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).
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).
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