title: Microscale purification of antigen-specific antibodies.
abstract: 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.
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Brown
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States.
authors.forename: Erica
authors.initials: E
authors.lastname: Normandin
authors.affiliation: Molecular and Cellular Biology Program, Dartmouth College, Hanover, NH 03755, United States.
authors.forename: Nana Yaw
authors.initials: NY
authors.lastname: Osei-Owusu
authors.affiliation: Ragon Institute of MGH, MIT, and Harvard University, Cambridge, MA 02139, United States.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Mahan
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States.
authors.forename: Ying N
authors.initials: YN
authors.lastname: Chan
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States.
authors.forename: Jennifer I
authors.initials: JI
authors.lastname: Lai
authors.affiliation: Animal Models and Vaccine Section, National Cancer Institute, Bethesda, MD 20814, United States.
authors.forename: Monica
authors.initials: M
authors.lastname: Vaccari
authors.affiliation: U.S. Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States.
authors.forename: Mangala
authors.initials: M
authors.lastname: Rao
authors.affiliation: Animal Models and Vaccine Section, National Cancer Institute, Bethesda, MD 20814, United States.
authors.forename: Genoveffa
authors.initials: G
authors.lastname: Franchini
authors.affiliation: Ragon Institute of MGH, MIT, and Harvard University, Cambridge, MA 02139, United States.
authors.forename: Galit
authors.initials: G
authors.lastname: Alter
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, United States. Electronic address: Margaret.e.ackerman@dartmouth.edu.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Ackerman
mesh_terms: D000906:Antibodies / Q000276:immunology* / Q000302:isolation & purification*; D000941:Antigens / Q000276:immunology*; D006031:Glycosylation; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology
chemical_list: D000906:Antibodies; D000941:Antigens; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
keywords: Antibody; Antigen; Effector function; Glycosylation; Purification