AAV Dip'n'Check
Rapid AAV Titer Determination
In both academia and industry, AAV is the most commonly used viral vector for the delivery of therapeutic transgenes. However, testing individual and crude AAV samples quickly during development and manufacturing is often difficult. Most accurate analytical AAV tools are not convenient for single sample measurements and/or require highly purified samples.
PROGEN's AAV Dip'n'Check lateral flow tests are a quick and easy in-process control for AAV capsid purification processes or for analytical pre-testing. A lateral flow sandwich assay for the semi-quantitative detection of fully assembled AAV capsids using PROGEN's exclusive AAV antibodies.
Available for AAV1, AAV2, AAV3, AAV5, AAV6, AAV8 and AAV9.
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Your AAV Dip'n'Check Benefits
Reliable in-process control: can be easily implemented both during AAV manufacturing or as a tool to analyze further downstream processes
Fast results: only takes 20 minutes from start to finish
Easy to use: an easy to follow 4-step protocol and easy to use lateral flow test
Suitable for all preparations: can be used with crude lysate or purified AAV material
Capsid-specific: AAV particle antibodies have a binding affinity to the epitopes present only on fully assembled AAV capsids
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How do the AAV Dip'n'Check Tests Work?
Lateral flow tests are based on the passing of a liquid sample through a series of capillary beds driven by capillary forces. The analysis of the presence of a specific substance in the sample follows the same principal as the affinity chromatography.
The surface of the tests contains reactive molecules that show either a positive or negative visual result depending on the presence or absence of the substance. Lateral flow tests generally use specific conjugates usually consisting of an antibody specifically binding the target molecule labelled with a visual tag, e.g. colloidal gold.
The samples to be analyzed are pre-incubated with the conjugates before the application of the sample to the test strip.
After the conjugate binds to the target molecule, the complex migrates further along the test strip until reaching the test line containing an immobilized reagent to capture the target-conjugate complex. With the binding of the complex, the visual tag accumulates at the test line and shows a visual change.
In the case of the Dip´n´Check AAV a purple-red color confirms the presence of the target molecule. In contrast to the test line, the control line should appear independently of the presence of the target molecule to confirm the validity of the test.
Easy Four Step Workflow
The AAV sample is pre-incubated with two differently conjugated AAV antibodies to allow coupling of the gold conjugate and for immobilization at the test line. The two antibody solutions contain the same antibody but labelled with two different conjugates.
The antibodies used for the Dip'n'Check AAV kits are AAV particle antibodies exclusively binding to fully assembled AAV capsids. Both AAV antibody conjugates delivered with this test are mixed in equal volumes with running buffer and the AAV sample, and incubated for 10 min to allow binding of the antibodies.
The test strip is then placed in the solution to allow complex formation with the gold nanoparticles at the conjugate pad and further migration by capillary force to the test and control lines.
The Dip´n´Check AAV quick test is a semi-quantitative assay with a detection range of 1.0E+08 – 1.0E+10 capsids. Within this range, a 2-fold concentration difference can clearly be detected. There is no difference in the antibody affinity to empty and full capsids.
Performance Data
AAV Dip'n'Check Lateral Flow Tests can be used for a broad range of applications, ultimately helping to facilitate and accelerate AAV-based gene therapy.
Lets take a look at the variety of applications that the AAV Dip'n'Check Lateral Flow Tests can be used for.
- Comparing capsid titers of two unknown samples
- Titer estimation using a reference standard
- Spot landing in the optimal dynamic range
- Testing antibody binding to a variant AAV
1. Comparing capsid titers of two unknown samples
When comparing two AAV9 preparations we could see a higher intensity for sample 1 (S1). This indicates that there is a higher capsid titer compared to sample 2 (S2). We were able to confirm this result using image analysis and our AAV ELISA (Figure 2). We used several serotypes to compare the AAV Dip'n'Check Tests against corresponding AAV ELISA data and the results showed a strong correlation between the two methods (Table 1).
2. Titer estimation using a reference standard
AAV Dip’n’Check tests can be used as a titer estimation for AAV preparations, which you can then compare both visually and digitally with an AAV reference material (Figure 3 & Table 2). In Figure 3 you can see the AAV9 sample (1:1,000) has an estimated titer of approx. 5.0E+09 capsids/ml (see red arrows). We were able to confirm this data across different serotypes using the corresponding AAV ELISA titers (Table 3).
