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. 2017 May 19;7:100. doi: 10.3389/fonc.2017.00100

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Copyright © 2017 Ellwanger, Reusch, Fucek, Knackmuss, Weichel, Gantke, Molkenthin, Zhukovsky, Tesar and Treder.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Binding affinity and specificity for EGFRvIII antigen and on cells. (A,B) Concentration-dependent binding of different His-tagged antibody fragments (A) or monoclonal antibodies (B) to recombinant EGFRvIII-Fc, EGFRvIII^trunc-Fc, or EGFR-Fc coated in ELISA. K_D values indicated in the graphs were calculated with the equation one site binding (hyperbola), least squares (ordinary) fit using GraphPad Prism. (C–E) Concentration-dependent cell binding of different antibody constructs analyzed by sigmoidal dose–response/four parameter logistic fit: (C) CHO cells stably transfected to overexpress EGFRvIII, (D) DK-MG cells expressing EGFRvIII and EGFR, (E) A431 cells overexpressing EGFR (cetuximab staining signals on A431 cells due to high intensity are plotted on the separate left axis, signals of all other antibodies are plotted on the right axis with different scale). (F) FACS histogram overlay of A431 cells stained with saturating concentrations of ch806 or cetuximab or detection antibodies alone. MFI, mean fluorescence intensity, ctrl., control antibody staining at single dose of 10 µg/mL.