Binding Sites and Reactivity
The A20 and A20R antibodies used for the AAV2 Titration ELISAs (PRATV & PRAAV2R) specifically recognize assembled AAV2 capsid, hence epitopes that result from a specific conformational assembly of the capsid proteins VP1, VP2 and VP3. Several publications cited below describe the analysis of binding sites for the A20 antibody using different techniques. Multiple amino acids have been identified, that are very likely to be part of the binding site, especially those that were identified with more than one method (green boxes).
The amino acids of each binding site are located in different parts of the protein chains and are recognized as the epitope of the antibody only in the assembled capsid where they are in close proximity to each other and in the correct conformation. Note that they do not react with denaturated AAV2-VP.
- McCraw et al. Structureof adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20. Virology (2012) 431:40-9.
- Wobus, C. E. et al. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection. J. Virol. 74, 9281–93 (2000).
- Huttner et al. Genetic modifications of the adeno-associated virus type 2 capsid reduce the affinity and the neutralizing effects of human serum antibodies. Gene Ther (2003) 10:2139-47.
- Lochrie et al. Mutations on the external surfaces of adeno-associated virus type 2 capsids that affect transduction and neutralization. J Virol. (2006) 80:821-34.
The A20 and the A20R antibody were tested for neutralization using different antibody concentrations. After the HeLa cells were seeded in a 96-well plate they were incubated for 48h with the AAV2 particles containing a GFP reporter and the indicated concentration of the A20 or A20R antibody, respectively.
The microscopic analysis of the cells clearly demonstrates the neutralizing activity of the A20 and A20R antibody by decreasing GFP expression with increasing antibody concentration.
- Moskalenko, M.et al. Epitope Mapping of Human Anti-Adeno-Associated Virus Type 2 Neutralizing Antibodies: Implications for Gene Therapy and Virus Structure. Jounal Virol.74, 1761–1766 (2000).
- Wobus, C. E. et al. Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection.J. Virol.74, 9281–93 (2000).
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