Cell-Based AAV Neutralizing Antibody Assay: A Colorimetric Technique to Detect Neutralizing Antibodies Against Specific Adeno-Associated Viruses in Serum

Recombinant adeno-associated viruses, rAAVs, genetically engineered, non-enveloped, single-stranded DNA viruses, are promising therapeutic gene carriers.

rAAVs attach to target cell membrane receptors and undergo clathrin-mediated endocytosis, facilitating nuclear delivery of the rAAV genome and therapeutic gene expression. However, neutralizing immune system antibodies directed against rAAVs bind to the specific rAAV epitope, interfering with cell membrane binding, cellular entry, and gene transfer.

To detect neutralizing antibodies against specific rAAVs in sheep serum, first, obtain appropriate concentrations of diluted serum. Add recombinant AAVs expressing the human placental alkaline phosphatase, hPLAP, gene. Allow neutralizing antibodies to bind and neutralize AAVs.

Transfer this mixture to the wells of a multi-well plate containing adherent human epithelial cell culture. Upon incubation, AAVs undergo cellular endocytosis. Transduced cells express cell membrane-bound hPLAP. However, neutralized AAVs cannot enter the cells.

Discard the medium from the wells. Use paraformaldehyde to fix the cells. Add pre-warmed buffer, and heat the cells to inactivate the endogenous cellular alkaline phosphatase; hPLAP is resistant to heat inactivation.

Add the hPLAP substrates - bromochloroindolyl phosphate, BCIP, and nitro blue tetrazolium, NBT. hPLAP dephosphorylates BCIP, which undergoes dimerization, forming a blue intermediate. The released hydrogen ions reduce NBT, producing an insoluble purple precipitate. Image to observe purple deposits surrounding the transduced cells.

A lower proportion of purple area suggests an increased concentration of AAV-neutralizing antibodies.

Start plating HT1080 cells on day one by diluting the cells to a concentration of 1 x 10⁵ cells/mL 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 10⁴ 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 x 10⁶ viral genome per microliter virus working solution. Mix the virus/serum dilutions by pipetting, and then, place the tubes containing the virus/serum mixtures in an incubator at 37 degrees 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 x 10⁴ cells per well, then, wrap the plate in a foil to place in an incubator at 37 degrees 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 degrees Celsius for 90 minutes to denature endogenous alkaline phosphatase activity.

Following incubation, add 50 microliters of the freshly prepared, dissolved BCIP/NBT into each well, and incubate the wrapped plates at room temperature for 2 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.