Adeno-associated viruses (AAV) are non-pathogenic ssDNA viruses, which are subject of intense studies as viral vectors for gene therapy. The virus transduces a variety of dividing and non-dividing cells showing longterm gene expression with low cellular immune response. Methods for the characterization of AAV preparations currently include titration ELISA, qPCR, ddPCR, DNA dot blot, cell based assays, SDS-PAGE or electron microscopy. PROGEN’s Dip’n’Check AAV1 and AAV6 is a lateral flow sandwich assay for the detection of fully assembled AAV1 or AAV6 capsids using an antibody specific for AAV1 and AAV6. The quick and reliable detection of AAV samples allows a rapid estimation and comparison of concentrations in various samples for further downstream analysis or purification steps (e.g. for finding the optimal dilution range for subsequent ELISA analysis).
This lateral flow test is a semi-quantitative assay with a detection range of 1.0E+08 – 1.0E+10 capsids of AAV1 and AAV6 in the final assay volume of 156 μl. Within this range, a 2-fold concentration difference is clearly detectable.
- Reliable in-process control - during AAV manufacturing or to analyze further downstream processes
- Fast results - 20 minutes from start to finish
- Easy to use - for a rapid estimate and comparison of AAV capsid titers
- Suitable for all preparations - crude lysate or purified AAV material
- Serotype-specific - analyse binding affinity of AAV particle antibodies to AAV capsid variants
The test is based on the specific binding of an anti-AAV1/AAV6 antibody to fully assembled viral capsids. Empty and full capsids show no difference in binding to this antibody.
The antibody is used with two different conjugates, one of which is bound to the immobilized streptavidin on the test line (antibody conjugate B) while the other (antibody conjugate A) is bound by a conjugate-specific antibody on the gold nanoparticles on the test strip (Figure 1).
Both AAV antibodies 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 antibodies to the capsids. Ideally, the AAV sample should be tested in different dilutions. Then, the test strip is placed in this solution to allow complex formation with the gold nanoparticles and upward migration of the solution by capillary force to the test and control lines.
Figure 1: Test principle of the Dip’n’Check AAV1 and AAV6
Preparation of Reagents
Prior to use, it is important to allow the AAV test strips to reach room temperature (RT, 20 – 26°C) in order to avoid condensation of moisture on the test strip. After taking out the required number of strips, immediately close the plastic container and avoid longer exposure of the strips to moisture and light. Please use gloves and only touch the handling zone at the top end above the waste pad (Figure 2).
It is possible to test crude lysates of HEK293 or insect cells, no matrix effects were detected in samples with a total protein concentration of 100 μg/ml in the final test volume. Other additives frequently used in buffers have also been tested. The tolerated concentrations for these compounds are listed in Table 1 of section 10 (Matrix effects).
Figure 2: Dip’n’Check AAV1 and AAV6 test procedure
The upper control line must be clearly visible in all cases (positive control, negative control, unknown samples) in order to confirm the migration of gold particle complexes over the whole detection area of the strip. If the staining of the control line is very weak or absent, the test is invalid and should be repeated with a higher dilution of the sample as mentioned above.
Figure 3: Possible results of Dip’n’Check AAV1 and AAV6
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