AAV Titration ELISA
Reliable Quantification of Assembled AAV Capsids
The PROGEN AAV Titration ELISA is the industry-standard assay for quantifying fully assembled AAV capsids. It provides reliable, reproducible, and accurate data, making it a trusted tool for AAV development from research to clinical manufacturing.
How does the AAV Titration ELISA work?
- Capture: A monoclonal antibody specific for a conformational epitope on assembled AAV capsids is coated onto a microtiter plate to capture fully assembled AAV particles from the specimen. Captured AAV particles are detected in a two-step process.
- Detection: A biotin-conjugated monoclonal AAV antibody binds to the captured AAV particles.
- Signal: Streptavidin-peroxidase reacts with the biotin, and after adding substrate, the streptavidin conjugated peroxidase catalyses the substrate reaction, producing a bright colour.
- Readout: Absorbance at 450 nm is measured photometrically, directly proportional to the number of bound viral particles.
Your AAV Titration ELISA Benefits
Easy-to-use: Same workflow across all AAV serotypes; no expensive equipment required. Flexible single-break strips.
Reliable: PROGEN’s exclusive particle antibodies detect native, non-denatured capsids (full & empty). An industry gold standard with established success in clinical trials.
Reproducible: Robust method with CVs <10% (See Figure 1).
Accurate: AAV2 & AAV8 assays traceable to ATCC reference standards, and all other ELISAS (AAV1, 3, 5, 6, 9, and rh10) use internal gold standards.
GMP-ready: Can be validated for GMP use for adherence to FDA guidelines for the development of AAV gene therapies.
Can your variant be detected by PROGEN antibodies?
The PROGEN AAV ELISA antibodies specifically recognize defined conformational epitopes present on fully assembled capsids of each AAV serotype.
• If your vector uses standard serotypes: Detection is ensured.
• If your vector is shuffled or sequence-modified: Binding affinity may be reduced or altered, affecting 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 AAV ELISA FAQs section: '"Do PROGEN AAV ELISAs recognize AAV shuffled vectors?".'
Performance Data
To ensure reliability, reproducibility, and comparability, each AAV ELISA undergoes comprehensive quality control.
- Data Comparability: Intra- & inter-assay variance and lot-to-lot consistency.
- Matrix Effects: Tolerance testing for additives (e.g., salt concentration, buffer composition).
- Internal gold standards: Established for all serotypes where international standards are unavailable.
Data Comparability
Intra-Assay Variance
The intra-assay variance is tested by applying three samples in 24 replicates to the same microtiter plate in a single run. Recovery has to be within our internal acceptance range of +/- 30% while observed CVs are usually < 5%.
Inter-Assay Variance
The inter-assay variance is determined by measuring a comprehensively characterized internal control in four ELISA plates from the same lot on six different days. Observed CVs are usually < 7%.
Lot-to-lot Consistency
We focus on ensuring the lot-to-lot consistency of our AAV ELISA kits by using independent internal controls.
Note: Values are representative; serotype-specific data available upon request.
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.
1 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)
Automating the AAV ELISA
Working to move from manual to fully automated workflows?
Using the DSX® Dynex® system, PROGEN has developed a high-throughput AAV9 ELISA protocol.
Download our latest poster to view a high-throughput protocol for the AAV9 ELISA using the DSX® Dynex®.
Key insights include::
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Plate homogeneity, intra-/inter-assay variance, and recovery data.
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Direct comparison of manual vs automated ELISA results.
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Practical learnings from our automation journey.
Watch PROGEN’s AAV ELISA in Action
In this 90-second video, learn how to implement PROGEN’s AAV ELISA kits to accurately determine the capsid titer of your AAV preparations. Discover reliable and reproducible AAV quantification in action, and how it can enhance the consistency of your experimental outcomes. No costly equipment, no special training needed.
AAV ELISA FAQs
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.
- 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.
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
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:
AAV Xpress ELISAs were developed based on the corresponding AAV Titration ELISAs.
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.
