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Figure 1

From: Targeted delivery of TLR3 agonist to tumor cells with single chain antibody fragment-conjugated nanoparticles induces type I-interferon response and apoptosis

Figure 1

Generation and functional validation of parental scFvs and mono-biotinylated scFv-BAPs. (a) Schematic representation of PSCA- and EGFRvIII-specific parental scFv and scFv-BAP antibody constructs consisting of a Igκ-leader secretion signal, a variable heavy (VH) and a variable light (VL) chain, a C-terminal c-myc epitope and a 6x histidine (His) tag. A biotin acceptor peptide (BAP) was introduced to scFv-BAPs for site-specific enzymatic mono-biotinylation. (b) Purity of scFv(h-AM1) (open arrowhead) and scFv(h-AM1)-BAP (black arrowhead) was analyzed using Coomassie-stained polyacrylamide gel under reducing conditions. (c) Size of scFv(h-AM1) and scFv(h-AM1)-BAP via c-myc epitope and site-specific biotinylation of scFv(h-AM1)-BAP were assessed by Western blot analysis. The full-length blots/gels are presented in Supplementary Fig. 1. (d) Binding studies for functional characterization of scFvs and scFv-BAPs via the c-myc epitope and (e) for validation of mono-biotinylated scFv(h-AM1)-BAP and scFv(MR1.1)-BAP were undertaken using flow cytometry. Open histograms represent staining controls using only secondary antibodies.

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