A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Preexisting antibodies that neutralize AAV pose a barrier to the advancement of AAV gene therapies. This assay is a screening tool to detect neutralizing antibodies against AAV. The main advantage is that it’s cost-effective, time efficient, easy to set up, and requires minimal technical skills, lab equipment, and reagents.

Start plating HT1080 cells on day one by diluting the cells to a concentration of 1 x 10 to the fifth cells per milliliter in pre-warmed complete DMEM media. Then seed 100 microliters of cells per well into clear 96-well flat bottomed plates to the concentration of 1 x 10 to the fourth cells per well. Incubate the plate at 37 degrees Celsius with 5%carbon dioxide overnight for 16 to 22 hours.

On day two, generate serial dilutions of the serum samples of interest in 1.5 milliliter microcentrifuge tubes using pre-warmed complete DMEM. To each tube with diluted serum samples, add 66 microliters of the 7.5×10 to the six viral genome per microliter virus working solution. Mix the virus serum dilution by pipetting and then place the tubes containing the virus serum mixtures in an incubator at 37 degree Celsius with 5%carbon dioxide for 30 minutes to allow potential neutralization to occur.

After 30 minutes, pipette 100 microliters of the virus serum mixture to each well on the 96-well plate containing 1×10 to the fourth cells per well, then wrap the plate in a foil to place in an incubator at 37 degree Celsius with 5%carbon dioxide overnight for 16 to 24 hours. On day three, aspirate the media from the wells of the 96-well plate using a fume hood vacuum without disrupting the adhered cells and add 50 microliters of 4%paraformaldehyde to each well. After wrapping the plate in foil, leave it for 10 minutes at room temperature.

Later, wash and aspirate the cells twice with 200 microliters of PBS at room temperature. After the second wash, pipette 200 microliters of pre-warmed PBS into each well and wrap the plate in foil followed by incubation at 65 degree Celsius for 90 minutes to denature endogenous alkaline phosphatase activity, following incubation at 50 microliters of the freshly prepared dissolved BCIP/NBT into each well and incubate the wrapped plates at room temperature for two to 24 hours. Later, take photos of each well using a 4X objective lens in a light microscope camera, ensuring the consistent use of the same exposure, white balancing, and light settings for all assays.

To analyze the image in ImageJ, select the file and click Open. For the colored images, convert to gray scale by selecting the Image, Type, and 8-bit tabs in order. Then go to the Image tab and select Adjust and Threshold to alter the threshold until all colored areas are colored and red but the background is not.

click on Analyze and Set Measurements and tick the check boxes for Area, Area fraction, Limit to threshold, and Display label before hitting OK.To determine the signal reading of a given well, click on Analyze and Measure so that the percent area column of the popup window displays the signal reading. The study established the optimal viral dosage by adding the AAV6-hPLAP reporter gene in the cells at a range of concentrations of the viral genome. A multiplicity of infection of 15, 000 conferred 36%plate coloration and was selected as the optimal viral dosage.

The representative analysis displays the correlation between coloration and multiplicities of infection. The highest tested concentration that did not affect the linear correlation between coloration and viral concentration was obtained. The study of neutralizing activity on an anti-AAV6 monoclonal antibody against adeno-associate virus or AAV6 at log 10 dilution showed 50%inhibition of AAV6 transduction or TI50 at a concentration of 10 nanograms per milliliter.

In the neutralizing antibody or NAb assay, the degree of AAV neutralization varied within the naive sample population with TI50 titre values ranging from 1/2 to 1/80. The assay results indicated that AAV inhibition TI50 titre values before AAV administration ranged from 1/4 to 1/80. After AAV administration, the TI50 titre increased to between 1/2000 and greater than 1/32, 000, demonstrating a clear contrast in titre values between pre-and post-AAV administration.

When taking photos of the wells, it is important to consistently use the same microscope settings throughout the assay and to also make sure that the photos are taken directly in the center of the wells.