Hitrap protein g sepharose column

Recombinant PspC2 protein lacking the LPxTG motif and the first 37 aa, which constitute a signal peptide region, was expressed and purified in E. coli (see above). Purified PspC2 was sent to Innovagen AB for rabbit immunization and production of polyclonal antibodies. Polyclonal anti-PspC2 IgG were purified by protein G affinity chromatography according to the manufacturer’s manual using HiTrap protein G-sepharose columns (GE Healthcare).

IgG was purified from sera or plasma using the HiTrap Protein G Sepharose columns (GE Healthcare Bio-sciences, USA). The samples were further dialyzed with Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific, USA) and concentrated with Amicon Ultra-15 centrifugal filters (Merck Millipore, Germany). Protein concentration was measured by Bradford assay (Bio-Rad, USA). For immunoblotting, proteins (5 µg per lane) were separated on 10% SDS polyacrylamide gels, transferred onto polyvinylidene fluoride membrane, detected using peroxidase-conjugated rabbit anti-human IgG (1:10,000, Dako, Denmark) and visualized using chemiluminescence (Advansta, USA). Pooled IgG isolated from AQP4–IgG-seropositive NMOSD patients were termed IgG_(AQP4+), pooled IgG isolated from AQP4–IgG- and MOG-IgG-seronegative NMOSD patients were termed IgG_(AQP4−), and pooled IgG isolated from healthy subjects were termed IgG_(Healthy).

The hybridoma cell line producing BB5.1 was re‐cloned and expanded; the antibody (mAb) was produced in large quantities using Integra flasks [Integra Biosciences (Tathcham, Berkshire, UK), Generon, CeLLine 1000 DC‐90005) in medium supplemented with ultralow IgG fetal bovine serum (ThermoFisher, Loughborough, UK), and purified under sterile conditions on a 5‐ml HiTrap Protein G sepharose column (GE Healthcare, Amersham, UK; #GE17‐0405‐01). Purity of the mAb was confirmed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and the isotype was tested using IsoStrips (#11493027001; Roche).