Frequently Asked Questions (FAQs)

AAV Titration ELISA

PROGEN AAV ELISAs are reliable quantification tools for total AAV capsid determination for gene therapy research and development. They are robust and accurate tools, which have been demonstrated to show low inter- and intra-assay variability. Currently the AAV ELISA appears to be the best format for the quantification of rAAV preparations in comparison to other quantification methods in terms of inter-assay variability and ease of use.

For application of AAV vectors in gene therapy, the determination of total capsid titer is essential, since most of the AAV preparations contain a significant number of empty capsids. Accurate characterization and quantification of purified AAV particle preparations represent a critical step for clinical application, to minimize immunogenic responses and maximize gene transfer to the target cells. Therefore, reliable and reproducible quantification of AAV titers is essential for safe and effective AAV gene therapy.

Current methods for the characterization of AAV preparations are Dot Blot and ELISA to determine the amount of intact (full & empty) viral capsids, as well as qPCR and ddPCR, which allow the measurement of packaged DNA. For usage of Dot Blot or ELISA for quantification of total capsid titers, the application of a suitable antibody, detecting assembled particles only, is indispensable.

The PROGEN AAV ELISA is based on the sandwich ELISA technique. Microtiter plates, pre-coated with a monoclonal antibody specific for a conformational epitope on assembled AAV capsids, are used to capture the AAV particles (full & empty). The following detection of AAV particles is a two-step process where a biotin-conjugated monoclonal AAV antibody is bound to the captured AAV particles, followed by a streptavidin peroxidase-conjugate reaction with the biotin molecules. After addition of the substrate, a color reaction represents the amount of specifically bound viral particles. Absorbance is measured photometrically at 450 nm and the AAV titer of unknown samples can be calculated based on the curve of the included AAV Kit Control.

For detailed information and short protocol see AAV ELISA.

PROGEN AAV ELISAs are currently available for AAV serotypes 1, 2, 5, 6, 8 and 9.

There are several methods to determine AAV titers, however these methods differ in quality, reliability and most important in the distinct AAV titer they measure. For example, qPCR and ddPCR detects viral DNA and is suitable for the determination of AAV DNA titers only. These methods give no indication of total AAV capsid titers, which is an essential parameter for the use of AAV in gene therapy. Even though qPCR and ddPCR are frequently used and highly sensitive methods for AAV titer determination, they show high variability. In contrast to qPCR/ddPCR, the PROGEN AAV ELISA determines the total capsid AAV titer, including full and empty capsids. This information is essential to minimize immunogenic responses and maximize gene transfer to the target cells, especially when AAV vectors are used for gene therapy. However, the PROGEN ELISA cannot be used for the measurement of AAV DNA titers. Compared to the high variability of qPCR/ddPCR, the PROGEN ELISA is a very robust and accurate method for AAV determination. The PROGEN AAV ELISA provides reliable and consistent data since the inter- and intra-assay variability is very low.

PROGEN recommends the use of different methods for AAV titer determination, including AAV DNA titer and AAV total capsid titer to ensure complete quantification and analysis of AAV preparations.   

PROGEN offers serotype-specific AAV ELISA kits for quantification of the AAV serotypes 1, 2, 5, 6, 8 and 9.

The recognition of shuffled AAV vectors depend on the specific capsid region which is affected by the shuffling. The capture antibodies used for PROGEN’s AAV ELISAs bind specific and, in some cases, well defined conformational epitopes. These epitopes are generated by the capsid assembly of the corresponding AAV serotypes. A first indication that the ELISA might recognize your shuffled AAV vector is the presence of the antibody-binding epitope. However, changes in the protein sequences of the capsid proteins might also influence conformation of the proteins, hence the conformation of the epitopes presented on the AAV capsid. This might influence binding affinity of the antibody and affect determination of the titer based on the (non-shuffled) Kit Control provided with the AAV ELISA kit. 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 assay needs to be tested and optimized for your specific AAV vector. PROGEN highly recommends the production and calibration of a suitable (shuffled) Kit Control, to ensure reliable titer determination of your individually shuffled AAV vector with PROGEN ELISA kits.

For more information on the antibody binding epitopes in AAV1, 2, 5 and 8, relevant for PROGEN’s AAV ELISA, see the following publications on the specific antibody binding epitopes:

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).

AAV2:

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).

AAV5:

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).

AAV8:

Gurda, B. L. et al. Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8. J. Virol. 86, 7739–7751 (2012).

Some of PROGEN´s AAV ELISA kits show cross-reactivity with other AAV serotypes. E.g. the AAV2 ELISA kit cross-reacts with AAV3. However, this does not mean that the AAV2 ELISA kit is suitable for quantification of AAV total capsid content of AAV3 preparations. The PROGEN ELISA kits are precisely calibrated for the specific AAV serotypes and provide only the serotype-specific Kit Control. Therefore, PROGEN can only ensure accurate quantification of the corresponding AAV serotype. This is true for all PROGEN´s AAV ELISA kits.

To learn more about cross-reactivities of the AAV antibodies see the following publications:

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).

