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.