AAV8 capsid assay principle

The Adeno-Associated Virus serotype 8 (AAV8) assay measures AAV8 capsid in cell supernatant or cell lysate. The assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. In presence of AAV8 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.

AAV8 capsid assay protocol

The AAV8 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 antibodies labeled with HTRF fluorophores were added before an overnight incubation. The assay can be run in up to a 1536-well format by simply resizing each addition volume proportionally.

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 % CV was calculated for each sample.

The excellent % of recovery obtained from these experiments shows the good dilution linearity of the assay. Samples were AAV8 empty capsids serially diluted with the kit DIL5 diluent.

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

5 µL of capsids were transferred into a white detection plate (384 low volume), followed by 5 µL of diluent then 10 µL of the HTRF AAV8 capsid detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

Signals were interpolated on the assay standard curve to interpolate concentrations. The assay is specific to AAV8 as other serotypes were not detected using this assay.

Validation of HTRF AAV8 capsid detection on full and empty AAV8 particles

To demonstrate the detection of both full and empty AAV8 capsids, recognition of full AAV8-CMV-eGFP and empty AAV8 capsids was analyzed in the assay. A large range of AAV8-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 and 10 µL of the HTRF AAV8 capsid detection reagents. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, the HTRF AAV8 capsid detection assay can detect in the same way both full and empty AAV8 capsids.