AAV ELISA Figure 1 Showing variance within laboratories

AAV Xpress ELISAs


Reliable AAV Capsid Titer determination in less than 2 hours

PROGEN's ELISA Kits have reference standard status in gene therapy centers worldwide. The AAV Xpress ELISA Kits have been developed based on the standard PROGEN ELISA Kits. 

Created in response to the increasing time pressure being endured industry wide. The kit components have been adjusted to reduce the Xpress ELISA assay time by more than 50%. Showing the same accuracy and reproducibility, whilst saving time and allowing a higher number of samples to be processed. 

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AAV ELISA Figure 1 Showing variance within laboratories

Your Benefits


Giving you the same accuracy and reproducibility as the PROGEN AAV ELISA - but quicker - with results in less than 2 hours. 


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

Accurate: Same accuracy and reproducibility as the standard PROGEN AAV Titration ELISAs. 

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. 

Reliable: PROGEN‘s exclusive capsid antibodies recognize native, non-denatured AAV capsids (full and empty) for the quantification of intact AAV particles.

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

GMP-ready: can be validated for use in a GMP setting and adhere to FDA guidelines for the development of AAV gene therapies. An industry gold standard with established success in clinical trials.
  

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What customers are saying



"Dip'n'Check AAV Kits from PROGEN are very easy to use.
Semi-quantative readout by analysing strips with image analysis software tracked
extremely well with qPCR-based viral genome titer assays in our hands."

 

Tomasz Witkos, AstraZeneca

 

"For rapid confirmation of AAV production,
the PROGEN Dip'n'Check Strips are fantastic."

 

Analytics Team Leader

"These Test strips were a perfect way to confirm which
PROGEN ELISA kit to use when dealing with serotypes that have variations.”"

 

AskBio

 

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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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