PMID: 9534915Apr 16, 1998Paper

Epitope mapping of the house-dust-mite allergen Der p 2 by means of site-directed mutagenesis

Allergy
G A HakkaartR van Ree

Abstract

Recombinant Der p 2, expressed in the baker's yeast Saccharomyces cerevisiae, was used as a tool to determine IgE- and monoclonal antibody (mAb)-binding sites on this allergen. For this purpose, mutant molecules were produced by application of site-directed mutagenesis. The amino-acid residues spanning cys21-cys27 and cys73-cys78 were deleted, thus preventing loop formation through disulfide bonds. Charged residues in three predicted antigenic sites (residues 45-48, 67 + 69, and 88-90) were replaced by alanine residues, IgE- and mAb reactivity to these mutants was compared to that to "wild type" Der p 2. Residues spanning cys73-cys78 were involved in the antigenic binding site for mAb alpha DpX. Mutations in the areas adjacent to this loop (i.e., 67 + 69 and 88-90) had similar effects on this mAb (10- to 20-fold decreases in reactivity were observed), supporting the suggestion that these areas are involved in this antigenic structure. The area of residues 45-48 was shown to be involved in an epitope for mAb 2B12. The reactivity of mAb 7A1 was influenced by substitutions of residues 45-48 as well as 88-90. Deletion of the residues spanning cys21-cys27 resulted in decreased reactivity to three mAbs (10E11, alpha DpX, and 7A1). Fr...Continue Reading

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