The nitrocellulose membrane was blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV9 (intact particle) mouse monoclonal, ADK9 (Cat. No. 690162) was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgA HRP was also diluted in blocking buffer (antibody concentration 250 ng/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using Pierce™ ECL Plus Western Blotting Substrate.
Neutralization of AAV9 with mouse monoclonal AAV9 antibody clone ADK9 (A) and human chimeric AAV9 antibody clone ADK9-h1 (B) by using AAV9-NanoLuc® viral particles from Promega. (A) anti-AAV9 (intact particle) mouse monoclonal, ADK9 (Cat. No. 610162) or (B) anti-AAV9, human chimeric, ADK9-h1 (Cat. No. 692378) were preincubated with AAV9-NanoLuc® viral particles for 30 min at RT at 300 rpm (antibody concentrations 0.2-3,000 ng/ml). HEK293 cells (100 µl) were plated at 200,000 cells/ml in DMEM + 1% FCS. Virus-antibody-mix (20 µl) was added to the cells and incubated for 16-24 h at 37°C. Extracellular NanoLuc Inhibitor and Nano-Glo® Live Cell Assay System (Promega) was added to the wells and incubated for 5 min at RT at 300 rpm. Luminescence was measured using an ID5-Reader and plotted with Softmax Pro 7.1 software to determine the EC50 values.
Comparison of binding affinity and neutralization activity of anti-AAV9 antibodies (mouse monoclonal ADK9, mouse recombinant ADK9-1R and human chimeric ADK9-h1).
For AAV9 the neutralization epitope has been identified and might give an additional indication about the binding residues of the neutralizing antibody ADK9 used for the corresponding AAV9 Titration ELISAs. ADK9 recognizes and neutralizes assembled AAV9 capsids.
Adachi, K., Enoki, T., Kawano, Y., Veraz, M. & Nakai, H. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat. Commun. 5, (2014).
In the study cited below, cryo-electron microscopy and image reconstruction were used to define the epitopes of anti-AAV9 monoclonal antibody, ADK9 on the capsid surface. 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.
Emmanuel, S. N. et al. Structurally Mapping Antigenic Epitopes of Adeno-associated Virus 9: Development of Antibody Escape Variants. J. Virol. 96, (2022).
The nitrocellulose membrane was blocked with 5% dry milk in PBST (PBS + 0.1% Tween 20) for 1 h at RT. The primary antibody anti-AAV9 (intact particle) mouse monoclonal, ADK9 (Cat. No. 690162) was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgA HRP was also diluted in blocking buffer (antibody concentration 250 ng/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using Pierce™ ECL Plus Western Blotting Substrate.
Neutralization of AAV9 with mouse monoclonal AAV9 antibody clone ADK9 (A) and human chimeric AAV9 antibody clone ADK9-h1 (B) by using AAV9-NanoLuc® viral particles from Promega. (A) anti-AAV9 (intact particle) mouse monoclonal, ADK9 (Cat. No. 610162) or (B) anti-AAV9, human chimeric, ADK9-h1 (Cat. No. 692378) were preincubated with AAV9-NanoLuc® viral particles for 30 min at RT at 300 rpm (antibody concentrations 0.2-3,000 ng/ml). HEK293 cells (100 µl) were plated at 200,000 cells/ml in DMEM + 1% FCS. Virus-antibody-mix (20 µl) was added to the cells and incubated for 16-24 h at 37°C. Extracellular NanoLuc Inhibitor and Nano-Glo® Live Cell Assay System (Promega) was added to the wells and incubated for 5 min at RT at 300 rpm. Luminescence was measured using an ID5-Reader and plotted with Softmax Pro 7.1 software to determine the EC50 values.
Comparison of binding affinity and neutralization activity of anti-AAV9 antibodies (mouse monoclonal ADK9, mouse recombinant ADK9-1R and human chimeric ADK9-h1).
For AAV9 the neutralization epitope has been identified and might give an additional indication about the binding residues of the neutralizing antibody ADK9 used for the corresponding AAV9 Titration ELISAs. ADK9 recognizes and neutralizes assembled AAV9 capsids.
Adachi, K., Enoki, T., Kawano, Y., Veraz, M. & Nakai, H. Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing. Nat. Commun. 5, (2014).
In the study cited below, cryo-electron microscopy and image reconstruction were used to define the epitopes of anti-AAV9 monoclonal antibody, ADK9 on the capsid surface. 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.
Emmanuel, S. N. et al. Structurally Mapping Antigenic Epitopes of Adeno-associated Virus 9: Development of Antibody Escape Variants. J. Virol. 96, (2022).
