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.