Extended Data Fig. 2: Normalized S-curves for determining the 30% OD450 ELISA endpoint and PRNT50 in age-panel sera.

(a) Sera were 5-fold diluted starting at a dilution of 1:10 and added to plates coated with a VACV-infected cell lysate. A positive reference control was included on every plate to generate an S-curve with minimum (0%) and maximum (100%) OD450 values. All sample OD450 values were normalized to the positive control on the respective plate and plotted as log (inhibitor) vs response curve with four parameter variable slope using GraphPad Prism 9.02. A 30% endpoint dilution was estimated by calculating the proportionate distance between two dilutions, from which a titer was determined. 30% endpoint titers are shown in Fig. 1a. (b) Sera were 2-fold diluted starting at a dilution of 1:10 in duplicate, followed by the addition of virus suspension leading to ± 500 plaques/well in infection controls. Percentage plaque reduction compared to the infection control was calculated per dilution and plotted as log (inhibitor) vs response curve with four parameter variable slope using GraphPad Prism 9.02. The dilution that would yield 50% reduction of plaques compared to the infection control was estimated by determining the proportionate distance between two dilutions, from which an endpoint titer was calculated. PRNT50 values are shown in Fig. 1b. Symbols indicate individual donors, gray lines represent samples from the age-panel, purple lines represent the controls from the different ELISA plates. All samples from this serum panel were run in the same ELISA or PRNT experiment, avoiding any possible batch effects.