Hi trap protein a high performance column

Five female BALB/c mice aged 6 to 8 weeks were immunized using the Repetitive IMmunization Multiple Sites (RIMMS) protocol (36 (link)). These received 100-μL injections of cleaved, monomeric CDTb antigen at the following doses, 20, 5, 5, and 2.5 μg, subcutaneously at multiple points on days 0, 4, 8, and 11 with GSK adjuvant system 02 (AS02). On day 13, lymph nodes were collected from the 5 mice. After grinding, cells were chemically fused with SP2/0-Ag14 myeloma at a 5:1 ratio using polyethylene glycol 1500 (PEG-1500; Roche) and plated in 96-well cell culture plates at a concentration of 50,000 cells/well. Cells were then cultivated in serum-containing medium at 37°C in a 5% CO₂ atmosphere. To generate IgG of clones selected for characterization, cell culture supernatants were dialyzed into phosphate-buffered saline (PBS) and sterile-filtered through a 0.22-μm membrane. Antibody content in cell culture supernatant was determined by analytical protein A affinity chromatography using a Hi-Trap Protein A High Performance column (Cytiva 17-0403-03). IgG concentration was determined via absorbance at 280 nm, which was used to calculate the concentration of IgG in the dialyzed supernatant. Dialyzed supernatant containing IgG was then stored at −80°C.

Briefly, infiltrated 7 dpi whole-leaf samples were homogenized using a Matstone 6-in-one juice extractor (Matstone, PH), protein were extracted using PBS, pH 7.4 containing cOmplete™ ULTRA tablets mini, and EASYpack protease inhibitor cocktail (Roche, CH). The supernatant was clarified by a series of centrifugation and filtration steps. The bNAbs were then purified by protein A affinity chromatography using a HiTrap^® Protein A High-Performance column (Cytiva, USA), coupled to a Äkta Avant 150 (Cytiva, USA), as per the manufacturer's instructions.