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Selection of Single Domain Antibodies from Immune Libraries Displayed on the Surface of E. coli Cells with Two β-Domains of Opposite Topologies

Figure 7

Determination of the equilibrium dissociation constant (KD) of sdAb VTIR1 by SPR and E. coli display.

(A) SPR sensograms monitoring real-time association and dissociation of purified sdAb VTIR1 (at the indicated concentrations) to biotinylated TirMEHEC immobilized onto a Streptavidin-SA sensor chip. The increase in resonance units (RU) is recorded along time (in seconds). Dissociation of VTIR1 is evaluated by injection of buffer at the time indicated with an arrow. (B) RU values at 220 seconds (labeled with a rectangle in A) are plotted versus the different concentrations of VTIR1. The curve was fitted by non-linear least squares regression. (C) The KD of VTIR1 was estimated by flow cytometry analysis of E. coli cells expressing NVTIR1 incubated with different concentrations of biotinylated TirMEHEC (1-20 nM) under equilibrium conditions. The mean fluorescent intensities (MFI) of bacteria, after labeling with Streptavidin-PE, were plotted versus the concentration of TirMEHEC used in the assays. The curve was fitted by non-linear least squares regression.

Figure 7