High-throughput Neutralization Assay of AAV-GFP Vectors Using Neutralizing Antibodies

In this experiment, the Celigo™ image cytometer simultaneously imaged and quantified GFP expression after target cells were treated with different concentrations of nAbs.

1. The target cells were seeded in a 96-well plate and allowed to adhere overnight
2. Cells were transduced with AAV-A vector to low Multiplicity of Infection (MOI)
3. Cells were pre-incubated overnight with different dilutions of nAbs and sera at 1X and 5X dilutions
4. The Celigo was used to image and analyze the cells in brightfield and GFP fluorescence to determine transduction efficiencies

Antibody samples exerted dose-dependent neutralization effects on transduction efficiencies

The Celigo was able to directly measure the GFP+ cell percentages in the presence or absence of different concentrations of nAbs. The GFP fluorescent images and analysis results are shown in Figure 1 and Figure 2 respectively. From the GFP images, the Celigo automatically segments individual cells, allowing rapid quantification of the GFP+ percentage of total cells when compared to brightfield. 

• The GFP fluorescent images clearly showed that 1X diluted nAb samples dramatically reduced GFP+ percentages (<1%)4
• The graph confirms that the percentage of GFP-expressing cells was slightly higher for the 5X dilution (~18% of cells)
• GFP+ percentages were comparable between the nAb and sera at both 1X and 5X dilutions