A rapid Focus-Forming Assay for quantification of infectious adenoviral vectors.

Autor: Elahi SM; Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: Mehdy.Elahi@cnrc-nrc.gc.ca., Nazemi-Moghaddam N; Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: Nazila.Nazemi-Moghaddam@cnrc-nrc.gc.ca., Gadoury C; Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: Christine.Gadoury@cnrc-nrc.gc.ca., Lippens J; Department of Immunobiology, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: Julie.Lippens@cnrc-nrc.gc.ca., Radinovic S; Department of Downstream Processing and Analytics, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: Stevo.Radinovic@cnrc-nrc.gc.ca., Venne MH; Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: mhvpilates@gmail.com., Marcil A; Department of Immunobiology, National Research Council Canada, Building Montreal, Montréal, Canada. Electronic address: Anne.Marcil@cnrc-nrc.gc.ca., Gilbert R; Department of Production Platforms & Analytics, National Research Council Canada, Building Montreal, Montréal, Canada; Department of Bioengineering McGill University, Montréal, Canada. Electronic address: Renald.Gilbert@cnrc-nrc.gc.ca.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2021 Nov; Vol. 297, pp. 114267. Date of Electronic Publication: 2021 Aug 23.
DOI: 10.1016/j.jviromet.2021.114267
Abstrakt: Currently available methods to titrate adenoviral vectors (AdV) in the absence of a gene reporter such as GFP, are either time-consuming or not very reproducible. A Focus-Forming Assay (FFA) for quantification of infectious AdV particles followed by automated focus counting was developed using new monoclonal antibodies (mAbs) against the human adenovirus type 5. Briefly, in this method, 96-well plates of HEK293A cells were infected with 2-fold dilutions of AdV at seeding time. Forty eight hours post-infection, the cells were fixed with methanol. The cells were then incubated with each mAb followed by a FITC conjugated anti-mouse antibody. The plates were scanned and positive cells counted using an automated fluorescence microscopy system. The results of the FFA were compared with the plaque assay and the TCID 50 assay. The titer of six different recombinant AdV were compared using the FFA along with a commercial kit. The results were similar, but in contrast to the commercial kit for which the stained cells are counted manually, the software automatically counts the positives cells in the FFA. The automatic counting of positive cells makes the FFA a more precise and reliable assay compared to the commercial kit for titration of AdV.
(Crown Copyright © 2021. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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