Recombinant protein quantification
Levels of IgG1 in culture supernatants were evaluated by ELISA. Standard
ELISA protocols were followed using 96-well Maxisorb plates (Thermo
Fisher Scientific) and signals measured using a plate reader (Molecular
Devices, Sunnyvale, CA) at 405 nm (reference at 492 nm). The primary
(coating) antibody used was a goat anti-human IgG (Fcγ
fragment-specific) (Jackson ImmunoResearch #109-005-098). The secondary
(detection) antibody used was a goat anti-human IgG (H+L
specific)-alkaline phosphatase antibody (Jackson ImmunoResearch
#109-055-088). Quantification was achieved by interpolation from a
standard curve obtained from 1.9 - 125 ng/mL dilutions of purified IgG.
The volumetric productivity was determined by the overall production of
IgG for each culture volume, while the specific productivity for each
culture was calculated based on the product concentration over the
integral viable cell density (IVCD) (Renard et al., 1988).