B Cell Epitope Mapping by High-Density Competition ELISAs.

Advances in the discovery and expression of large libraries of monoclonal (MAb) and single-domain (VH) antibodies against protein antigens necessitate a means by which to rapidly bin antibodies before embarking on resource-intensive epitope mapping methods such as cryogenic electron microscopy (EM) and X-ray crystallography. In this chapter we describe the use of competition enzyme-linked immunosorbent assays (ELISAs) for the purpose of epitope binning antibodies from any given species. In its simplest form, a competition ELISA compares the ability of a query MAb (or VH) to bind to an immobilized target antigen in the presence of a competitor MAb (or VH) whose epitope is already known. Detection of the query MAb is achieved through an enzyme-linked, species-specific secondary antibody and developed using a colorimetric substrate. While useful, the indirect competition ELISA has several drawbacks that may limit its applications for large screens, including artifacts associated with immobilization of protein antigens on polystyrene microtiter plates. We discuss alternative competition ELISA strategies involving the capture of soluble antigen without and with biotin tagging. We also describe how competition ELISAs, once optimized, can be employed along with large antibody collections to yield fine resolution epitope maps of protein antigens.