AAV5 Capsid assay principle

The Adeno-Associated Virus serotype 5 (AAV5) assay measures AAV5 capsid in cell supernatant or cell lysate. The assay uses two antibodies anti-AAV5: one labelled with d2 (acceptor) and the other to biotin that binds to Streptavidin Eu-cryptate (donor) in a premix. In presence of AAV5 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.

AAV5 capsid assay protocol

The AAV5 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 detection plate for detection by HTRF® reagents. The Biotin antibody anti-AAV5 was pre-mixed with Streptavidin labeled with donor before being added. The assay can be run in up to a 1536-well format by simply resizing each addition volume proportionally.

AAV5 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.

Validation of HTRF AAV5 capsid detection on full and empty AAV5 particles

To demonstrate the detection of both full and empty AAV5 capsids, recognition of full AAV5-CMV-eGFP and empty AAV5 capsids was analyzed in the assay. A large range of AAV5-CMV-eGFP concentration (GC/mL) was calculated in 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), followed by 5 µL of diluent then 10 µL of the HTRF AAV5 capsid detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature. As expected, the HTRF AAV5 capsid detection assay can detect in the same way both full and empty AAV5 capsids.