Microscale purification of antigen-specific antibodies

J Immunol Methods. 2015 Oct:425:27-36. doi: 10.1016/j.jim.2015.06.005. Epub 2015 Jun 12.


Glycosylation of the Fc domain is an important driver of antibody effector function. While assessment of antibody glycoform compositions observed across total plasma IgG has identified differences associated with a variety of clinical conditions, in many cases it is the glycosylation state of only antibodies against a specific antigen or set of antigens that may be of interest, for example, in defining the potential effector function of antibodies produced during disease or after vaccination. Historically, glycoprofiling such antigen-specific antibodies in clinical samples has been challenging due to their low prevalence, the high sample requirement for most methods of glycan determination, and the lack of high-throughput purification methods. New methods of glycoprofiling with lower sample requirements and higher throughput have motivated the development of microscale and automatable methods for purification of antigen-specific antibodies from polyclonal sources such as clinical serum samples. In this work, we present a robot-compatible 96-well plate-based method for purification of antigen-specific antibodies, suitable for such population level glycosylation screening. We demonstrate the utility of this method across multiple antibody sources, using both purified plasma IgG and plasma, and across multiple different antigen types, with enrichment factors greater than 1000-fold observed. Using an on-column IdeS protease treatment, we further describe staged release of Fc and Fab domains, allowing for glycoprofiling of each domain.

Keywords: Antibody; Antigen; Effector function; Glycosylation; Purification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / immunology*
  • Antibodies / isolation & purification*
  • Antigens / immunology*
  • Glycosylation
  • Humans
  • Immunoglobulin Fc Fragments / immunology
  • Immunoglobulin G / immunology


  • Antibodies
  • Antigens
  • Immunoglobulin Fc Fragments
  • Immunoglobulin G