Glycine hcl buffer

In the direct binding assay, LOX-1 was immobilized on a carboxymethylated dextran sensor chip (CM-5; GE Healthcare) to a level of approximately 2,000 response units (RU). An unmodified surface was used as the reference channel. For single-injection experiments, the NadA protein samples were injected at a 100 nM concentration over the surface at a flow rate of 30 µl/min for 120 s, followed by a dissociation time of 120 s. To remove the remaining nondissociated samples, the surface was regenerated with a 5-s injection of 10 mM glycine-HCl buffer, pH 1.7, at 30 µl/min (GE Healthcare). For determination of the equilibrium dissociation constant (K_D) and kinetic rate constants for LOX-1 binding to wild-type NadA3 24–170 and the NadA3 Y42A mutant protein, a multiinjection titration series of five consecutive injections (at 40 μl/min for 120 s) of purified NadA protein was performed with increasing concentrations (7.8 to 125 nM for the wild type and 0.25 to 4 µM for the Y42A mutant). Surface regeneration was performed as described above.

Binding competition between mAb059c and pembrolizumab/nivolumab was evaluated by a label-free biolayer interferometry assay on an Octet Red 384 (Pall ForteBio, USA). All experiments were performed at 25 °C in PBS buffer with 0.005% Tween-20 (PBST). PD1-hFc was loaded onto AHC sensors at a concentration of 2 µg/ml for 300 s in Stage 1, and then the 1^st antibody (100 nM) was loaded onto the biosensor for 600 s to obtain saturation in Stage 2. The second antibody was loaded on the biosensor at a concentration of 100 nM for another 600 s in Stage 3. The above experiments were repeated in the reverse order of antibody loading in Stages 2 and 3. PBST buffer was used as the negative control. The competition between mAb059c and PD-L1 was evaluated in similar way except that mAb059c and PD-L1 were loaded onto the biosensor sequentially in Stages 2 and 3. All sensors were regenerated using 10 mM glycine-HCl buffer (pH 1.7, GE Healthcare), and recharging was done in 10 mM NiCl2 buffer. The real-time binding response was measured during the course of the experiment. The binding responses of the two commercial antibodies and mAb059c to PD-1 were compared with the response from the negative control (PBST), and competitive/noncompetitive behavior was determined.