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AAVrh10 Titration ELISA

Cat. No. PRAAV10

Available, delivery time 1-7 days

€552.00*

Key Features
  • ELISA for quantitation of AAVrh10 capsids
  • Detection of full and empty AAVrh10 capsids
  • ADK8 antibody is used as capture and detection antibody
Product description
Quantity 96 tests
Reactivity AAVrh10
Storage 2-8°C
Intended use Research use only
Application ELISA
Product description Unique microtiter plate enzyme immunoassay for the quantitation of virions and assembled empty capsids of AAVrh10. The monoclonal capture-antibody detects a conformational epitope not present on unassembled capsid proteins.
Kit Control/Standard Empty capsid preparation of AAVrh10
Form supplied Reagent kit, 12 x 8-well strips
Background

The assay represents a fast, sensitive and reproducible method for titration of intact AAVrh10 wt virions, AAVrh10 recombinant virions or assembled and intact empty AAVrh10 capsids.

The ELISA principle:

The assay is based on the sandwich ELISA technique where a monoclonal antibody (mab) specific for a conformational epitope on assembled AAV capsids is coated onto the plate and is used to capture AAV particles from the specimen.

The detection of captured AAV particles is a two-step process.

  1. A biotin-conjugated mab is bound to the captured AAV particles.
  2. A streptavidin peroxidase conjugate reacts with the biotin molecules. The addition of the substrate results in a color reaction which is proportional to the amount of specifically bound viral particles.

Comparison of AAV Quantification Methods:

Each of the commonly used quantification methods has its pros and cons:

  • qPCR is widely used, but suffers from several issues such as sample preparation, primer design, or PCR efficiency that can lead to high inter-laboratory variation of results.
  • Digital droplet PCR methods overcome some of the limitations of qPCR. However, variations between labs can still occur due to different sample processing protocols.
  • Dot blot is a simple and quantitative method, if reliable reference material is used. However, it suffers from the limited linearity and dynamic range of western blotting in general.

Given the practical drawbacks of the aforementioned techniques, a conventional sandwich ELISA currently appears to be superior in terms of inter- and intralaboratory variation as well as ease of use. Therefore, it represents the best format for reliable and reproducible quantification of total rAVV capsid titers.

Use of shuffled/mutated AAV:

The recognition of shuffled/mutated AAV vectors depend on the specific capsid region which is affected by the shuffling/mutation. The capture antibodies used for PROGEN’s AAV ELISAs bind specific and, in some cases, well defined conformational epitopes. These epitopes are generated by the capsid assembly of the corresponding AAV serotypes. A first indication that the ELISA might recognize your shuffled/mutated AAV vector is the presence of the antibody-binding epitope. However, changes in the protein sequences of the capsid proteins might also influence conformation of the proteins, hence the conformation of the epitopes presented on the AAV capsid. This might influence binding affinity of the antibody and affect determination of the titer based on the (non-shuffled) Kit Control provided with the AAV ELISA kit. Since these properties strongly depend on the specific shuffling/mutation performed, PROGEN cannot guarantee successful and precise quantification of your shuffled/mutated AAV vector. Even if the antibody binding epitopes are still present on your shuffled/mutated AAV capsid, your assay needs to be tested and optimized for your specific AAV vector.

PROGEN highly recommends the production and calibration of a suitable (shuffled/mutated) Kit Control, to ensure reliable titer determination of your individually shuffled/mutated AAV vector with PROGEN ELISA kits.

Limited Use Label License: Research Use Only
Product is exclusively owned by PROGEN Biotechnik GmbH. The use of these products for the development, manufacturing and sale of secondary products/derivatives which are based on the purchased products and/or which include the purchased product require a royalty based sub-license agreement.

 

 

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FAQs
There are several methods to determine AAV titers, however these methods differ in quality, reliability and most important in the distinct AAV titer they measure. For example, qPCR and ddPCR detects viral DNA and is suitable for the determination of AAV DNA titers only. These methods give no indication of total AAV capsid titers, which is an essential parameter for the use of AAV in gene therapy. Even though qPCR and ddPCR are frequently used and highly sensitive methods for AAV titer determination, they show high variability. In contrast to qPCR/ddPCR, the PROGEN AAV ELISA determines the total capsid AAV titer, including full and empty capsids. PROGEN´s ELISA cannot be used for the measurement of AAV DNA titers. Compared to the high variability of qPCR/ddPCR, the PROGEN ELISA is a very robust and accurate method for AAV determination. The PROGEN AAV ELISA provides reliable and consistent data since the inter- and intra-assay variability is very low.
PROGEN recommends the use of different methods for AAV titer determination, including AAV DNA titer and AAV total capsid titer to ensure complete quantification and analysis of AAV preparations.  
The Kit Control provided with each AAV Titration ELISA consists of fully characterized, empty particles and is used to generate the standard curve for the ELISA. Based on the standard curve, the total capsid titer of an unknown AAV sample can be determined.  
PROGEN´s AAV2 and AAV8 Titration ELISAs are calibrated based on the international standard material provided by the ATCC.
  
