AAV Titration ELISAs
ELISAs for quantification and detection of full and empty capsids.
AAV ELISA Controls
Reliable positive controls in detections and quantification assays.
AAV ELISA Short Protocols
Download your short protocol for the AAV ELISA Titration kits and the AAV Xpress ELISA kits.
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 capture antibodies used for PROGEN’s AAV ELISAs bind specific and, for some of the antibodies, well defined conformational epitopes. These epitopes are generated by the capsid assembly of the corresponding AAV serotypes.
A first indication that the capture 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.
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.
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:
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AAV1:
- 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).
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).
For AAV particle purification we recommend OptiPrep produced by Serumwerk Bernburg AG. You can find alternative density gradient media on our density gradient media page.
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 incubation time for the AAV Xpress ELISAs from 3 hours and 15 minutes to 1 hour and 20 minutes. 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) and 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.
