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Immunology and Gene Therapy

2A. Transduction inhibition based neutralizing antibody assay (AAV and AdV Vectors)

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Neutralizing antibodies

Principle: Virus neutralization assays are performed to assess the potential of antibody to block virus transduction of susceptible cell lines. The ability of antibodies to block adenovirus infection of Hela cells or AAV2 infection of 84-31 (293 cells stably expressing Ad-E4) in vitro is analyzed utilizing Green Fluorescent Protein as a reporter*. For the assay, various dilutions of antibodies pre-incubated with moi of 100 for adenovirus and 1000 for AAV reporter viruses for 1 hour at 37C, is added to 90% confluent cell cultures. Cells are incubated for 20 hours. Expression of GFP is quantitated by manually counting under a UV-microscope and LacZ by an automated luminometer. The neutralizing titer of antibody is calculated by the highest dilution of the sera where 50% of the cells are green.

*or LacZ

 

2B. Infection inhibition based on neutralizing antibody assay (AAV WT Virus)

The principle of this assay is the same as the transduction inhibition neutralizing antibody assay but in the infection inhibition assay we use wild type virus and A549 cells. Infection is quantified by immunostaining using a goat anti-hexon antibody raised against a conserve region present in all identified Adeno types.

Infection inhibition neutralizing antioby assay using polyclonal rabbit antisera

Figure : Infection inhibition neutralizing antibody assay to HAdv5 and SAdV24 using polyclonal rabbit antisera to HAdv5, SAdV23, SAdV24 and Human pooled IgG. Neutralization titers are highlighted.