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. 2015 Aug 21;26(11):767–776. doi: 10.1089/hum.2015.097

Figure 4.

Figure 4.

(A) Schematic representation of the self-complementary fluorescent reporter construct used for in vitro neutralization assay. ITR, inverted terminal repeat; smCBA, small chicken β-actin promoter; mCherry, red fluorescent reporter gene; p(A), polyadenylation signal. (B) Diagrammatic workflow of in vitro neutralization experiments. (C) Neutralization of AAV2 (left) and QuadYF-TV (right) vectors by serum containing high (Sero +ve) or low (Sero –ve) levels of neutralizing antibodies raised against AAV2 at dilutions ranging from 1:40 to 1:320 as assessed by flow cytometry. A high percentage of neutralization (90–100%) indicates that transduction of the ARPE-19 cells by the vector has been effectively inhibited, that is, little to no transgene expression was observed. It is clear that despite the introduction of five capsid mutations, circulating antibodies raised toward wild-type AAV2 recognize the QuadYF-TV vector capsid.