# The impact of Fc glycosylation on IgG susceptibility to hinge region chemical reduction: implications for the development of immunoassays

## TL;DR

Removing sugar molecules from antibodies improves their chemical modification, leading to more sensitive and consistent immunoassays.

## Contribution

This study shows that deglycosylation of IgGs enhances hinge region reduction and standardizes bioconjugation protocols.

## Key findings

- Deglycosylation increases hinge region accessibility to reducing agents, improving rIgG production.
- Deglycosylated IgGs show a 20-fold increase in ELISA sensitivity compared to glycosylated IgGs.
- Deglycosylation reduces batch-to-batch variability in IgG reduction, enabling consistent yields.

## Abstract

Antibodies are glycoproteins, and Fc glycosylation plays a critical structural role in maintaining the proper folding of the CH2 domain. Deglycosylated IgGs exhibit an open CH2 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.

Image 1

•Fc glycosylation influences IgG susceptibility to chemical reduction hinge region.•IgG deglycosylation enhances hinge region chemical reduction yields.•IgG deglycosylation overcomes IgG batch variability in chemical reduction.•IgG deglycosylation standardizes chemical reduction in bioconjugation protocols.

Fc glycosylation influences IgG susceptibility to chemical reduction hinge region.

IgG deglycosylation enhances hinge region chemical reduction yields.

IgG deglycosylation overcomes IgG batch variability in chemical reduction.

IgG deglycosylation standardizes chemical reduction in bioconjugation protocols.