Serum antibody screening using glycan arrays

Abstract

Humans and other animals produce a diverse collection of antibodies, many of which bind to carbohydrate chains, referred to as glycans. These anti-glycan antibodies are a critical part of our immune systems' defenses. Whether induced by vaccination or natural exposure to a pathogen, anti-glycan antibodies can provide protection against infections and cancers. Alternatively, when an immune response goes awry, antibodies that recognize self-glycans can mediate autoimmune diseases. In any case, serum anti-glycan antibodies provide a rich source of information about a patient's overall health, vaccination history, and disease status. Glycan microarrays provide a high-throughput platform to rapidly interrogate serum anti-glycan antibodies and identify new biomarkers for a variety of conditions. In addition, glycan microarrays enable detailed analysis of the immune system's response to vaccines and other treatments. Herein we review applications of glycan microarray technology for serum anti-glycan antibody profiling.

Figure 1.

Representative glycan epitopes for serum anti-glycan antibodies. (A) ABO blood group and Lewis antigens. (B) N-linked glycan antigens. (C) O-linked glycan antigens. (D) Glycolipid glycans. (E) Bacterial, fungal, and other microbial or non-human glycans. (F) Sialic acids. Note that some structures may fit into multiple groups (ex., glycolipid and non-human glycan). Glycoglyph was used to produce glycan structures.

Figure 2.

Preparation of Glycan Microarrays. Glycan microarrays may be generated using free glycans that are either chemo-enzymatically synthesized or derived from nature. Synthetic glycans may have a ‘linker’ of some type at the reducing end (e.g., aliphatic amine), or they can be derivatized with linkers that have amines. Glycans may also be chemically linked to proteins (neoglycoproteins) or to lipids (neoglycolipids). Glycan-amine derivatives or neoglycoproteins may then be covalently linked to glass slides functionalized with N-hydroxysuccinimide (NHS) or epoxide chemistry, and neoglycolipids can be non-covalently linked to slides functionalized with nitrocellulose. Depicted here is a ‘defined glycan microarray’ in which all glycan structures are known. Because of the difficulty in acquiring many defined glycans, “shotgun glycan microarray” approaches have been developed in which natural glycans are fractionated and then structurally defined after being recognized by an antibody or lectin, as described in the text.