Fig. 5.

The superior rigidity of IgG2 CH1-hinge. a A diagram showing anti-mCD40 antibody TR-FRET. Anti-mCD40 antibody molecules mixed with CD40-Tb and CD40-D2 (mCD40 labeled with Tb donor and D2 acceptor fluorochromes, respectively) can simultaneously bind CD40-Tb and CD40-D2, and emit TR-FRET signal quantified as the relative ratio of detected 665 nm fluorescence to 620 nm fluorescence (Em665/Em620) upon stimulation. b A diagram of the model showing that hinge flexibility of hIgG anti-mCD40 antibodies correlates with TR-FRET signal levels. Left, anti-mCD40 antibodies with little hinge flexibility do not trigger TR-FRET signal due to the large distance between CD40-Tb and CD40-D2; middle, hinge flexibility can bring CD40-Tb and CD40-D2 close enough to trigger TR-FRET signal; right, anti-mCD40 antibodies with large hinge flexibility give rise to stronger TR-FRET signal due to more molecules with closer CD40 binding sites. c–e TR-FRET signal levels of anti-mCD40 antibodies of indicated constant domains. Shown are relative TR-FRET signal levels (Em665/Em620) plotted against the concentration of control IgG or the anti-mCD40 antibodies of indicated constant domains. Bars represent the mean ± SEM. ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001, two-way ANOVA with Holm–Sidak’s post hoc. Source data (c–e) are provided as a Source Data file. A representative of two independent experiments is shown