The use of antigen fragments generated by specific proteolytic cleavage is a relatively simple "library" approach for epitope mapping in which possible overlapping fragments are screened with the antibody on Western blots. Proteolytic fragmentation with numerous proteases having different cleavage specificites can be carried out on native and denaturated proteins, generating a small and large number of fragments, respectively. To determine the antigenic site of a monoclonal antibody, we have examined the limited proteolytic digestion of the transducin alpha -subunit with four different proteases and detected antibody binding to fragments by Western blot. Using this approach, the epitope for this antibody was localized within the amino-terminal 17 residues of transducin alpha -subunit.
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[Show abstract][Hide abstract] ABSTRACT: Protective antibodies play an essential role in immunity to infection by neutralizing microbes or their toxins and recruiting microbicidal effector functions. Identification of the protective B-cell epitopes, those parts of microbial antigens that contact the variable regions of the protective antibodies, can lead to development of antibody therapeutics, guide vaccine design, enable assessment of protective antibody responses in infected or vaccinated individuals, and uncover or localize pathogenic microbial functions that could be targeted by novel antimicrobials. Monoclonal antibodies are required to link in vivo or in vitro protective effects to specific epitopes and may be obtained from experimental animals or from humans, and their binding localized to specific regions of antigens by immunochemical assays. The epitopes are then identified with mapping methods such as X-ray crystallography of antigen-antibody complexes, antibody-inhibition of hydrogen-deuterium exchange in the antigen, antibody-induced alteration of the nuclear magnetic resonance spectrum of the antigen, and experimentally-validated computational docking of antigen-antibody complexes. The diversity in shape, size and structure of protective B-cell epitopes, and the increasing importance of protective B-cell epitope discovery to development of vaccines and antibody therapeutics are illustrated through examples from different microbe categories, with emphasis on epitopes targeted by broadly neutralizing antibodies to pathogens of high antigenic variation. Examples include the V-shaped Ab52 glycan epitope in the O-antigen of Francisella tularensis, the concave CR6261 peptidic epitope in the hemagglutinin stem of influenza virus H1N1, and the convex/concave PG16 glycopeptidic epitope in the gp120 V1/V2 loop of human immunodeficiency virus type 1. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Abstract MEDI546 is an antagonist human monoclonal antibody that targets interferon alpha receptor 1 (IFNAR1). MEDI546 has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of MEDI546 using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that MEDI546 recognizes the SD3 subdomain of IFNAR1 with the critical residue R(279). Further, we solved the crystal structure of MEDI546 Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the MEDI546 Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that MEDI546 sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of MEDI546 and may provide insights towards designing antibody therapies against IFNAR1.