Description
Adeno-associated virus (AA) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases. AAV Serotype 1 (AAV1) is an efficient vector for gene delivery to skeletal muscle or the central nervous system. The AAV1 kit is designed to detect and quantify AAV1 particles in an easy-to-use, no-wash format. The simple and robust procedure benefits from increased throughput compared to ELISA.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
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.
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.
