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-AAV5 (intact particle) mouse monoclonal, ADK5b was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 200 ng/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Plus Western Blotting Substrate.
Neutralization of AAV5 with mouse monoclonal AAV5 antibody clone ADK5a (A) and mouse monoclonal AAV5 antibody clone ADK5b (B) by using AAV5-NanoLuc® viral particles from Promega. (A) anti-AAV5 (intact particle) mouse monoclonal, ADK5a or (B) anti-AAV5 (intact particle) mouse monoclonal, ADK5b were preincubated with AAV5-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-AAV5 antobodies (mouse monoclonal ADK5a, ADK5b and human chimeric ADK5b-h1 and ADK5b-h2).
In the publication cited below multiple contact sites and footprint residues for ADK5a and ADK5b have been identified, that are very likely to be part of the binding site. 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.
*The residues in the VR are underlined. Those involved in AAV5 receptor binding are bold and italicized.
Tseng et al. Adeno-Associated Virus Serotyp 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in PArvovirus Antigenic Reactivity. J. Virol. 89:1794-1808 (2015).
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-AAV5 (intact particle) mouse monoclonal, ADK5b was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1 h at RT. The secondary antibody goat anti-mouse IgG HRP was also diluted in blocking buffer (antibody concentration 200 ng/ml) and incubated for 1 h at RT. The bands were visualized by chemiluminescent detection using PierceTM ECL Plus Western Blotting Substrate.
Neutralization of AAV5 with mouse monoclonal AAV5 antibody clone ADK5a (A) and mouse monoclonal AAV5 antibody clone ADK5b (B) by using AAV5-NanoLuc® viral particles from Promega. (A) anti-AAV5 (intact particle) mouse monoclonal, ADK5a or (B) anti-AAV5 (intact particle) mouse monoclonal, ADK5b were preincubated with AAV5-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-AAV5 antobodies (mouse monoclonal ADK5a, ADK5b and human chimeric ADK5b-h1 and ADK5b-h2).
In the publication cited below multiple contact sites and footprint residues for ADK5a and ADK5b have been identified, that are very likely to be part of the binding site. 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.
*The residues in the VR are underlined. Those involved in AAV5 receptor binding are bold and italicized.
Tseng et al. Adeno-Associated Virus Serotyp 1 (AAV1)- and AAV5-Antibody Complex Structures Reveal Evolutionary Commonalities in PArvovirus Antigenic Reactivity. J. Virol. 89:1794-1808 (2015).
- Purified, lyophilized
- Mouse monoclonal
- Suitable for ELISA, ICC and neutralization assays
- Reacts with AAV5 intact capsids
- Isotype: IgG2b kappa
Product description
| Quantity | 50 µg |
|---|---|
| Antibody Type | Monoclonal |
| Host | Mouse |
| Isotype | IgG2b kappa |
| Conjugate | Unconjugated |
| Application | Dot blot, ELISA, ICC/IF, IP, Neutralization assay |
| Purification | Affinity chromatography |
| Reactivity | AAV5 |
| No reactivity | AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, AAV9, AAVDJ, AAVrh10, AAVrh74 |
| Storage before reconstitution | 2-8°C until indicated expiry date |
| Storage after reconstitution | Up to 3 months at 2-8°C; long term storage in aliquots at -20°C; avoid freeze/thaw cycles |
| Intended use | Research use only |
| Clone | ADK5b |
| Immunogen | AAV5 capsids |
| Concentration | 50 µg/ml after reconstitution with 1 ml dist. water |
| Formulation | Lyophilized; reconstitute in 1 ml dist. water (final solution contains 0.09% sodium azide, 0.5% BSA in PBS buffer, pH 7.4) |
| Binding affinity | KD value (AAV5) = 6.0E-11 M |
| Synonym | Adeno-associated Virus 5, AAV-5 |
Applications
| Tested applications | Tested dilutions |
|---|---|
| Immunocytochemistry (ICC)/ Immunofluorescence (IF) | 1:20 |
| Dot Blot | 1:500 (0.1 µg/ml; non-denaturing conditions) |
| ELISA | Assay dependent |
| Immunoprecipitation (IP) | 1:5 |
| Neutralization Assay | EC50 ~9 ng/ml (AAV5) - assay dependent |
Background
For characterization of different stages of infection and very useful for the analysis of the AAV5 assembly process. ADK5b specifically reacts with intact adeno-associated virus 5 particles, empty and full capsids. Recognizes a conformational epitope of assembled capsids, not present in denatured capsid proteins and native but unassembled capsid proteins. The antibody cannot be used for immunoblotting.
The antibody is usefull 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 (7)
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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.
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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- Consists of fully assembled, empty AAV5 capsids
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- Fully assembled, empty AAV5 capsids
- Serotype-specific conformational epitopes
- Reliable positive control for dot blot, western blot, ELISA
- Quality controlled for purity, AAV titer and filling grade
- Alignment to internal reference standard material
For US shipment, the packages are only sent out for delivery on Mondays and Tuesdays, in the EU from Monday until Wednesday.
- Fully assembled AAV5 capsids filled with an eGFP reporter gene controlled by a CMV promoter
- Serotype-specific conformational epitopes
- Reliable positive control for dot blot, western blot, ELISA, PCR and cell-based assays
- Quality controlled for purity, filling grade, aggregation, total capsid and genome titer
- Alignment to internal reference standard material
For US shipment, the packages are only sent out for delivery on Mondays and Tuesdays, in the EU from Monday until Wednesday.
- ELISA for quantitation of AAV5 capsids
- Detection of full and empty AAV5 capsids
- ADK5a antibody is used as capture and detection antibody
- get results in less than 2 hours and increase your daily sample throughput
- easy workflow & process integration
- same accuracy and reproducibility as the standard AAV5 Titration ELISA
- ADK5a antibody is used as capture and detection antibody
- Mouse monoclonal
- Suitable for affinity chromatography, dot blot, ICC/IF, IP and WB
- Reacts with denatured VP1, VP2, VP3 proteins of AAV1, 2, 3, 5, 6, 7, 8, 9, rh10, DJ
- Isotype: IgG1
- Rabbit polyclonal
- Suitable for ELISA, ICC/IF, IP and WB
- Reacts with AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVDJ and AAVrh74, weakly with AAV4
- Strong affinity for denatured capsid proteins
- Weak affinity for native capsids
- Purified, lyophilized
- Mouse monoclonal
- Suitable for dot blot, ELISA and neutralization assay
- Reacts with AAV5 intact capsids
- Isotype: IgG2a kappa
- Purified, lyophilized
- Mouse monoclonal
- Suitable for ELISA, ICC and neutralization assays
- Reacts with AAV5 intact capsids
- Isotype: IgG2b kappa
- Rabbit polyclonal
- Suitable for Affinity Chromatography and WB
- Reacts with AAV5
- Recombinant chimeric (human Fc region)
- Suitable for dot blot, neutralization assay and serology ELISA
- Reacts with AAV5 intact capsids
- Isotype: IgG1
- Recombinant chimeric (human Fc region)
- Suitable for dot blot, neutralization assay and serology ELISA
- Reacts with AAV5 intact capsids
- Isotype: IgG2
- Rapid lateral flow test for the detection of AAV5 capsids
- Semi-quantitative AAV titer determination in cell lysates and purified preparations
- Detection of full and empty AAV5 capsids
- Highly effective density gradient media for the separation of full and empty AAV particles
- Designed for the purification of viruses (such as AAV)
- Useful to isolate different cell types and organelles
- Ready-made, sterile and endotoxin-tested solution of iodixanol