AAV ELISA

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Reliable AAV Capsid Titer determination

Recombinant Adeno-Associated Virus (rAAV) vectors are increasingly used for in vivo gene transfer to target inherited genetic diseases. Yet it is essential to carry out a reliable quantification of rAAV capsid titers to ensure a safe gene transfer to the target cell.

As the industry gold standard you can rely on PROGEN’s AAV ELISAs to consistently deliver dependable capsid titer determination. You can select from a portfolio of AAV titration ELISAs and Xpress ELISAs for the most commonly used AAV serotypes. These are based on PROGEN‘s exclusive portfolio of AAV particle antibodies.

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Your AAV ELISA Benefits

Easy-to-use: all assays use a similar workflow for all AAV serotypes and do not require expensive equipment or special training. Includes single-break strips that can be used separately for different test runs.

Quick: the ELISA Kits are ready to be shipped and easy to implement into all workflows. The Xpress ELISA Kits generate results in less than 2 hours without compromising performance.

Reliable: PROGEN‘s exclusive particle antibodies recognize native, non-denatured AAV capsids (full and empty) for the quantification of intact AAV particles. An industry gold standard with established success in clinical trials.

Reproducible: an established and robust method that generates reproducible data (See Figure 1). Regular AAV ELISA users can easily achieve low inter- & intra-assay variance with a coefficient of variation (CV) below 10%.

Accurate: AAV2 and AAV8 ELISAs are based on the ATCC international reference standard material and all remaining ELISAs (AAV1, 3, 5, 6, 9 and rh10) adhere to internal gold standards.

GMP-ready: can be validated for use in a GMP setting and adhere to FDA guidelines for the development of AAV gene therapies.

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What customers are saying

We’ve been using several kits every week and have had no trouble at all with them. We’ve been getting very good reproducibility and low variability between repeats, assays and users. Everyone is enjoying the speed of the assay and it has increased our throughput too.

Jennie Gullick, PhD, R&D Bioprocessing, Pall Corporation

AAV Xpress ELISAs

Results in less than 2 hours

AAV Xpress ELISAs were created in response to the increasing need industry wide to get results faster and save time. The kit components have been adjusted to reduce the assay time by more than 50%. The Xpress ELISA kits have been developed based on the standard PROGEN ELISA kits, so they show the same accuracy and reproducibility. The only difference is you will be able to process a higher number of samples in less time.

Both PROGEN's AAV ELISA and AAV ELISA Xpress Kits have reference standard status in gene therapy centers worldwide.

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Reliable and Reproducible Data

Watch our short 90 second video and find out how you can easily implement AAV ELISAs to reliably determine the capsid titer of your AAV preparation, with no expensive equipment required.

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 quantifying 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:

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.

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:

Contact sides and footprint residues for ADK1a/1b

Contact sides and footprint residues for ADK5a/5b

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

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).

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, AAVrh10 and AAVrh74) 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.

Yes, for AAV particle purification we recommend OptiPrep produced by Serumwerk Bernburg AG. You can find more information on our OptiPrep site.

Our Dip'n'Check AAV lateral flow assays are perfectly suitable to estimate the approximate titer quick and easy just before performing the PROGEN ELISA. The result within 20min will guide you to the optimal dilution for your sample to hit the dynamic range. More information about our Dip’n’Check can be found on our Poster: Potential Applications for AAV Lateral Flow Tests

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. We recommend to dilute the sample at least 1:10 in ASSB 1x to get a reliable result. 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. AAVrh74 capsids can be quantified with a combination of AAVrh10 ELISA and AAVrh74 kit control as standard, since the antibody used in the AAVrh10 ELISA also recognizes AAVrh74.