AAV3B capsid assay principle

The Adeno-Associated Virus serotype 3B (AAV3B) assay measures AAV3B capsid in cell supernatant or cell lysate. The assay uses two anti-AAV3B antibodies: one coupled to HRP that binds to anti-HRP d2 (acceptor) in premix 1, and the other coupled to biotin, that binds to Streptavidin Eu-cryptate (donor) in premix 2. In presence of AAV3B capsid in a cell extract or supernatant, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor, and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the capsid present in the sample and provides a means of assessing any changes caused by experimental variability under a no-wash assay format.

AAV3 Capsid assay protocol

The AAV3B Capsid assay protocol using a 384-well small volume white plate is described on the right. 5 µL of sample or standard and 5 µL of diluent are dispensed directly into the plate for detection by HTRF® reagents. The Biotin anti-AAV3B antibody is pre-mixed with Streptavidin labeled with the donor, and HRP anti-AAV3B is pre-mixed with anti-HRP labeled with the acceptor. 5 µL of each premix are added. The assay can be run in up to a 1536-well format by simply resizing each addition volume proportionally.

AAV3B Capsid assay specifications

Intra-assay

Each of the 3 samples was measured 24 times, and % CV was calculated for each sample.

Inter-assay

Each of the samples was measured in 3 independent experiments performed by different operators, and the % CV was calculated for each sample.

Cross reactivities

Cross reactivities were assessed using other serotypes from the AAVs family. Standard curves were generated for each serotype using AAVs capsids diluted in the kit diluent.

5 µL of capsids were transferred into a white detection plate (384 low volume), and 5 µL of diluent followed by 10 µL of the HTRF AAV3B capsid detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

The assay shows differential affinities depending on the serotype, but does not detect AAV8 and AAV9.

Validation of HTRF AAV3B capsid detection on full and empty AAV3B particles

To demonstrate the detection of both full and empty AAV3B capsids, recognition of full AAV3B-CMV-eGFP and empty AAV3B capsids was analyzed in the assay. A large range of AAV3B-CMV-eGFP concentrations (GC/mL) was converted into VP/mL using an independent sample quantitation assay. 5 µL of full or empty capsids diluted in the kit diluent were then transferred into a white detection plate (384 low volume), and 5 µL of diluent followed by 10 µL of the HTRF AAV3B capsid detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. As expected, the HTRF AAV3B capsid detection assay could detect both full and empty AAV3B capsids in the same way.