Figure 2.

Thiol reactivity and site-specific conjugation of h4G3cys. (A) Thiol reactivities of h4G3 and h4G3cys were evaluated using 4-PDS, and expressed as molar ratios of free thiol groups per antibody. Data represent mean ± SD (n = 3). (B) h4G3 and h4G3cys were conjugated using maleimide-PEG₂-biotin, and biotin was detected by Western blotting using SHRP. Human IgG-HRP was used as a loading control. Binding traces of FITC-labeled h4G3cys against CLDN3 on the cell membrane. (C) and(D) TOV-112D or CLDN3/TOV-112D cells were sequentially treated with FITC-labeled h4G3cys at 3 and 9 nmol/L. The cells were then replaced with fresh medium in the dissociation phase. The fluorescence signal was calculated by subtracting the value of the reference cells from that of target cells, and normalized (n = 3). The obtained binding curves were fitted by ‘one-to-one two-state’ model (C) and ‘one-to-two’ model (D). Three independent experiments curves are shown in red, green, and blue. The fitting curves are shown in black.