IgG Production and Secretion Assay
ExpiCHO-S™ cells stably expressing Rheb[WT], [T23M] or [E40K] were seeded at a density of 4 x 106cells/mL in 125 mL shaker flasks and allowed to grow for 16 h to a density of 10 x 106 cells/mL. Cells were then diluted to 6 x 106 cells/mL and transfected with pcDNA3.2 vector encoding heavy and light chains of rabbit IgG1 at 2:1 light:heavy chain ratio via ExpiFectamine™ CHO Transfection Kit according to the manufacturer’s instructions. ExpiFectamine™ CHO/plasmid complexes were prepared by diluting 12 µg plasmid in 1 mL OptiPRO™ medium and 80 µL of ExpiFectamine™ CHO Reagent in 920 µL OptiPRO™ medium. Diluted ExpiFectamine™ reagent was added to diluted DNA and incubated at room temperature for 5 min before ExpiFectamine™ CHO/DNA complex was added to cells. Cells were incubated at 37°C and 8% CO2 on an orbital shaker at 125 rpm at 19 mm shaking diameter. After 24 h, 150 µL of ExpiCHO™ Enhancer and 6 mL of ExpiCHO™ Feed were added to cells. 20 µL of medium were removed each day in order to determine rate of secretion. 10-days post transfection, cells were pelleted via centrifugation at 1000 rpm for 10 min and supernatant collected.
IgG concentration in growth medium was determined via Easy-Titre™ Rabbit IgG Assay Kit as per the manufacturer’s instructions. Growth medium containing rabbit IgG1 were diluted 1:1000 in PBS. 20 µL of Anti-IgG Sensitized Beads were transferred to a 96-well plate and 20 µL of diluted cell medium containing rabbit IgG added. Plates were mixed on a plate mixer for 5 min before 100 µL of blocking buffer was added to each well. Absorbance was measured at 405 nm and IgG concentration calculated from a standard curve of know IgG concentration generated from 1:2 serial dilutions starting at 500 ng/mL.