610.000.001.8310.7511.61
19A0.950.043.970.001.3413.5518.89
19F1.293.960.000.002.6312.4213.80
23F0.530.006.310.002.8911.5913.86
  • a Primary and secondary antibody (Ab) incubation times, 60, 90, and 120 min; primary and secondary Ab incubation temperatures, 18, 20, and 25°C. Factorial design of experiments (DoEs) was performed to evaluate assay robustness. The following factors were studied: day (analyst), primary antibody incubation time and temperature, and secondary antibody incubation time and temperature. Primary and secondary antibody incubation times were evaluated in a range from 60 to 120 minutes and primary and secondary incubation temperature in a range from 18 to 25oC. Percent relative standard deviation (%RSD) was estimated for variations in test sera concentrations due to the factors evaluated. For each serotype, a variance component analysis was carried out to estimate the variability of each factor. The individual components of %RSD are not additive, because the effects of each component have a different weight on the total %RSD. The %RSD total is calculated by using the following formula (36): RSD = loge(1 + %RSD2).