Table 2. Assays used to measure anti-AAV neutralizing antibodies.

Assay	Advantages	Limitations
Binding antibody assay Capsid particles or pep-tides are coated onto test wells and binding antibodies measured with a secondary anti-Ig antibody	-Easy to setup and validate -Equally sensitive for all AAV serotypes -Allows to measure antibody subclasses -Used in clinical trials to prescreen subjects prior to enrollment	-Binding antibodies are not always neutralizing, which increases the risk of excluding subjects who have no neutralizing antibodies
In vitro cell-based assay The neutralizing antibody titer (NAb) is determined by measuring the residual activity of a reporter vector after incubation with a test sample	-Relatively easy to setup -Used in several clinical trials to prescreen subjects prior to enrollment -Directly reflects the neutralization activity of the sample	-Most serotypes do not efficiently transduce cells in vitro -Not very sensitive in detecting low-titer NAb -Validation may be challenging -Uses a reporter vector, not the therapeutic vector, which may have different infectivity
In vivo neutralization assay Residual expression of the vector is measured in mice passively immunized with the test sample	-Works well for most serotypes -Faithfully represents the effect of antibodies on the efficiency of transduction of a target tissue -Has been used in clinical trials to prescreen subjects	-Variability results, need large number of animals -Time consuming and expensive -Difficult to validate