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Int Arch Allergy Immunol. 2012;157(4):323-30. doi: 10.1159/000330108. Epub 2011 Nov 23.

Validation of a phage display and computational algorithm by mapping a conformational epitope of Bla g 2.

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1
Department of Pediatrics, Child Health Research Center, University of Texas Medical Branch, Galveston, TX 77555-0366, USA.

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.

Comment in

PMID:
22123204
PMCID:
PMC3242705
DOI:
10.1159/000330108
[Indexed for MEDLINE]
Free PMC Article
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