AAV Xpress ELISA

Results in Less Than 2 Hours

The AAV Xpress ELISA was designed for researchers who need faster, reliable, and reproducible quantification of fully assembled AAV capsids.

Based on PROGEN’s standard ELISA kits, Xpress maintains the same accuracy while reducing assay time by over 50%, enabling higher throughput in less time.

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How does AAV Xpress ELISA work?

Xpress ELISA works with the same principle as AAV Titration ELISA, based on the sandwich ELISA technique:

Capture: A monoclonal antibody specific for a conformational epitope on assembled AAV capsids coats the microtiter plate.
Detection: Biotin-conjugated monoclonal antibody binds the captured capsids.
Signal: Streptavidin-peroxidase reacts with biotin and catalyzes the substrate to produce a bright color.
Readout: Absorbance at 450 nm is measured photometrically, proportional to bound viral particles.

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What are the benefits of AAV Xpress ELISA

Time-saving: Complete assays in <2 hours, 50% faster than standard ELISA.
High-throughput: Process more samples in parallel with single-break strips.
Reliable: Same PROGEN antibodies and quality standards as the standard ELISA.
Reproducible: CVs <10%, traceable standards maintained.
Easy integration: Fits seamlessly into existing AAV workflows without additional equipment.

AAV Xpress ELISA Short Protocol

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Variant detection with PROGEN antibodies

Same principle as the Titration ELISA: detection depends on preserved conformational epitope. The PROGEN AAV ELISA antibodies specifically recognize defined conformational epitopes present on fully assembled capsids of each AAV serotype.

Standard serotypes: Detection ensured.
Shuffled/modified capsids: May affect binding and titer determination.

A first indication is whether the antibody-binding epitope is preserved in your variant. More details on binding sites can be found in our FAQs section: Do PROGEN AAV ELISAs recognize AAV shuffled vectors?

Performance Data

AAV-based gene therapy has expanded rapidly over the past thirty years. This growth has led to a surge in demand from pharmaceutical companies and a boost in innovative technologies within the field. Consequently, more AAV gene therapy products are progressing from preclinical stages to clinical trials, highlighting concerns about their reliability, reproducibility, and comparability for production and regulatory bodies.

Data Comparability: Each AAV ELISA Kit has to pass a comprehensive quality control. This includes an analysis of intra- and inter-assay variances, as well as lot-to-lot consistencies. This quality control process ensures comparability of results obtained with PROGEN AAV ELISAs.
Matrix Effects: There are differences in buffer composition in the analyzed samples due to differences in company production processes. To ensure the best possible results, we analyzed tolerated concentrations of different additives in our AAV ELISAs.
Internal Gold Standards: International standard material is only available for serotypes AAV2 and AAV8. PROGEN has established internal gold standards to ensure we can provide AAV ELISA kits with consistent quality for each serotype.

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Matrix Effects of Additives

Determining AAV capsid titers can be influenced by several conditions. For example, the composition of your lysis buffer can have an impact. If the buffer has a high salt concentration, it can interfere with accurately detecting capsids.¹

For more information, you can refer to the table below, which shows the analysis of matrix effects of different additives on the AAV ELISAs. Data on cumulative effects have not been collected; therefore, tolerated concentrations could differ from the concentrations indicated in this table.

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 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.

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.

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" 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

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.