Enzyme Immunoassay for the Quantitative Determination of AAV Serotypes 1, 2, 3, 5, 6, 8 and 9 Particles in Cell Culture Supernatants and Purified Virus Preparations. Immunotitration by PROGEN's AAV Titration ELISAoffers a fast, sensitive and reproducible method for titration of intact AAV wild type virions, AAV recombinant virions or assembled and intact empty AAV capsids.
The principle of the assay is based on the sandwich ELISA technique. A monoclonal antibody specific for a conformational epitope on assembled AAV capsids is coated onto microtiter strips and is used to capture AAV particles from the specimen.
Captured AAV particles are detected in two steps. First a biotin-conjugated monoclonal antibody to AAV2 is bound to the immune complex. In the second step streptavidin peroxidase conjugate reacts with the biotin molecules. Addition of substrate solution results in a color reaction which is proportional to the amount of specifically bound viral particles. The absorbance is measured photometrically at 450 nm.
Quantification methods for rAAV vector preparations include quantitative PCR (qPCR), digital droplet PCR (ddPCR) for measuring DNA, dot blot, and enzyme-linked immunosorbent assay (ELISA) for intact viral capsids proteins. Other methods using protein chromatography, flow cytometry, or virus particle counting instruments have also been described, but are generally only applicable for highly skilled users and/or require specialised and expensive equipment. Electron microscopy can be used to determine the ratio of empty and full viral capsids, but is not useful for an absolute quantification of particels.
Each of the mentioned techniques has its pros and cons: qPCR is widely used, but suffers from several issues such as sample preparation, primer design, or PCR efficiency that can lead to high inter-laboratory variation of results for identical samples. Digital droplet PCR methods overcome some of the limitations of qPCR. There is no need of a dilution series of standard DNA with known concentrations to measure an unknown sample, and limited PCR-efficiency is not an issue as it is for qPCR. However, variations between labs can still occur due to different sample processing protocols. Dot blot is a relatively simple and quantitative method, but works only with reliable reference material, while suffering from the limited linearity and dynamic range of western blotting in general.
Given the practical drawbacks of the aforementioned techniques, a conventional sandwich ELISA currently appears to be the best format for the quantification of rAVV preparations.