AAV ELISA FAQs
In house validation shows that our PROGEN AAV ELISA kits can be used under GMP conditions. However, it is also necessary to validate kits under your individual conditions. If you need support with your validation you can contact us at support@progen.com.
Performance data is available here.
Performance data is available here.
Some of PROGEN´s AAV ELISA kits show cross-reactivity with other AAV serotypes. For example, the AAV2 ELISA kit cross-reacts with AAV3.
However, the AAV2 ELISA kit is not suitable for quantifiying the total AAV capsid content of AAV3 preparations. We recommend using the corresponding ELISA for your specific AAV serotype to ensure reliable capsid titer quantification, due to the ELISA's serotype-specific calibration.
However, the AAV2 ELISA kit is not suitable for quantifiying the total AAV capsid content of AAV3 preparations. We recommend using the corresponding ELISA for your specific AAV serotype to ensure reliable capsid titer quantification, due to the ELISA's serotype-specific calibration.
To learn more about cross-reactivities of the AAV antibodies take a look at the table below and the corresponding publications:
- Mietzsch, M. et al. OneBac: Platform for Scalable and High-Titer Production of Adeno-Associated Virus Serotype 1–12 Vectors for Gene Therapy. Hum. Gene Ther.25, 212–222 (2014).
- 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).
- Kuck, D., Kern, A. & Kleinschmidt, J. A. Development of AAV serotype-specific ELISAs using novel monoclonal antibodies. J. Virol. Methods140, 17–24 (2007).
The recognition of shuffled AAV vectors depends on the specific capsid region which is affected by the shuffling. The antibodies used for PROGEN’s AAV ELISAs bind to specific conformational epitopes. These epitopes are generated by the capsid assembly of the corresponding AAV serotypes.
A first indication that the antibody recognizes your shuffled AAV vector is the presence of the antibody-binding epitope. However, changes in protein sequences of the capsid proteins might also influence conformation of the proteins and furthermore the conformation of the epitopes presented on the AAV capsid. Thus, binding affinity of the antibody and determination of the titer based on our Kit Control (non-shuffled) might be influenced and affected.
To test if your shuffled variant is detected by one of our PROGEN antibodies you can either use our antibody sample size and perform a dot blot or you can easily check the binding affinity with our Dip'n'Check lateral flow assay.
Since these properties strongly depend on the specific shuffling performed, PROGEN cannot guarantee successful and precise quantification of your shuffled AAV vector. Even if the antibody binding epitopes are still present on your shuffled AAV capsid, your ELISA assay needs to be tested and optimized for your specific AAV vector. PROGEN recommends the production and calibration of a suitable (shuffled) Kit Control, to ensure reliable titer determination of your individually shuffled AAV vector.
AAV1:
AAV2
AAV5
AAV8
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:
- 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.
Yes, PROGEN offers serotype-specific AAV ELISA Controls for the standardization of your AAV titer determination.
The serotype-specific positive controls are fully characterized empty AAV capsids standardized with PROGEN´s internal gold standards* (AAV1, AAV3, AAV5, AAV6 & AAV9) or the ATCC international gold standard material (AAV2 & AAV8).
*For more information on PROGEN´s internal gold standard, take a look at our posters "Developing Reliable AAV Standards for ELISA" and "The New AAV3 Titration ELISA" describing the establishment and characterization of AAV5 and AAV3 internal gold standards.
The serotype-specific positive controls are fully characterized empty AAV capsids standardized with PROGEN´s internal gold standards* (AAV1, AAV3, AAV5, AAV6 & AAV9) or the ATCC international gold standard material (AAV2 & AAV8).
Yes, for AAV particle purification we recommend OptiPrep produced by Serumwerk Bernburg AG. You can find more information on our OptiPrep site.
PROGEN’s AAV ELISA kits are provided with one pre-coated microtiter plate containing 12 x 8-well-strips.
PROGEN recommends the use of seven serial dilutions of the provided Kit Control (standard curve), two blank controls and 2-3 dilutions of the unknown AAV samples to ensure a reliable quantification of AAV titers. The Kit Control and the AAV samples need to be tested in duplicates to provide accurate results (for further information take a look at the instructions provided with the AAV ELISA kit).
Bearing this in mind, once you have applied the standard curve, forty duplicate measurements can be applied.
Bearing this in mind, once you have applied the standard curve, forty duplicate measurements can be applied.
Due to the high affinity and specificity of the AAV capture antibodies used for the PROGEN AAV ELISA kits, the detection of AAV particles from cell extracts is possible. However, detection is only possible within the reading range of the ELISA kits and reliable quantification depends on adequate titration of the corresponding samples. Furthermore, the detection of AAV capsids from cell extract can be influenced by several conditions, e.g. the composition of your lysis buffer. For example, high salt concentrations in your buffer might inhibit adequate capsid detection. For more information, see the following publication:
Grimm, D. et al. Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Ther.6, 1322–30 (1999).AAV Xpress ELISAs were developed based on the corresponding AAV Titration ELISAs.
The only difference is the adjustment of the kit components to shorten the assay time for the AAV Xpress ELISAs from ~4.5h to ~2h. The process is the same for both the AAV Xpress ELISAs and the AAV Titration ELISAs.
All the AAV Titration ELISAs and the AAV Xpress ELISAs have been calibrated based on the ATCC standard material (AAV2 & AAV8) or the PROGEN internal gold standard material (AAV1, AAV3, AAV5, AAV6, AAV9 & AAVrh10), respectively.
PROGEN offers AAV Titration ELISA kits for the quantification of AAV serotypes 1, 2, 3, 5, 6, 8, 9, and rh10 and AAV Xpress ELISA kits for AAV serotypes 2, 5, 6, 8, and 9.
AAV Antibodies FAQs
Yes, PROGEN offers several AAV virus particle specific, neutralizing antibodies against AAV serotypes 1, 2, 3, 4, 5, 6, 8, and 9.
For more information on the neutralizing activity of our antibodies check out the internally collected data and publications for the corresponding serotypes. Just click the button for your specific serotype after following the link.
Yes, PROGEN offers a selection of AAV antibodies in sample sizes. Find a list of AAV antibodies here: AAV Antibodies
PROGEN offers a wide range of AAV antibodies in different quantities and formats (e.g. lyophilized and supernatant). A selection of AAV antibodies are also available in 10µg lyophilized sample sizes. For more information on the different AAV antibodies please see AAV Antibodies.
Anti-Adeno-Associated Virus Capsid Proteins Antibodies
For more information on the antibody binding epitopes of the capsid protein antibodies A1, B1 and A69 see the table below summarizing the published data from the following publication on the specific antibody binding sites:
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).
Orders & Shipment FAQs
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