Cd3d cd3e heterodimer

The affinities and kinetics of the TF-TCB binding to CD3D × CD3E and TF antigen were evaluated by surface plasmon resonance (Biacore 8K, GE Healthcare Life Sciences). TF-011, an anti-TF antibody sharing the same TF binding moieties with the TF-TCB, was used as control²¹ (link). CD3D × CD3E heterodimer (Sino Biological, Beijing, China) or the TF antigen (Peprotech, Rocky Hill, NJ, USA) was immobilized to CM5 chip surface using routine 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide amine coupling protocols. The immobilization buffer was 10 mmol/L sodium acetate (pH 4.5, GE Healthcare Life Sciences). Surface densities after immobilization ranged from 50 to 100 RU. Two-fold serial dilutions of antibodies (TF-TCB or TF-011) ranging from 0.318 to 162.8 nmol/L in HBS-EP running buffer (GE Healthcare Life Sciences) were used to analyze binding. Running buffer alone was used as a zero reference. The antibodies were injected for 200 s followed by 180 s of dissociation time. Surface was regenerated by 0.1 mol/L glycine (pH 1.5) for 30 s. Data were analyzed using a 1:1 Langmuir binding model to calculate the kinetics and binding constants.