Fig. 1: Optimized pre-incubation conditions and constant serum concentration yield an improved AAV neutralization assay across different serotypes.

A Neutralization curve for an anti-AAV9 antibody (ADK9) tested against AAV9. Vertical error bars denote 95% credible intervals, and the vertical dashed arrow pointing to the horizontal bar indicates the ND50 estimate and its uncertainty range (“Methods” section). B–D Transduction curves for seven human sera tested against AAV9, AAV1, and AAV5. Each color represents a different donor; the same color denotes the same donor across panels; the x-axis shows 2-fold serum dilutions, and the y-axis shows transduction efficiency relative to an antibody-free control. Solid lines (Constant) stay near 100% for non-neutralizing samples, whereas dashed lines (Variable) can exceed 100% due to changing total serum. The lavender, teal, and plum curves report neutralizing activity sufficient to reach the ND50 within the tested dilution range. ND50 dilutions for neutralizing samples are marked by vertical dashed arrows pointing to horizontal bars indicating 95% credible intervals (“Methods” section). Throughout panels, Constant refers to the CSC method, where total serum remains fixed at 10%, while Variable refers to the VSC method, where total serum concentration increases with less dilution. E Schematic overview of two pre-incubation strategies under antibody-free serum conditions. Top: “Serum on AAV” denotes pre-incubation of AAV with different concentrations of FBS while cells are pre-incubated in DMEM only. Bottom: “Serum on cells” denotes pre-incubation of cells with different concentrations of FBS, while AAV is pre-incubated in DMEM only. F Effect of FBS content during pre-incubation on transduction efficiency, normalized to the 0% FBS condition. Under “Serum on AAV”, luminescence increases sharply and plateaus near 5% FBS, whereas under “Serum on cells,” luminescence decreases as FBS content increases. Vertical error bars indicate standard deviations from the mean. G, H Schematic illustration of design differences between variable serum concentration (VSC, Variable in legends) versus constant serum concentration (CSC, Constant in legends) assay formats. In VSC, total serum changes with each dilution step because test serum is added on top of a fixed FBS level; in CSC, total serum is held at 10% throughout, differing only in the proportion of test serum to FBS. I–K Bar plots comparing neutralization titers (ND50) for the neutralizing sera against AAV9, AAV1, and AAV5 (as shown on B–D). Constant (filled bars) yields significantly higher titers than Variable (striped bars). Asterisks denote significant difference (“Methods” section). For samples exhibiting minimal (ND50 < 1/4) or no neutralization, a 1/1 titer is used as a placeholder. L Sensitivity comparison of Constant vs. Variable using a dilution series of ADK9 against AAV9. Constant shows significantly greater sensitivity (“Methods” section). (E) was created with BioRender.com.