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. 2016 Sep 9;5(10):e1220467. doi: 10.1080/2162402X.2016.1220467

Figure 5.

Figure 5.

Binding of the purified recombinant mAb1, Fab1, scFv to mPD-1. Binding of purified mAb1, Fab1 and scFv to mPD-1-positive EL4 cells (A). Murine T lymphoma EL4 cells were incubated with commercially available J43 (positive control), hamster IgG (negative control), Fab1, monomeric scFv, mCD80-hFc-6xHis (His-tagged positive control, CD80 binds to PD-L1 expressed by EL4 cells) or hErbB2-hFc-6xHis (His-tagged negative control). Binding of mAbs and 6xHis-tagged proteins was detected by flow cytometry using either FITC-conjugated mouse anti-hamster IgG antibody or PE-conjugated mouse anti-His tag antibody. Competition between purified recombinant mAb1, Fab1, scFv (monomeric and dimeric fractions), J43 and mPD-L1 (B and C). Binding of biotinylated mPD-L1-hFc to immobilized mPD-1, or binding of unlabeled mPD-L1-hFc to EL4 cells, in presence of increasing concentrations of competitors (J43, mAb1, Fab1, scFv) or negative control (Hamster IgG) was measured in ELISA (B) or flow cytometry (C) assays. PD-L1 was detected using either streptavidin-HRP or anti-human-Fc-PE. The signal obtained with the lowest concentration of hamster IgG was set as 100%. Represented values are the mean of two normalized measurements.