3. Spot landing in the optimal dynamic range
Most accurate capsid titer determination methods, for example the AAV ELISA, have a specific dynamic range. It can be challenging to find the correct dilution of unknown samples. But, by using the AAV Dip'n'Check tests to determine the titer of an unknown sample, you are able to hit the optimal dynamic range in the first attempt. In Figure 4, the 1:1,000 dilution of the AAV6 sample shows a similar intensity to the reference material, indicating that the undiluted sample has a capsid titer of approx. 5.0E+12 capsids/ml. Therefore, a dilution range between 1:25,000 to 1:100,000 allows you to hit the optimal dynamic range to determine the accurate titer required for the AAV ELISA.
4. Testing antibody binding to a variant AAV
Before using antibody-based methods (e.g. ELISA) for AAV capsid variants, you need to confirm the binding of AAV antibodies to the engineered capsid. Pre-testing the antibody binding using AAV Dip’n’Check Tests allows you to determine which AAV ELISA is most suitable. The binding capabilities of AAVrh10 and AAVrh74 to the ADK8 were confirmed by testing different dilutions of both serotypes with the AAV8 Dip’n’Check Tests (Figure 5, A & B). In contrast, the binding affinity was analyzed by testing similar dilutions of AAV8, AAVrh10 and AAVrh74 and comparing the signal intensities (Figure 5, C). The results from the AAV Dip’n’Check Tests show the AAV8 ELISA is suitable for use with AAVrh10 and AAVrh74. However, it also indicates that it is necessary to adapt the AAV ELISA due to the lower affinity of the antibody to the capsid variants.
"The Dip’n’Check Kits are very easy and fast and also seem quite accurate for a semi-quantitative assay! So that is great!"
Senior R&D Scientist, Hemotune AG
"These test strips were a perfect way to confirm which PROGEN ELISA kit to use when dealing with serotypes that have variations."
AskBio
"For rapid confirmation of AAV production, the PROGEN Dip'n'Check Strips are fantastic."
Analytics Team Leader
"Dip'n'Check Kits 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."
Tomasz Witkos, AstraZeneca
“They’re fairly quick, easy to do, and affordable. We could test after every step of processing to make sure there were still some AAVs there.”
— Elissa Hudspeth, NC State University
Potential Applications for AAV Dip'n'Check
AAV is the most commonly used viral vector for the delivery of therapeutic transgenes in academia and industry. However, the gene therapy community is facing major challenges, such as the high demand for effective and reliable analytical AAV tools for gene therapy manufacturing and develompent
This poster shows the potential applications of AAV lateral flow assays and how they can help accelarate your next AAV gene-therapy based project.
1. Comparing the capsid titer of two unknown samples
2. Estimating the titer using a reference standards
3. Spot landing in the optimal dynamic range
4. Testing antibody binding to variant AAV serotypes
Fast and Easy In-Process Control
Watch our 90 second video and find out how to determine the capsid titer of your AAV prep in 20 min using the Dip´n´Check AAV Lateral Flow Tests.
AAV Dip'n'Check FAQs
No, the Dip’n’Check assay detects all assembled capsids independent of filling grade.
The Dip’n’Check assay can be used with crude lysate. To find out more about the concentrations of tested tolerated buffer additives take a look in the product manual and go to Section 10 - Matrix Effects or you can also find Table 1 - Concentrations of tolerated buffer additives on the individual product website pages.
The AAV Dip'n'Check test can be used for a variety of applications:
- To compare AAV titers from different AAV preparations. With this information you can select the most promising sample for further processing.
- To estimate AAV titers in crude and purified AAV preparations. To either control efficiency of your processes or to pre-test AAV concentrations to hit the dynamic range of downstream analytics.
- To test the binding of common AAV antibodies to an AAV capsid variant. Allowing you to confirm which antibody-based methods are suitable for further analysis.
More information about the determination of the Dip’n’Check can be found on our poster: Potential Applications for AAV Lateral Flow Tests
The lateral flow assay detects 1E+08 to 1E+10 total capsids in the assay volume, which corresponds to a concentration of 1E+09-1E+11 capsids/ml of the starting material.
