High-Throughput Tools for Characterization of Antibody Epitopes

Antibodies are molecules of tremendous importance. In their primary role, they protect our bodies against disease. However, in recent decades, scientists have harnessed the binding capabilities of antibodies and have applied them widely in research, diagnostics and therapeutics. Consequently, it is important to characterize antibodies thoroughly. In parallel to the characterization of antibodies, it is also important to characterize the binding area that is recognized by the antibody, known as an epitope. With the development of new technologies, such as high-throughput sequencing (HTS), it is important to determine how these methods can improve our understanding of antibodies and their epitopes. The overall objective of the presented studies was to investigate how emerging technologies (specifically HTS coupled with phage display and next-generation peptide microarrays) could be used for epitope mapping.
In Chapter 1, it was examined whether combining phage display, a traditional epitope mapping approach, with HTS would improve the method. The developed approach was successfully used to map Ara h 1 epitopes in sera from patients with peanut allergy. Notably, the sera represented difficult biological samples due to the rarity of the relevant antibodies and the polyclonal nature of serum. The inclusion of control samples enabled the development of a bioinformatic approach that identified peptide motifs of interest based on clustering and contrasting. A widespread problem in phage display, which is the unintended selection of peptides that are target-unspecific, was examined by comparing patient and control samples. The experiments highlighted that HTS can potentially improve on phage display by enabling the analysis of complex biological samples. Coupling the two methods furthermore has the capacity to omit traditional clone picking and functional testing which is a laborious part of phage display.
In the following study, Chapter 2, it was described how the approach developed in Chapter 1 could be utilized for a different application of phage display, specifically the identification of peptide binders. In this study, phage display screenings were used to identify peptides that could inhibit a major toxin in cobra snake venom, α-cobratoxin. Peptide inhibitors were successfully identified. Importantly, HTS enabled the identification of toxin inhibitors that were not discovered by traditional phage display.
Phage display coupled with HTS was again used in Chapter 3 in an attempt to map the epitopes of a therapeutic target injected into animals. The animals were immunized with a therapeutic target and the expectation was that they develop antibodies, which can be used in therapy. While no epitopes could be definitively identified, the study demonstrated the potential of the MiSeq HTS platform. Sequencing of the phage library also showed that many of the target-unrelated phages identified in the previous chapters, were frequent in the original library, thus indicating that they held proliferation advantages.
Finally, in Chapter 4, a different emerging technology, next-generation peptide microarrays, was applied for epitope mapping of major peanut allergens using sera from allergic patients. New developments in the peptide microarray have enabled a greatly increased throughput. In this study, these improvements were utilized to characterize epitopes at high resolution, i.e. determine the importance of each residue for antibody binding, for all major peanut allergens. Epitope reactivity among patients often converged on known epitope hotspots, however the binding patterns were somewhat heterogeneous when examined at the residue level. A high degree of correlation between IgE and IgG4 epitope binding patterns were observed, possibly indicating a common clonal origin. Finally, since the patients had been sampled over time it could be confirmed that the epitope binding patterns were stable over multiple years.
Taken together, the presented studies demonstrated new applications for the investigated techniques focusing on their utilization in epitope mapping. In the process, new insights were obtained into how antibodies recognize their targets in a major disease, i.e. food allergy.