Abstract
Antibodies are glycoproteins, and Fc glycosylation plays a critical structural role in maintaining the proper folding of the C(H)2 domain. Deglycosylated IgGs exhibit an open C(H)2 domain conformation that structurally affects also the neighboring hinge region. Selective reduction of disulfide bonds in this region using mild reducing agents generates monovalent thiol-containing IgGs (rIgGs), which can be immobilized on modified surfaces to orient the Fab fragment outward, enhancing antigen binding efficiency and assay sensitivity. This study investigates the effect of Fc glycosylation on IgG chemical reduction and its implications for in-house ELISA and commercial immunoassays. IgGs were enzymatically deglycosylated with Endo S and then reduced to rIgGs by 2-mercaptoethylamine. Deglycosylation and reduction efficiency were verified by non-reducing SDS-PAGE. Glycosylated and deglycosylated rIgGs were tested in in-house ELISA and commercial immunoassays. Results showed that deglycosylation significantly improves rIgG production, probably by increasing hinge-region accessibility to reducing agents. This led to a 20-fold increase in ELISA sensitivity compared to glycosylated rIgGs. Deglycosylation also mitigates batch-to-batch variability in IgG reduction, enabling consistent rIgG yields. These findings highlight the capability of deglycosylation to standardize rIgG production, broadening its applications in diagnostic immunoassays and biosensing technologies.