Protein a chromatography column

Genes encoding heavy chains and light chain were inserted separately into pcDNA3.4 and amplified in E. coli DH5α. PureLink™ HiPure Plasmid Miniprep Kit (Invitrogen) was used for low endotoxin plasmid preparation. Monoclonal antibodies were transiently expressed by co-transfecting ExpiCHO-S cells (ThermoFisher) with heavy chain and light chain plasmids using an ExpiCHO™ Expression System (Gibco). Cell culture was harvested after an 8–14 day of incubation at 37 °C with humidified atmosphere of 8% CO₂ with shaking. Full-length IgG was obtained by affinity purification utilizing a Protein A chromatography column (GE Healthcare) in AKTA avant (Cytiva). For long-term storage, antibodies were kept in a solution containing 10 mM Histidine-HCl, 9% trehalose, and 0.01% polysorbate 80.

IgE antibodies were purified from the serum pool of 15 healthy controls and 30 allergic patients, respectively. First, polyvinylpyrrolidone (PVP) 40000 was added to the serum at a final concentration of 3% (W/V), which was stirred at 4°C for four hours using a magnetic stirrer. Next, the PVP-treated serum was centrifugated at 17,000 g for 10 minutes to remove β-lipoprotein and globulin from the pellet. The retained supernatant was immediately replaced with antibody binding buffer using ÄKTA pure FLPC system (GE Healthcare, Sweden) with HiPrep 26/10 Desalting Column (GE Healthcare, Sweden). Then, following a previous protocol, IgG/M/A was removed from the solution prepared in the previous step with Protein A chromatography column (GE Healthcare, Sweden) (27 (link)), and the flow-through containing IgE was collected. After purification of the Protein L chromatography column (GE Healthcare, Sweden), purified IgE from the healthy controls (cIgE) and allergic patients (pIgE) was obtained, respectively. The purified IgE was visualized with SDS-PAGE gel staining with Coomassie brilliant blue R-250. In addition, the protein concentration was measured by the BCA method.