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Fig. 2 | BMC Biotechnology

Fig. 2

From: A novel synthetic trivalent single chain variable fragment (tri-scFv) construction platform based on the SpyTag/SpyCatcher protein ligase system

Fig. 2

Analysis of the HER3 binding Tri-scFv and monomeric scFv-SpyCatcher to recombinant HER3 and a HER3-postive cell line. Biolayer interferometry kinetic analysis of anti-HER3 Tri-scFv and anti-HER3 scFv-SpyCatcher against recombinant HER3. a HER3 sensors were generated by covalently immobilizing HER3 on AR2G sensors. HER3 sensors were placed in wells containing concentrations of Anti-HER3 Tri-scFv (i) and anti-HER3 scFv-SpyCatcher (ii), ranging from 20 nM to 500 nM. b anti-HER3 Tri-scFv and anti-HER3 scFv-SpyCatcher sensors were generated by covalently immobilizing Anti-HER3 Tri-scFv (i) and anti-HER3 scFv-SpyCatcher (ii) on AR2G sensors. Tri-scFv and monomeric scFv loaded sensors were then placed in wells containing concentrations of HER3, ranging from 20 nM to 500 nM. c Comparison of koff (i), kon (ii), and KD (iii) between anti-HER3 Tri-scFv and scFv-SpyCatcher with HER3 using HER3 and scFv sensors. Data analysis and fitting were performed using ForteBio’s Data analysis software version 8.1. Error bars represent the percentage of fitting error as calculated by FortéBio Data Analysis software. d Flow cytometry analysis of binding of anti-HER3 Tri-scFv and scFv-SpyCatcher to the HER3-positive FaDu cell line. A fixed concentration (0.5 μM) of IRDye680RD-labeled anti-HER3 Tri-scFv-IRDye680 (HER3 Tri-scFv) and anti-HER3 scFv-SpyCatcher-IRDye680 (HER3-scFv) were incubated with 1 × 105 FaDu cells. Anti-Maltose Binding Protein scFv-SpyCatcher-680 (MBP scFv-SpyCatcher) was used a negative control. The mean fluorescent intensity (MFI) was 592 for FaDu cells, 696 for the MBP-scFv, 3834 for the scFv, 4636 for the bivalent-scFv, and 6919 for the Tri-scFv. The Labeling efficiency of the Tri-scFv-IRDye680, bivalent-scFv-IRDye680, scFv-SpyCatcher-IRDye680 and MBP-scFv-IRDye680 was 2.1, 1.7, 1.6, and 1.5 dyes per antibody fragment, respectively

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