Cm5 sensor chip

SPR analysis of SA binding to His-PP2AA1 or His-PP2AA3 was performed with a Biacore T200 instrument (GE Healthcare). A synthesized active SA analog, SA-f, was immobilized on a CM5 sensor chip (GE Healthcare) first: the carboxyl group of the CM5 sensor chip was activated using a mixture of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxy-succinimide (NHS) for 7 min at a flow rate of 5 μL/min. After activation, 1 mM of SA-f dissolved in 0.1 M borate buffer (pH 10) was passed over for a period of 3 min at 5 μL/min for immobilization. Then excess reactive groups were inactivated by flowing ethanolamine hydrochloride-NaOH pH 8.5 for 7 min, at 5 μL/min. 1 × PBS buffer (GE Healthcare) was used as running buffer in all assays. To test SA binding of His-PP2AA1 or His-PP2AA3, proteins were diluted in 1 × PBS buffer, and then flowed through the flow cell of sensor chip with SA-f immobilized or through the reference cell. The binding signal was generated by subtracting the signal of reference cell from that generated with the SA-f flow cell. The flow cells were regenerated with flowing 250 mM NaOH solution. Details about the chemical synthesis of SA derivatives are described in the Supplemental Information.

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 R_max value was considered good.

Purified sdAbs identified by conventional panning and the highest affinity binders identified via ABTAG screening after one and two rounds of panning were subjected to standard SPR analysis. Following purification by immobilized-metal affinity chromatography (18 (link)), sdAbs and sdAb-ABTAG fusions were further purified by size exclusion chromatography (SEC) using Superdex S75 and Superdex S200 Increase columns (GE Healthcare), respectively. CEACAM6 was subjected to Superdex S75 chromatography to remove possible aggregates.
sdAbs from conventional panning and from ABTAG round 2 screening were immobilized on CM5 sensor chips (GE Healthcare) at 50 µg/mL in 10 mM acetate buffer, pH 4. Multiple cycle kinetics were performed on a Biacore 3000 (GE Healthcare) in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA, and 0.005% P20 by flowing appropriate concentrations of CEACAM6 at a flow rate of 40 µL/min. Purified sdAb-ABTAG fusions from ABTAG round 1 screening were immobilized on CM5 sensor chips (GE Healthcare) at concentrations of 12–25 µg/mL in 10 mM acetate buffer, pH 3.5. Single cycle kinetics (SCK) were performed on a Biacore 3000 (GE Healthcare) in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA, and 0.005% P20 by flowing appropriate concentrations of CEACAM6 at a flow rate of 25 µL/min. Data were analyzed using BIAevaluation v4.1.1 Software (GE Healthcare).