- Purified, liquid
- Mouse monoclonal
- Suitable for dot blot, ELISA, ICC and neutralization assay
- Reacts with AAV9 intact particles
- Isotype: IgA kappa
Product description
| Quantity | 1 ml (50 µg/ml) |
|---|---|
| Antibody Type | Monoclonal |
| Host | Mouse |
| Isotype | IgA kappa |
| Conjugate | Unconjugated |
| Application | Dot blot, ELISA, ICC/IF, Neutralization assay |
| Purification | Affinity chromatography |
| Reactivity | AAV9 |
| No reactivity | AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV11, AAV12, AAVDJ, AAVrh10, AAVrh74 |
| Storage | Up to 1 month: 2-8°C; long term storage in aliquots at -20°C; avoid freeze/thaw cycles |
| Intended use | Research use only |
| Clone | ADK9 |
| Immunogen | AAV9 capsids |
| Concentration | 50 µg/ml |
| Formulation | Liquid; PBS with 0.09% sodium azide and 0.5% BSA |
| Synonym | Adeno-associated virus 9; AAV-9 |
Applications
| Tested applications | Tested dilutions |
|---|---|
| Immunocytochemistry (ICC)/ Immunofluorescence (IF) | Assay dependent |
| Dot Blot | 1:100-1:1,000 (0.05-0.5 µg/ml; non-denaturing conditions) |
| ELISA | Assay dependent |
| Neutralization Assay | EC50 ~2 ng/ml (AAV9) - assay dependent |
Background
The antibody is also useful for neutralizing experiments.
Limited Use Label License: Research Use Only
Product is exclusively licensed to PROGEN Biotechnik GmbH. The use of these products for the development, manufacturing and sale of secondary products/derivatives which are based on the purchased products and/or which include the purchased product require a royalty based sub-license agreement.
References/Publications (6)
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FAQs
The concentration of purified antibodies is mentioned on the datasheet.
For prediluted antibodies the concentration may vary from lot to lot. The concentration of these antibodies is not mentioned on the datasheet and can be requested at support@progen.com.
The supernatant format contains FCS proteins from cell culture medium supplemented with FCS.
The serum antibodies contain other proteins present in serum.
- Supernatant and supernatant concentrate: This format contains hybridoma cell culture supernatant. The antibody is not purified and the antibody concentration is not determined. The antibody concentration may vary from lot to lot. Therefore we recommend to titrate the optimal concentration for the application used for each new lot.
- Lyophilized, purified: This format contains purified antibody in lyophilized form. The reconstitution of this antibody is described in the datasheet. The buffer composition after reconstitution is also mentioned on the datasheet.
- Liquid, purified: This format contains purified antibody in liquid format. The concentration is mentioned on the datasheet.
- Prediluted, purified: This format contains purified antibody in liquid format. Most antibodies in this format are diluted to be ready-to-use for IHC with standard tissue. But some antibodies of this format need further dilution for IHC. This is mentioned on the datasheet.
Most of our liquid antibodies and reconstituted lyophilized antibodies may be stored for short term storage (up to 3 month) at 2-8°C. For long term storage we recommend to store the antibody at -20°C in aliquots. Please avoid freeze and thaw cycles.
Most of our conjugated antibodies should be stored at 2-8°C.
The individual storage conditions are mentioned on the datasheet.
There is no difference in performance.
Anti-Adeno-Associated Virus Capsid Proteins Antibodies
Anti-Adeno-Associated Virus Particle Antibodies
For more information on the antibody binding epitopes on assembled AAV1, 2, 5, 8 and 9 capsids, see the tables below summarizing the published data from the following publications on the specific antibody binding sites:
-
AAV 1
- Tseng, Y.-S. et al. Adeno-Associated Virus Serotype 1 (AAV1)-and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity. J. Virol. 89, 1794–1808 (2015).
- 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.
- Tseng, Y.-S. et al. Adeno-Associated Virus Serotype 1 (AAV1)-and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity. J. Virol. 89, 1794–1808 (2015).
- Gurda, B. L. et al. Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8. J. Virol. 86, 7739–7751 (2012).
EM structures of assembled AAV1, 2, 4, 5, 6, and 8 capsids with Fab-fragments are available in the following publications:
- Emmanuel, S. N. et al. Parvovirus Capsid-Antibody Complex Structures Reveal Conservation of Antigenic Epitopes Across the Family. Viral Immunolgoy. 34(1) (2021).
- McCraw, S. M. et al. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20. Virology. 432(1-2):40-9 (2012).
- Tseng, Y. et al. Adeno-Associated Virus Serotype 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in Parvovirus Antigenic Reactivity. J Virol. 89(3):1794-1808 (2015).
- Jose, A. et al. High-Resolution Structural Characterization of a New Adeno-associated Virus Serotype 5 Antibody Epitope toward Engineering Antibody-Resistant Recombinant Gene Delivery Vectors. J Virol. 93(1):e01394-18 (2019).
- Silveria, M. A. et al. The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors. Viruses. 12(11):1326 (2020).
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