AAV2: ATCC-VR-1616 rAAV2 RSS reference material

AAV8: ATCC-VR-1816 rAAV8 RSS reference material

The remaining AAV Titration ELISAs are calibrated based on our internal gold standards. These standards are highly pure, full AAV capsid preparations. Characterization was performed by qPCR or ddPCR by different labs and electron microscopy (EM). The Kit Controls provided with our AAV ELISAs are adjusted to these internally characterized standards. For more information on the characterization of our internal gold standards download our posters “Developing reliable AAV standards for ELISA” and “The new AAV3 Titration ELISA – continued tradition of reliable AAV titer determination”.
Please note, that the internal gold standards used for the calibration of PROGEN´s AAV Titration ELISAs cannot be purchased or provided in any way since they are produced exclusively for internal use to calibrate PROGEN´s ELISAs.
No, PROGEN does not offer sample size ELISA kits. Each kit contains one 96-well microtiter plate for testing.
The recognition of shuffled AAV vectors depend 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

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).
PROGEN’s AAV ELISA is based on the sandwich ELISA technique. Microtiter plates are pre-coated with a monoclonal antibody specific for a conformational epitope on assembled AAV capsids and thereby capturing the AAV particles (full & empty). The detection of AAV particles is a two-step process, (i) a biotin-conjugated monoclonal AAV antibody binds to the captured AAV particles, followed by (ii) a streptavidin peroxidase-conjugate reaction with the biotin molecules. After addition of the substrate, a color reaction represents the amount of specifically bound viral particles. Absorbance is measured photometrically at 450 nm and the AAV titer of unknown samples can be calculated based on the standard curve generated with the corresponding Kit Control.
For detailed information and short protocol see AAV ELISA.
PROGEN AAV Titration ELISAs are currently available for AAV serotypes 1, 2, 3, 5, 6, 8, 9, and rh10. For faster analysis of your AAV capsid titers PROGEN offers AAV Xpress ELISAs for AAV serotypes 2, 8, and 9.
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, please check out 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.

No, PROGEN does not offer an AAV ELISA based quantification service. PROGEN certainly offers the tools for reliable and reproducible AAV quantification by ELISA by providing serotype-specific ELISA kits. An AAV ELISA kit contains one microtiter plate coated with the corresponding AAV specific antibody (ready-to-use), suitable Kit Control (lyophilized AAV particles), anti-AAV Biotin conjugate, streptavidin peroxidase conjugate, assay buffer (ASSB), as well as substrate and stop solution.

PROGEN is distributor for density gradient media produced by Axis-Shield Diagnostics and offers amongst others OptiPrep™ for AAV particle purification. For a complete list of density gradient media offered by PROGEN, please visit our Density Gradient Website .
Please note, distribution of the Axis-Shield products in PROGEN’s portfolio is restricted to DACH countries only. 
PROGEN AAV ELISAs are reliable quantification tools for total AAV capsid determination for gene therapy research and development. They are robust and accurate tools, which have been demonstrated to show low inter- and intra-assay variability. Currently the AAV ELISA appears to be the best format for the quantification of rAAV preparations in comparison to other quantification methods in terms of inter-assay variability and ease of use.
The PROGEN ELISA kits are precisely calibrated for the specific AAV serotypes and provide only the serotype-specific Kit Control. Therefore, PROGEN can only ensure accurate quantification of the corresponding AAV serotype. This is true for all PROGEN´s AAV ELISA kits.
PROGEN’s AAV ELISA kits are provided with one pre-coated microtiter plate containing 12 x 8-well-strips. To ensure a reliable quantification of AAV titers, 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. The Kit Control and the AAV samples need to be tested in duplicates to provide accurate results (for detailed instructions, see the work instructions provided with the AAV ELISA kit). Therefore, after having applied the standard curve, 40 duplicate measurements can be applied.
PROGEN offers serotype-specific AAV Titration ELISA kits for quantification of the AAV serotypes 1, 2, 3, 5, 6, 8, 9, and rh10 as well as AAV Xpress ELISA kits for AAV serotypes 2, 8, and 9.
Some of PROGEN´s AAV ELISA kits show cross-reactivity with other AAV serotypes. E.g., the AAV2 ELISA kit cross-reacts with AAV3. However, this does not mean that the AAV2 ELISA kit is suitable for quantification of AAV total capsid content of AAV3 preparations. Due to the serotype-specific calibration of the ELISAs PROGEN recommends using the corresponding ELISA for your specific AAV serotype for reliable capsid titer quantification.
To learn more about cross-reactivities of the AAV antibodies see the following table 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 AAV Xpress ELISAs for the AAV serotypes 2, 8, and 9 were developed based on the corresponding AAV Titration ELISAs. The difference between the AAV Titration ELISAs and the AAV Xpress ELISAs is the adjustment of the kit components to shorten the incubation times from 3:15 hrs to 1:20 hrs. Despite the adjustments, the kit components of the AAV Xpress ELISAs are the same as provided with the AAV Titration ELISAs. Also the processing of the ELISA stays exactly the same except for the shortened incubation times for the AAV Xpress ELISAs.
All of PROGEN´s AAV ELISAs, including the AAV Titration ELISAs as well as 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
For application of AAV vectors in gene therapy, the determination of total capsid titer is essential, since most of the AAV preparations contain a significant number of empty capsids. Accurate characterization and quantification of purified AAV particle preparations represent a critical step for clinical application, to minimize immunogenic responses and maximize gene transfer to the target cells. Therefore, reliable and reproducible quantification of AAV titers is essential for safe and effective AAV gene therapy.
The determination of AAV capsid titers from e.g. cell extract can be influenced by several conditions such as the composition of your lysis buffer. For example, high salt concentrations in your buffer might inhibit adequate capsid detection. For more information, refer to the table below showing the analysis of matrix effects of different additives on the AAV ELISAs. For more information visit our AAV-ELISA page

table matrix effects

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