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. Author manuscript; available in PMC: 2009 Feb 6.
Published in final edited form as: Nat Methods. 2007 Jan 7;4(2):143–145. doi: 10.1038/nmeth993

Figure 2.

Figure 2

Figure 2

Figure 2

Figure 2

Evaluation of scFv binding and internalization properties. (a) Western blotting of full-length, secreted scFv. Batch secretion yields for each clone are listed above the blot. (b) Equilibrium binding attributes of scFvA and scFvD. Left panel: binding isotherm for scFvA interaction with live RBE4 cells. The plot shows the fitted monomeric equilibrium binding functions and experimental data from two independent experiments. Right panel: binding isotherm for dimerized scFvD interaction with RBE4 cells. The plot shows the fitted monomeric equilibrium binding functions used to generate an apparent affinity (avidity) and experimental data from two independent experiments. Insets indicate raw flow cytometry histograms that were used to generate the binding curves. (c) Yeast display immunoprecipitation of antigens for scFvA (A), scFvD (D), scFvJ (J) and O×26 scFv (O). Irrelevant anti- hen egg lysozyme scFv (N) was used as a negative control. The immunoprecipitation products were resolved by either nonreducing (NR) or reducing (R) gel electrophoresis and were probed with an anti-biotin antibody or an anti-transferrin receptor antibody (O×26). (d) Binding and internalization characteristics of scFv. RBE4 cells were labeled with purified, pre-dimerized scFvA, scFvD, irrelevant scFv 4-4-20 or O×26 monoclonal antibody at 4°C for cell surface labeling and then shifted to 37°C to promote cellular trafficking. The cells were then labeled with AlexaFluor 555 conjugated anti-mouse IgG (red) at 4°C followed by AlexaFluor 488 conjugated anti-mouse IgG (green) with or without cell permeabilization by saponin (SAP) treatment. Merged images of the AlexaFluor-labeled images are shown. Scale bar: 20 μm.