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).