Reliable and Reproducible Data

Enzyme Immunoassay for the Quantitative Determination of AAV Serotypes 1, 2, 3, 5, 6, 8, 9, and rh10 Particles in Cell Culture Supernatants and Purified Virus Preparations. Immunotitration by PROGEN's AAV Xpress ELISA & AAV Titration ELISA offers a fast, sensitive and reproducible method for titration of intact AAV wild type virions, AAV recombinant virions or assembled and intact empty AAV capsids.

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Principle of the AAV ELISA

The principle of the assay is based on the sandwich ELISA technique. A monoclonal antibody specific for a conformational epitope on assembled AAV capsids is coated onto microtiter strips and is used to capture AAV particles from the specimen. Captured AAV particles are detected in two steps. First a biotin-conjugated monoclonal antibody to AAV is bound to the immune complex. In the second step streptavidin peroxidase conjugate reacts with the biotin molecules. Addition of substrate solution results in a color reaction which is proportional to the amount of specifically bound viral particles. The absorbance is measured photometrically at 450 nm.

ELISA Capture Antibodies



The capture antibodies used for PROGEN’s AAV ELISAs bind specific and defined conformational epitopes. These epitopes are generated by the capsid assembly of the corresponding AAV serotypes. Changes in the protein sequences of the capsid proteins, e.g. in shuffeled vectors, might influence the conformation of the proteins, hence influencing the conformation of the antibody binding epitopes presented on the AAV capsid. This will influence the binding affinity of the antibody and affect determination of the titer based on the (non-shuffled) Kit Control provided with the AAV ELISA kit. A first indication that the ELISA might recognize your shuffled AAV vector is the presence of the antibody-binding epitope. Find more information about the potential antibody binding sites of PROGEN´s AAV antibodies in the FAQ section "Do the PROGEN AAV ELISAs recognize AAV shuffled vectors".

PROGEN´s AAV ELISAs are reliable quantification tools for total AAV capsid determination for research and development. They are robust and accurate tools, which have been demonstrated to show low inter- and intra-assay variability. For application of AAV vectors in therapeutic context, 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 application of AAV in patients.

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Matrix Effects of Additives

The determination of AAV capsid titers from e.g. cell extract can be influenced by several conditions such as the composition of your lysis buffer. For example, high salt concentrations in your buffer might inhibit adequate capsid detection (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).). For more information, refer to the table below showing the analysis of matrix effects of different additives on the AAV ELISAs. Please note, that data on cumulative effects have not been collected, therefore tolerated concentrations might differ from the concentrations indicated in the table.

Simple Plex Viral Titer Assays


PROGEN´s well-established AAV particle antibodies have teamed up with the automated immunoassay system of ProteinSimple. PROGEN's AAV particle antibodies exclusively detect fully assembled, intact capsids thus allowing a robust and reliable quantification of total capsid titers.

The new Simple Plex assays, available for the AAV serotypes AAV1, AAV2, AAV6 and AAV8, combine the efficiency & reproducibility of PROGEN´s established ELISAs, using the unique AAV particle antibodies with the convenient workflow and robustness of the Ella platform. The microfluidic cartridge offers a broad dynamic range and hands-free automation to accelerate AAV process development.


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AAV ELISA Short Protocols

Download your short protocol for the AAV ELISA Titration kits and the AAV Xpress ELISA kits.  

     

 

AAV ELISA Titration

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AAV Xpress ELISA

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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.
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. 

To learn more about cross-reactivities of the AAV antibodies take a look at the table below and the corresponding publications:

Table:crossreactivity of AAV Antibodies

  1. 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).
  2. 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).
  3. 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:

Residues for A20 and ADK8 binding

Contact sides and footprint residues for ADK1a/1b

Contact sides and footprint residues for ADK5a/5b

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.
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.

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.

AAV shortprotocols
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.


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