Anti human igg fc antibody

K_D values were determined by SPR technology using a Biacore3000 instrument (GE Healthcare), as described previously²⁶ . The anti-human IgG Fc antibody (GE Healthcare) was amine-coupled to a CM-5 sensor chip for use. MAb C12H5 was then captured on the sensor surface at a 30 μL/min flow rate in HBS buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% Tween-20, pH 7.4). The kinetics of C12H5 binding with HA and its mutants were measured at 30 μL/min flow rate in HBS buffer, delivered at 2-fold serially diluted concentrations (160, 80, 40, 20, and 10 nM). The flow durations were 200 s for the association stage and 10 min for dissociation. Association rates (k_a), dissociation rates (k_d), and affinity constants (K_D) were calculated using BIAcore evaluation software. All experiments were repeated twice. The mean k_a, k_d, and K_D values are reported.

The binding kinetics of H_CL_C or H₃L₂ to rhDLL4 were analyzed using an Surface Plasmon Resonance (SPR)-based assay on Biacore X100 system (GE Healthcare, Buckinghamshire, UK). An anti-human IgG Fc antibody (GE Healthcare, Buckinghamshire, UK) was firstly immobilized onto a CM5 biosensor chip. Then, appropriate concentration of H_CL_C or H₃L₂ was captured to the surface with an increase of Resonance Unit (RU) up to 2,000. Finally, various concentrations of rhDLL4 were passed through the chip. After each reaction, the captured H_CL_C or H₃L₂ antibody and analyte were removed by Regeneration (3 M magnesium chloride). The whole reaction was conducted at 25 °C and flow rate of 30 μl/min. Sensorgrams of each concentration were obtained and analyzed by Biacore evaluation software (GE Healthcare, Buckinghamshire, UK). The equilibrium constant K_D was calculated from ratio of dissociation rate constant k_d to association rate constant k_a (k_d/k_a).

Surface plasmon resonance, SPR device (Biacore T200, GE Healthcare) was used for evaluating the affinity of IMM40H to recombinant human CD70 trimer. Following the amine coupling methodology described by the manufacturer, the anti-human IgG(Fc) antibody (GE Healthcare, Cat# BR-1008–39) was diluted to 25 µg/mL in 10 mM sodium acetate (pH 5.0) and then attached to a CM5 biosensor (GE Healthcare, Cat#BR100530). The SPR experiment was performed at 25°C in 1xHEPES running buffer (pH 7.4, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005%Tween-20). The samples, which were diluted to 10 µg/mL in 1xHEPES (pH 7.4), were captured on the anti-hIgG(Fc) antibody surface. A concentration series of 3.125–0.049 nM trivalent human CD70 was injected over the captured antibodies at 30 µL/min to measure association and dissociation. The anti-hIgG(Fc) antibody capture surface was regenerated between test cycles with 30 s injections of 3 M magnesium chloride. Biacore T200 Evaluation Software v.3.1 was used to fit the rate constants ka (kon, association rate) and kd (koff, dissociation rate) from the reference flow cell and 0 nM blank-subtracted sensorgrams to a 1:1 binding model.