AAV ELISAs

Yes, some of the antibodies used in our ELISA kits show cross-reactivity with other serotypes. 

For more information on cross-reactivity of our antibodies, please visit our AAV particle antibody webpage.

For example, the AAV8 ELISA kits cross-react with AAVrh10. However, the AAV8 ELISA kit is not suitable for quantification of the total AAV capsid content of AAVrh10 preparations e.g. due to its serotyp-specific kit control. For quantification of AAVrh10 PROGEN offers an ELISA optimized for AAVrh10 containing AAVrh10 kit control. 

With a suitable kit control an ELISA kit can be validated for a specific serotyp. For example, PROGEN offers the AAVrh74 Kit control, which can be used in combination with our AAVrh10 ELISA kit to quantify AAVrh74 capsids. This specific applications needs to be qualified by the user. 

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. 

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:

 

 

 

 

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

• 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.

 

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" in the download area of the product page. The posters describe 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

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.