High concordance of ELISA and neutralization assays allows for the detection of antibodies to individual AAV serotypes

Figures

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  Figure 1. Correlation between ELISA and neutralization assay for rhesus macaque samples (A) Fifty rhesus macaque samples were screened for preexisting antibodies against the indicated AAV serotype using ELISA (left) and neutralization assay (right). For the ELISA, sera samples were diluted 1:20 and added to plates coated with the indicated serotype. Binding antibodies were determined using an anti-rhesus IgG secondary antibody. For the neutralization assay, sera samples were diluted 1:10 and mixed with an equal volume of the indicated vector. HEK293T cells were then added to the mixture, and luciferase was quantified after 24 h. See also Table S1. (B) Correlation plots comparing the screen results from the ELISA and neutralization for the indicated serotype. Pearson r values, p values (two tailed), and R2 values are included for each comparison.

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  Figure 2. Correlation between ELISA and neutralization assay for human samples (A) Twenty human samples were screened for preexisting antibodies against the indicated AAV serotype using ELISA (left) and neutralization assay (right). Both the ELISA and neutralization assays were performed as described in Figure 1A. See also Table S2. (B) Correlation plots comparing the screen results from the ELISA and neutralization for the indicated serotype. Note that sample h368409 is indicated as the only sample for which binding antibodies were not detected, yet the sample had neutralizing activity. Pearson r values, p values (two tailed), and R2 values are included for each comparison.

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  Figure 3. Screening of anti-AAV antibodies by ELISA after AAV1 or AAV8 inoculation in rhesus macaques (A) Sera samples from three rhesus macaques were screened using ELISA for preexisting binding antibodies to the indicated serotypes pre- and post-AAV1 intramuscular vector inoculation. ELISAs were performed as described in Figure 1. (B) Similar to (A), except the nine rhesus macaques were inoculated with an AAV8 vector.

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  Figure 4. Screening of anti-AAV antibodies by neutralization assay after AAV1 or AAV8 inoculation in rhesus macaques (A) The same sera samples from three rhesus macaques in Figure 3 were screened using neutralization assay for preexisting neutralizing antibodies to the indicated serotypes pre- and post-AAV1 intramuscular vector inoculation. Neutralization assays were performed as described in Figure 1. (B) Similar to (A), except the same nine rhesus macaque samples from Figure 3 were inoculated with an AAV8 vector.

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  Figure 5. Correlation between EISA and neutralization assays for rhesus macaque samples pre- and post-AAV inoculation Correlation plots for ELISA and neutralization assay results presented in Figures 3 and 4 for AAV1, AAV8, and AAV9. All 24 data points are included, 12 from pre-AAV inoculation and 12 from post-AAV inoculation. Pearson r values, p values (two tailed), and R2 values are included for each comparison.

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  Figure 6. Correlation between anti-AAV1 binding and neutralizing antibodies on changes in 4L6 concentrations after an AAV1 booster inoculation Correlation plots comparing the change in 4L6 concentrations from nine rhesus macaques that received AAV1 booster inoculations with AAV1 binding antibodies (ELISA; left) or neutralizing antibodies (neutralization; right). Pearson r values, p values (two tailed), and R2 values are included for each comparison. Note the negative Pearson r values indicates that higher change in 4L6 concentrations were observed with lower binding or neutralizing antibodies.