Validation of a Phage Display and Computational Algorithm by Mapping a Conformational Epitope of Bla g 2

Abstract

Background: Bla g 2, one of the major cockroach allergens, induces a strong IgE response against conformational epitopes, and on reexposure, sensitized individuals often display symptoms of allergic rhinitis and asthma. The aim of the current study was to perform a test of the efficacy of a modified phage display screening, characterization of selected phages and an automated algorithm, EpiSearch, in locating an important conformational epitope. Methods: The monoclonal antibody 7C11, which partially inhibits the binding of patient IgE antibodies to Bla g 2, was used to screen a random peptide phage library. After 3 rounds of panning, 32 phage clones were isolated and the amino acid sequences of their peptides were determined. The relative affinity and specificity of the binding of these peptides to 7C11 were tested in ELISAs. The amino acid composition of these peptides was then matched with clusters of residues on the surface of the 3-dimensional (3D) structure of Bla g 2, using our EpiSearch algorithm. Results: The amino acid sequences of the peptides on selected phages differed at only one position, occupied by 1 of 2 negatively charged residues. The two 12-mer sequences bound to 7C11 with similar avidity and specificity. There was good concordance between the residues in the 3D clusters identified from our phage display/computational method with the co-crystal structural analysis. Conclusion: Conformational epitopes may be mapped through screening of clones from random peptide phage display libraries and EpiSearch.

Epitope Mapping with Random Phage Display Library

“…For instance while mapping for the major cockroach allergens Bla g 2 epitope, we sequenced 40 clones and obtained 32 clean sequences. Twenty-four out of 32 were SMMKADFDEEPR, and other 8 were SMMKADFEEEPR [3] ( see Note 3 ).…”

“…EpiSeach (http://curie.utmb.edu/episearch.html) has been recently modified, and we successfully used it in combination with crystal structure analysis [2, 3]. …”

Conformational IgE epitopes of peanut allergens Ara h 2 and Ara h 6

“…Therefore, the phage display strategy is ideally suited for determination of individual polyclonal epitope recognition patterns. Phage display technology has been used to identify conformational epitopes in Ara h 1 and other allergens [42,[44][45][46][47].…”

Multiple IgE recognition on the major allergen of the Parietaria pollen Par j 2

“…However, despite the pivotal role established for IgE in allergic reactions, little is known about the interaction between allergens and antibodies, mainly due to the low concentration of this class of immunoglobulin in the sera of patients and to the fact that IgE-producing B cells have been poorly characterized [reviewed by (Gadermaier et al, 2014)]. Most of the data related to this aspect are derived from in vitro assays using mouse monoclonal antibodies and/or recombinant chimeric human IgE derived from mouse antibodies (Tai et al, 2013;Tiwari et al, 2012). In this respect, it has been demonstrated that human allergen-specific IgE may interact in a different way from mouse antibodies (Aalberse and Crameri, 2011); therefore, these strategies may not be able to fully elucidate the overall spectrum of antibody recognition.…”