Fig. 3. AAV6 luciferase and negative control bridging.

A Serial dilutions of pooled serum from positive healthy donors were evaluated using the existing lot of AAV6-luciferase at 4e4 MOI (black solid circle and line) and the new lot of AAV6-luciferase at MOI of 7e4 (light blue square and dashed line), 8e4 (lavender diamond and dashed line) and 9e4 (magenta triangle and dashed line); error bars indicate the SD for the respective sample dilution and condition. B More refined serial dilutions of positive pooled serum were evaluated using the existing lot of AAV6-luciferase at a 4e4 MOI in two separate dilution curves (back solid circle and line) and the selected new AAV6-luciferase lot at 8e4 MOI determined from 3A (magenta triangle and dash line). C 30 healthy donors and 6 assay quality controls were evaluated using the existing and new AAV6-luciferase lot at 4e4 and 8e4 MOI, respectively. A linear plot (slope = 0.9662 and R² = 0.9531) of NR response from both reagent lots shows comparable results with 100% concordance in seropositivity. Nine samples within the gray box are seropositive samples. D Bridging of a new negative healthy donor serum pool using the existing and new AAV6-luciferase lots (n = 16 samples). EN/EV existing negative/existing AAV6-luciferase (black), NN/EV new negative/existing AAV6-luciferase (magenta), NN/NV new negative/new AAV6-luciferase (green). The mean NR of the negative control in EN/EV was calculated and individual values were normalized to the mean NR to obtain %CV for EN/EV; all other conditions were normalized to mean NCS from EN/EV for %CV calculation. E 30 healthy donors were evaluated using the existing and new negative control serum lots. A linear plot (slope = 0.9359 and R² = 1.068) of NR response from both reagent lots shows comparable results with 100% concordance in seropositivity. Nine samples within the gray box are seropositive samples. CV coefficient of variation, NCS negative control serum, PC positive control (pool serum), R² R-squared.