Glycine hcl

The binding kinetics of the antibodies against human HER2 and human CD3 were assessed at pH 7.4 at 37 °C using the Biacore T200 (Cytiva, Marlborough, MA, USA). Anti-mouse IgG (Fc) antibody (Mouse antibody capture kit) was immobilized onto all flow cells of a CM4 chip using an amine coupling kit (Cytiva). Anti-HER2 and anti-CD3 antibodies and analytes were prepared in PB-P+ (Na-phosphate buffer 0.05 mol/L, NaCl 0.15 mol/L, 0.05 w/v% P-20) at pH 7.4. Antibodies were adjusted to concentrations corresponding to 200 RU for assessing binding with recombinant human HER2 (Sino Biological, 10004-H08H) and 630 RU for recombinant human CD3ε-CD3δ heterodimeric protein (CD3εδ) (In-house prepared, Table S1). Human HER2 was prepared by two-fold serial dilutions (6.3 nmol/L to 100 nmol/L). Human CD3 was prepared by two-fold serial dilutions (1.2 nmol/L to 4800 nmol/L) for mUCHT1 and mSP34, (18.8 nmol/L to 19,200 nmol/L) for mHIT3a and mOKT3, and (75 nmol/L to 76,800 nmol/L) for m12F6. The sensor surface was regenerated with Glycine 1.5 with 10 mmol/L glycine–HCl, pH 1.5 (Cytiva). Kinetic parameters were determined by processing and fitting the data to the 1:1 binding model for m4D5, m2C4, mUCHT1, and mSP34 and the steady state model for mHIT3a, mOKT3, and m12F6 using Biacore T200 Evaluation software, version 3.0 (Cytiva, Marlborough, MA, USA).