Dot blot analysis of AAV5 native (1E+09-1E+10 capsids) and denatured (1E+10 capsids, denatured at 95°C for 10 min in sample buffer) capsids with mouse monoclonal AAV5 antibodies clones ADK5a and ADK5b and human chimeric AAV5 antibodies clones ADK5b-h1 and ADK5b-h2.
The nitrocellulose membrane was blocked with 5% milk in PBST for 45 min at RT. The primary antibody anti-AAV5 (intact particle) mouse monoclonal, ADK5a (Cat. No. 610148) or ADK5b (Cat. No. 610149) or anti-AAV5, human chimeric, ADK5b-h1 (Cat. No. 692349) or ADK5b-h2 (Cat. No. 692347) was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1.5 h at RT. The secondary antibody anti-mouse IgA goat polyclonal, HRP conjugate or anti-human IgG goat polyclonal, HRP conjugate was also diluted in blocking buffer (antibody concentration 200 ng/ml) and incubated for 1.5 h at RT. The bands were visualized by chemiluminescent detection using Pierce™ ECL Western Blotting Substrate.
Dot blot analysis of AAV1-AAV9, AAVrh10, AAVrh74, AAVDJ native (1E+09-1E+10 capsids) and denatured (1E+10 capsids, denatured at 95°C for 10 min in sample buffer) capsids.
The nitrocellulose membrane was blocked with 5% milk in PBST for 45 min at RT. The primary antibody anti-AAV5, human chimeric, ADK5b-h1 (Cat. No. 692349) was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1.5 h at RT. The secondary antibody anti-human IgG goat polyclonal, HRP conjugate 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 Pierce™ ECL Western Blotting Substrate.
Neutralization of AAV5 with mouse monoclonal AAV5 antibody clone ADK5b (A) and human chimeric AAV5 antibody clone ADK5b-h1 (B) by using AAV5-NanoLuc® viral particles from Promega. (A) anti-AAV5 (intact particle) mouse monoclonal, ADK5b (Cat. No. 610149) or (B) anti-AAV5, human chimeric, ADK5b-h1 (Cat. No. 692349) 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).
Dot blot analysis of AAV5 native (1E+09-1E+10 capsids) and denatured (1E+10 capsids, denatured at 95°C for 10 min in sample buffer) capsids with mouse monoclonal AAV5 antibodies clones ADK5a and ADK5b and human chimeric AAV5 antibodies clones ADK5b-h1 and ADK5b-h2.
The nitrocellulose membrane was blocked with 5% milk in PBST for 45 min at RT. The primary antibody anti-AAV5 (intact particle) mouse monoclonal, ADK5a (Cat. No. 610148) or ADK5b (Cat. No. 610149) or anti-AAV5, human chimeric, ADK5b-h1 (Cat. No. 692349) or ADK5b-h2 (Cat. No. 692347) was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1.5 h at RT. The secondary antibody anti-mouse IgA goat polyclonal, HRP conjugate or anti-human IgG goat polyclonal, HRP conjugate was also diluted in blocking buffer (antibody concentration 200 ng/ml) and incubated for 1.5 h at RT. The bands were visualized by chemiluminescent detection using Pierce™ ECL Western Blotting Substrate.
Dot blot analysis of AAV1-AAV9, AAVrh10, AAVrh74, AAVDJ native (1E+09-1E+10 capsids) and denatured (1E+10 capsids, denatured at 95°C for 10 min in sample buffer) capsids.
The nitrocellulose membrane was blocked with 5% milk in PBST for 45 min at RT. The primary antibody anti-AAV5, human chimeric, ADK5b-h1 (Cat. No. 692349) was diluted in blocking buffer (antibody concentration 100 ng/ml) and incubated for 1.5 h at RT. The secondary antibody anti-human IgG goat polyclonal, HRP conjugate 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 Pierce™ ECL Western Blotting Substrate.
Neutralization of AAV5 with mouse monoclonal AAV5 antibody clone ADK5b (A) and human chimeric AAV5 antibody clone ADK5b-h1 (B) by using AAV5-NanoLuc® viral particles from Promega. (A) anti-AAV5 (intact particle) mouse monoclonal, ADK5b (Cat. No. 610149) or (B) anti-AAV5, human chimeric, ADK5b-h1 (Cat. No. 692349) 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).
