Current approaches to fine mapping of antigen–antibody interactions

Abstract

A number of different methods are commonly used to map the fine details of the interaction between an antigen and an antibody. Undoubtedly the method that is now most commonly used to give details at the level of individual amino acids and atoms is X-ray crystallography. The feasibility of undertaking crystallographic studies has increased over recent years through the introduction of automation, miniaturization and high throughput processes. However, this still requires a high level of sophistication and expense and cannot be used when the antigen is not amenable to crystallization. Nuclear magnetic resonance spectroscopy offers a similar level of detail to crystallography but the technical hurdles are even higher such that it is rarely used in this context. Mutagenesis of either antigen or antibody offers the potential to give information at the amino acid level but suffers from the uncertainty of not knowing whether an effect is direct or indirect due to an effect on the folding of a protein.Other methods such as hydrogen deuterium exchange coupled to mass spectrometry and the use of short peptides coupled with ELISA-based approaches tend to give mapping information over a peptide region rather than at the level of individual amino acids. It is quite common to use more than one method because of the limitations and even with a crystal structure it can be useful to use mutagenesis to tease apart the contribution of individual amino acids to binding affinity.

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…HX-MS is increasingly being used for the purpose of B cell epitope identification (47,49,50,52,53). The general principles of HX-MS and how the technique relates to other epitope mapping strategies have been covered in several recent reviews (45,54). In a particularly informative study, Malito and colleagues subjected a single MAb directed against factor H binding protein from Neisseria meningitidis to epitope mapping by peptide array, HX-MS, and X-ray crystallography (50).…”

Protein Structure Facilitates High-Resolution Immunological Mapping

“…Biochemical assays, namely, the enzyme-linked immunosorbent assay (ELISA), immunoblotting, and surface plasmon resonance (SPR) analysis, have been used to demonstrate immune response, antigen reactivity, and binding affinity. More recently, structural techniques such as X-ray crystallography and, as described in the studies reported in this issue of Clinical and Vaccine Immunology by Mantis and coworkers, hydrogen-deuterium exchange mass spectrometry (HD-X MS) (1, 2) have been used to resolve antibody and antigen complexes (24). The shift from methods that predominantly identify linear epitopes via denatured antigens or small peptides to native, conformational epitopes limits artifacts that arise in solid-phase binding of antigen, as noted by Mantis and coworkers.…”

“…Additionally, classification of indirect, structural residues and those involved in direct binding has illuminated interactions involved in neutralization (24). Understanding antibody-antigen contacts at the molecular level will ultimately aid in the engineering of antibodies of higher affinity to their antigens, which has been correlated with neutralization (27,28) On the other hand, crystal structures of antibodyantigen complexes are not always available and can limit the solid-phase binding analysis to a static snapshot of a complex.…”

“…These methods focused on primary sequences or linear epitopes, while other mapping strategies such as alanine-scanning mutagenesis required cautious interpretation due to potential secondary structure disruption (24). Mantis and coworkers utilized HD-X MS to identify dominant epitopes within ricin for serum analysis of antibody response using competitive ELISA.…”

Antisera preparation and epitope mapping of a recombinant protein comprising three peptide fragments of the cystic fibrosis transmembrane conductance regulator

“…For instance, short CFTR consensus sequences that are homologous to proteins expressed by bacteria or viruses can induce autoimmunity via molecular mimicry [12]. Accordingly, epitope mapping of CFTR is important for understanding autoimmune and immune responses [13], gaining insight into mechanisms of therapeutic antibodies [14,15], and securing and protecting intellectual property [16].…”