k_off

Binding

Rate Constant of Dissociation (koff)

1/s

Surface Plasmon Resonance (SPR)

None

None

None

None

None

None

scAb expression and characterization
For soluble antibody purification, antibody genes were subcloned into pMopac16 vector and expressed as scAb fragments in E.coli Jude-1 cells13 and then purified by Ni-NTA chromatography according to the manufacturer’s instructions (Thermo Scientific). After elution, proteins were dialyzed in PBS, then loaded onto a Hiload 16/600 Superdex 75 pg column (GE Healthcare) and antibody-containing fractions were pooled and concentrated to 2 mg/ml.
For affinity validation, SPR was performed for each antibody clone using a BIAcore 3000 biosenor (Biacore). About 400 RU (response units) of scAbs were immobilized on the CM5 sensor chip (GE Healthcare) using amine coupling chemistry. All binding experiments were done in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% P20 surfactant) (GE Healthcare). Ricin A chain was injected at con- centrations of 25, 50, 100, 200, and 400 nM with a flow rate of 30 mL/min for 2 min and a dissociation time of 10 min. The chip was regenerated after each injection by 100 mM citric acid, pH 3.0. The response generated by flowing ricin A chain over a BSA-coupled surface was used as control and consequently subtracted. Experiments were carried out in triplicates. All kinetic parameters were determined in BIAevaluation 3.0 software using a 1:1 Langmuir model and were reported as the average of the 3 technical replicates.