- Recombinant chimeric (human Fc region)
- Suitable for dot blot, neutralization assay and serology ELISA
- Reacts with AAV5 intact capsids
- Isotype: IgG1
Product description
| Quantity | 1 ml |
|---|---|
| Antibody Type | Monoclonal |
| Host | Recombinant chimeric (human Fc region) |
| Isotype | IgG1 |
| Conjugate | Unconjugated |
| Application | Dot blot, Neutralization assay, Serology ELISA |
| Purification | Affinity chromatography |
| Reactivity | AAV5 |
| No reactivity | AAV1, AAV2, AAV3, AAV4, AAV6, AAV8, AAV9, AAVDJ, AAVrh10, AAVrh74 |
| Storage | 2-8°C |
| Intended use | Research use only |
| Clone | ADK5b-h1 |
| Immunogen | AAV5 capsids |
| Concentration | 50 µg/ml |
| Formulation | PBS, pH 7.4 with 0.09% sodium azide and 0.5% BSA |
| Binding affinity | KD value (AAV5) = 8.1E-11 M |
| Synonym | Adeno-associated virus 5; AAV-5 |
Applications
| Tested applications | Tested dilutions |
|---|---|
| Serology Assay | 1-4 µg/ml (1:12.5-1:50; AAV5 ELISA) |
| Dot Blot | 0.1 µg/ml (1:500; non-denaturing conditions) |
| Neutralization Assay | EC50 ~4 ng/ml (AAV5) - assay dependent |
Background
Our human chimeric AAV antibodies are derived from our mouse monoclonal AAV antibodies and are a combination of the mouse antigen binding region and a human Fc region. Therefore, it provides the well-known characteristics of the corresponding mouse monoclonal antibody (anti-AAV5, ADK5b, Cat. No. 610149) like cross-reactivity and neutralization activity combined with a human Fc region allowing the use in an anti-human secondary antibody detection system.
Many humans in the general population have developed antibodies against AAV as a result of naturally acquired infections, which might affect efficacy and safety of the gene transfer using AAV vectors. Therefore, testing for pre-existing AAV antibodies in patient sera is an indispensable step for the selection of patients for AAV gene therapy clinical trials.
Our human chimeric AAV antibodies are close to the human derived samples, making them the ideal positive control for reproducible and comparable serological assays.
Limited Use Label License: Research Use Only
Product is exclusively owned 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.
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FAQs
The human chimeric AAV antibodies have the same variable region as our mouse monoclonal antibodies but instead a human constant region.
Anti-AAV5, ADK5b-h1 is an IgG1 antibody whereas ADK5b-h2 is an IgG2 antibody.
Yes, they show the same cross-reactivity as their mouse monoclonal versions. The table below shows where cross-reactivity takes place.
| Human Chimeric Antibody | Clone | AAV1 | AAV2 | AAV3 | AAV4 | AAV5 | AAV6 | AAV7 | AAV8 | AAV9 | AAVrh10 | AAVrh74 | AAVDJ |
| Anti-AAV1 | ADK1a-h1 | X | X | n.a.* | |||||||||
| Anti-AAV2 | A20-h1 | X | X | n.a.* | |||||||||
| Anti-AAV5 | ADK5b-h1 | X | n.a.* | ||||||||||
| Anti-AAV5 | ADK5b-h2 | X | n.a.* | ||||||||||
| Anti-AAV8 | ADK8-h1 | X | X | X | X | X | |||||||
| Anti-AAV9 | ADK9-h1 | n.a.* | X |
*n.a.: not analyzed
The binding affinity of the human chimeric antibodies to different AAV serotypes was tested and compared to the corresponding mouse monoclonal antibodies using biolayer interferometry (BLI) analysis. The table below shows the results.
| Serotype | Antibody Clone | Binding Affinity (KD Value) |
| AAV1 | ADK1a | <1.0E-12 M |
| ADK1a-h1 | 8.8E-12 M | |
| AAV2 | A20 | 2.6E-11 M |
| A20-h1 | 1.2E-10 M | |
| A20R | <1.0E-12 M | |
| AAV3 | A20 | <1.0E-12 M |
| A20-h1 | 9.3E-11 M | |
| A20R | <1.0E-12 M | |
| AAV5 | ADK5a | 4.9E-11 M |
| ADK5b | 6.0E-11 M | |
| ADK5b-h1 | 8.1E-11 M | |
| ADK5b-h2 | 3.6E-11 M | |
| AAV6 | ADK1 | <1.0E-12 M |
| ADK1a-h1 | 5.9E-11 M | |
| AAV8 | ADK8 | <1.0E-12 M |
| ADK8-h1 | <1.0E-12 M | |
| AAV9 | ADK9-1R | <1.0E-12 M |
| ADK9-h1 | 7.0E-11 M | |
| AAVrh10 | ADK8 | <1.0E-12 M |
| ADK8-h1 | <1.0E-12 M | |
| AAVrh74 | ADK8 | <1.0E-12 M |
| ADK8-h1 | <1.0E-12 M |
The lower the KD value the higher the binding affinity. The detection limit is 1.0E-12 M.
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