(T1130-01-08) Labelling AAV5 Capsid Reduced Its Binding Affinity to the Anti-AAV5 Antibody in Non-primate Human Serum Samples

Purpose: a. The adeno-associated viruses (AAVs) are the most popular “messenger” vector for gene therapy so far. Anti-AAV antibodies produce an immune barrier to the AAV transduction with resultant adverse effects. Thus, it is necessary to prescreen Anti-AAV5 antibodies prior to the treatment.
b. Current commercial monoclonal Anti-AAV5 Antibodies specifically react with intact AAV5 particles, either empty or full capsid. Most antibodies only recognize the conformational epitope of assembled capsid, not denatured or native but unassembled capsid. Polyclonal Anti-AAV5 Antibodies have lower binding affinity to the AAV5 capsid compared to monoclonal antibodies.
c. To identify the affinity of conjugated-AAV5 capsid to various antibodies, biotinylated or ruthenylated-AAV5 capsids were applied to detect various Anti-AAV5 Antibody in the non-human primate serum samples using the classic ADA bridging assay.

Methods: a. The AAV5 binding affinity to various commercial monoclonal or polyclonal Anti-AAV5 antibodies was tested using a generic sequential ELISA ligand binding assay.
b. The binding activity of biotin or ruthenium conjugated AAV5 capsid to various Antibodies were tested in the classic ADA bridging assays.

Results: a. The unlabelled AAV5 has the higher binding affinity to Monoclonal anti-AAV5 Antibody compared to Polyclonal Anti-AAV5 Antibody. The appropriate assay buffer (Assay buffer 1) improved the AAV5 capsid binding activity to various Anti-AAV5 antibodies in the ligand binding assay.
b. The biotinylated or ruthenylated-AAV5 capsids attenuated the binding affinity to various Anti-AAV5 antibodies in non-primate human serum samples by sequential and one step ADA bridging assay.

Conclusion: In summary, the surface conformational epitope changes of the AAV5 capsid after conjugation may have occurred resulting in the reduced binding affinity to the various Anti-AAV5 Antibodies in non-human primate serum samples in the classic ADA bridging assay. Our observation helped us to develop an alternative method for the detection of Anti-AAV5 total Antibody in the non-human primate samples in the near future.


The binding affinity of Biotinylated or Ruthenylated AAV5 Capsid to various Anti-AAV5 Abs