Microscale purification of antigen-specific antibodies

Area of Science:

• Immunology
• Glycobiology
• Biotechnology

Background:

• Antibody Fc domain glycosylation significantly influences effector functions.
• Assessing total IgG glycosylation reveals clinical condition differences, but antigen-specific antibody glycosylation is often more relevant.
• Analyzing antigen-specific antibody glycosylation in clinical samples is difficult due to low prevalence, high sample needs, and lack of high-throughput purification.

Purpose of the Study:

• To develop a microscale, automatable method for purifying antigen-specific antibodies from polyclonal sources.
• To enable high-throughput glycosylation screening of antigen-specific antibodies from clinical samples.
• To facilitate population-level glycosylation analysis of antibodies relevant to specific diseases or vaccinations.

Main Methods:

• A robot-compatible 96-well plate-based purification method for antigen-specific antibodies.
• Demonstration across multiple antibody sources (purified plasma IgG and plasma) and antigen types.
• On-column IdeS protease treatment for staged release of Fc and Fab domains for domain-specific glycoprofiling.

Main Results:

• Achieved high-throughput purification of antigen-specific antibodies suitable for population screening.
• Demonstrated method utility across diverse antibody sources and antigen types.
• Observed enrichment factors greater than 1000-fold for antigen-specific antibodies.
• Enabled staged release of Fc and Fab domains for differential glycoprofiling.

Conclusions:

• The developed method allows for efficient, high-throughput purification and glycoprofiling of antigen-specific antibodies.
• This approach overcomes historical challenges in analyzing low-prevalence antibodies from clinical samples.
• Facilitates detailed investigation of antibody glycosylation in disease and vaccination contexts.