Site-Specific Photocrosslinking to Immunoglobulin G Using Photoreactive Antibody-Binding Domains.

Abstract

The high specificity and strong binding affinity of antibodies, most commonly immunoglobulin G (IgG), have led to their use in a wide range of research, diagnostic and therapeutic applications. Many of these applications require the antibody to be labeled with additional chemical or biological moieties. Here, we describe a method for the rapid and site-specific labeling of nearly any "off-the-shelf" IgG. Our method utilizes small photoreactive antibody-binding domains (pAbBDs) that are produced by modifying the IgG-binding domains of Protein A and Protein G with the unnatural amino acid benzoylphenylalanine (BPA). The pAbBDs are covalently linked to IgG heavy chains upon exposure to ultraviolet light. Fusion of pAbBDs to a given protein of interest or conjugation of pAbBDs with drugs, fluorophores, and/or other chemical moieties, enables the facile production of a diverse range of antibody conjugates.