Surface plasmon resonance-based antibody competition was conducted on a Biacore T200 instrument (GE Healthcare) as described previously (32 (link)). Briefly, soluble gp350-ECD123-6His was immobilized on the CM5 sensor chip (GE Healthcare) by amine coupling at pH 5.5 until the response unit (RU) value reached ~500. Then, 50 µl of the serum samples (1/40 dilution) was injected at 30 µl/min, and the RU values were recorded as RU of serum. The chip was regenerated by one injection (50 µl) of 3 M MgCl2. Then, 50 µl (100 µg/ml) of mAb72A1 was injected at 30 µl/min before injecting of serum samples, and subsequently, the chip was regenerated by MgCl2. The RU values were documented as RU of mAb72A1. Competitive binding ability of serum samples to gp350 was calculated by an equation: Competitive percentage% = (RU of sera − RU of mAb72A1)/RU of sera.
For affinity kinetics analysis between recombinant gp350 and mAb72A1 or MBP-hCR2 SCR1-2, the three recombinant gp350s were immobilized on the CM5 sensor chip (GE Healthcare) at pH 5.5 with low RU value to avoid avidity effect. Then, mAb72A1 or MBP-hCR2 SCR1-2 was injected at 30 µl/min, and the chip was regenerated by 10 mM glycine–HCl, pH 1.5. Surface bound-kinetics fit (1:1 fit) was applied for data analysis, and χ2 less than 10% of Rmax value was considered good.
For affinity kinetics analysis between recombinant gp350 and mAb72A1 or MBP-hCR2 SCR1-2, the three recombinant gp350s were immobilized on the CM5 sensor chip (GE Healthcare) at pH 5.5 with low RU value to avoid avidity effect. Then, mAb72A1 or MBP-hCR2 SCR1-2 was injected at 30 µl/min, and the chip was regenerated by 10 mM glycine–HCl, pH 1.5. Surface bound-kinetics fit (1:1 fit) was applied for data analysis, and χ2 less than 10% of Rmax value was considered good.
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