SPR binding and kinetics measurement were carried out at 25°C on a BIAcore T200 instrument (BIAcore/GE Healthcare) as described earlier (42, 67, 68). The CM5 sensor chip (GE Healthcare) was immobilized with DENVrEDIII (for serotype 2) diluted in 10 mM sodium acetate (pH 4.5) using standard amine coupling chemistry. Serum samples collected at day 28 from immunized mice were injected at 1:50 to 1:400 dilution by using twofold dilution steps at a flow rate of 30 μl min−1. The contact time for the serum samples to interact with ligand immobilized on the sensor chip was 60 s, and dissociation time for the sample was 60 s. After each cycle, the sensor chip was regenerated with 50 mM NaOH with a specified contact time for 60 s. The experimental data were fit using 1:1 Langmuir model for determining the binding kinetics, and analysis was performed using BIAcore T200 Evaluation software version 2.0. To analyze antigen (DENVrEDIII)–specific antibody-binding avidity, maximal response unit (RUmax) and dissociation rates (kd) were measured. Avidity scores were determined as mentioned earlier (avidity score = maximum binding response/kd in RU.s) (61).

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