AAV1 Capsid assay principle

The Adeno-Associated Virus serotype 1 (AAV1) assay measures AAV1 capsid in cell supernatant or cell lysate. The assay uses two anti-AAV1 antibodies: one coupled to HRP and which binds to anti-HRP d2 (acceptor) in premix 1, and the other coupled to biotin and which binds to Streptavidin Eu-cryptate (donor) in premix 2. In presence of AAV1 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.

AAV1 Capsid assay protocol

The AAV1 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 antibody anti-AAV1 is pre-mixed with Streptavidin labeled with the donor, and HRP anti-AAV1 was pre-mixed with anti-HRP labeled with the acceptor. 5 µL of each premix were added. The assay can be run in up to a 1536-well format by simply resizing each addition volume proportionally.

standard curve Spike S1 kit

Intra assay (n=24)

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

Inter assay (n=4)

Each of the samples was measured in 3 independent experiments performed by different operators, and CV percentages were 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 AAV1 capsid detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. The assay showed differential affinities depending on the serotype, but did not detect AAV8 and AAV9.

Validation of HTRF AAV1 capsid detection on full and empty AAV1 particles

To demonstrate the detection of both full and empty AAV1 capsids, recognition of full AAV1-CMV-eGFP and empty AAV1 capsids were analyzed in the assay. A large range of AAV1-CMV-eGFP concentrations (GC/mL) were 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 AAV1 capsid detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature. As expected, the HTRF AAV1 capsid detection assay could detect both full and empty AAV1 capsids in the same way.