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).

Kuck, D., Kern, A. & Kleinschmidt, J. A. Development of AAV serotype-specific ELISAs using novel monoclonal antibodies. J. Virol. Methods140, 17–24 (2007).

It is possible to apply 40 dublicates to one ELISA plate after application of the recommended serial dilutions of the standard curve.

The PROGEN AAV ELISA kits are provided with one precoated microtiter plate containing 12 x 8-well-strips. To ensure a reliable quantification of AAV titers, PROGEN recommends the use of seven serial dilutions of the provided Kit Control (standard curve) including two blank controls as well as 2-3 dilutions of the unknown AAV samples. The Kit Control and the AAV samples need to be tested in duplicates to provide accurate results (for detailed instructions, see the manual provided with the AAV ELISA kit). Therefore, after having applied the standard curve, 40 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).

PROGEN´s AAV2 and AAV8 ELISA kits are calibrated based on the international standard material provided by the ATCC.

AAV2: ATCC-VR-1616 rAAV2 RSS reference material

Lock, M. et al. Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Hum Gene Ther. 10, 1273-85 (2010).

AAV8: ATCC-VR-1816 rAAV8 RSS reference material

Ayuso, E. et al. Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material. Hum Gene Ther. 11, 977-87 (2014).

The remaining AAV ELISA kits are calibrated based on internal gold standards. These standards are highly pure, full AAV capsid preparations. Quantification was performed by qPCR or ddPCR by different labs and electron microscopy (EM). The Kit Controls provided with the AAV ELISAs are adjusted to these internally produced standards. Please note, that the internal gold standards used for the calibration of PROGEN´s AAV Titration ELISAs cannot be purchased or provided in any way since they are produced exclusively for internal use to calibrate PROGEN´s ELISAs.

No, PROGEN does not offer sample size ELISA kits. Each kit contains one 96-well microtiter plate for testing. After opening of the AAV2 Titration ELISA can be used for up to two weeks and the AAV1, 5, 6, 8 and 9 Titration ELISA kits for up to four weeks.

No, PROGEN does not offer an AAV ELISA based quantification service. PROGEN certainly offers the tools for reliable and reproducible AAV quantification by ELISA by providing serotype-specific ELISA kits. An AAV ELISA kit contains one microtiter plate coated with the corresponding AAV specific antibody (ready-to-use), suitable Kit Control (lyophilized AAV particles), anti-AAV Biotin conjugate, streptavidin peroxidase conjugate, assay buffer (ASSB), as well as substrate and stop solution.

*Please note that shipping costs are calculated according to the dimensional weight of the package and the destination country.

PROGEN is distributor for density gradient media produced by Axis-Shield Diagnostics and offers amongst others OptiPrep™ for AAV particle purification. For a complete list of density gradient media offered by PROGEN, please visit our shop.

Please note, distribution of the Axis-Shield products in PROGEN’s portfolio is restricted to DACH countries only.  

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.

Yes, PROGEN offers a selection of AAV antibodies in sample sizes. Find a list of antibodies available in sample size of 10 µg (lyophilized) here: AAV Antibodies 

The following PROGEN AAV antibodies have been reported to have neutralizing activity:

For more information on the neutralizing activity of these antibodies see the following publications:

ADK1a:

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).

A20:

Moskalenko, M. et al. Epitope Mapping of Human Anti-Adeno-Associated Virus Type 2 Neutralizing Antibodies: Implications for Gene Therapy and Virus Structure. Jounal Virol.74, 1761–1766 (2000).

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).

ADK5b:

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).

ADK6:

Bennett, A. D. et al. AAV6 K531 serves a dual function in selective receptor and antibody ADK6 recognition. Virology518, 369-376 (2018)

ADK8:

Gurda, B. L. et al. Mapping a Neutralizing Epitope onto the Capsid of Adeno-Associated Virus Serotype 8. J. Virol.86, 7739–7751 (2012).

Raupp, C. et al. The Threefold Protrusions of Adeno-Associated Virus Type 8 Are Involved in Cell Surface Targeting as Well as Postattachment Processing. J. Virol.86, 9396–9408 (2012).

Sonntag, F. et al. The assembly activating protein (AAP) promotes capsid assembly of different AAV serotypes. J. Virol. 85, 12686-97 (2011)

ADK9:

Sonntag, F. et al. The assembly activating protein (AAP) promotes capsid assembly of different AAV serotypes. J. Virol. 85, 12686-97 (2011)

Tseng, Y.S. et al. Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies. J. Virol. Meth. 236, 105-110 (2016)

Varadi, K. et al. Novel random peptide libraries displayed on AAV serotype 9 for selection of endothelial cell-directed gene transfer vectors. Gene Ther. 19, 800-9 (2012).

Orders and Shipment

Yes, you can order directly via our webshop or by sending us an e-mail.

PROGEN sells and ships worldwide. Please note that regional import conditions and legislation have to be considered. Please contact usfor specific requests.

*Please note that shipping costs are calculated according to the dimensional weight of the package and the destination country.

Please find a complete list of distributors and countries here.