Zeba spin 7k mwco

Glycan present on anti-PSA antibody was oxidized by using a slightly modified mild chemical oxidation procedure as described previously (Chen et al., 2007 (link)). Shortly, monoclonal anti-fPSA (c = 2 mg mL^-1) was diluted by 150 mM NaIO₄ in 150 mM sodium acetate pH 5.5 solution down to 0.1 mg mL^-1 and incubated in the dark for 30 min at 4 °C. After a desalting procedure with previously equilibrated desalting columns (Zeba spin 7 K MWCO, Thermo Scientific), 2mM solution of propionic acid hydrazide in 150 mM sodium acetate pH 5.5 was added to the oxidized anti-fPSA in 1+1 ratio. The mixture was incubated in the dark for 2 h at RT. After the desalting procedure, the oxidized antifPSA (with concentration of 0.05 mg mL^-1) was stored in the form of aliquots at −80 °C. Standard enzyme-linked lectin binding assay (ELLBA, according to protocols published in our previous work (Chocholova et al., 2018a (link); Chocholova et al., 2018b ) revealed successful glycan oxidation and its modification by propionic acid hydrazide suppressing lectin binding.

The binding affinity of purified ZIKV E or ZIKV DIII protein with ZIKV mAbs was monitored by BLI using an Octet-Red96 device (Pall ForteBio). Briefly, 100 μg of each antibody was mixed with biotin (EZ-Link-NHS-PEG4-Biotin, Thermo Fisher) at a molar ratio of 20:1 biotin:protein and incubated at room temperature for 30 min. The unreacted biotin was removed by passage through a desalting column (5 ml Zeba Spin 7K MWCO, Thermo Fisher). The antibodies were loaded onto streptavidin biosensors (ForteBio) until saturation, typically 2 μg/ml for 3 min, in 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 3% BSA. Association and dissociation were measured at 25°C for all mAbs. The real-time data were analy zed using Biaevaluation 4.1 (GE Healthcare). Association and dissociation profiles, as well as steady-state equilibrium concentration curves, were fitted using a 1:1 binding model.