A Novel Multipeptide Microarray for the Specific and Sensitive Mapping of Linear IgE-Binding Epitopes of Food Allergens

Abstract

Background: The identification of B-cell epitopes of food allergens can possibly lead to novel diagnostic tools and therapeutic reagents for food allergy. We sought to develop a flexible, low-tech, cost-effective and reproducible multipeptide microarray for the research environment to enable large-scale screening of IgE epitopes of food allergens. Methods: Overlapping peptides (15-mer, 4 amino acid offset) covering the primary sequence of either peanut allergen Ara h 1 or all 3 subunits of the soybean allergen Gly m 5 were simultaneously synthesized in-house on a porous cellulose matrix. Identical peptide microarrays created with up to 384 duplicate peptide-cellulose microspots each were investigated for specificity and sensitivity in IgE immunodetection and in direct experimental comparison to the formerly established SPOT™ membrane technique. Results: The in-house microarray identified with 98% reproducibility the same IgE-binding peptides as the SPOT™ membrane technique. Additional IgE-binding peptides were identified using the microarray. While the sensitivity was increased between 2- and 20-fold, the amount of human serum required was reduced by at least two thirds over the SPOT™ membrane technique using the microarray. After subtraction of the potential background, we did not observe non-specific binding to the presented peptides on microarray. Conclusions: The novel peptide microarray allows simple and cost-effective screening for potential epitopes of large allergenic legume seed storage proteins, and it could be adapted for other food allergens as well, to study allergenic epitopes at the individual subject level in large paediatric and adult study groups of food allergic subjects.

Clinical utility of microarray B‐cell epitope mapping in food allergies: A systematic review

“…More recently, Kühne and colleagues compared peanut allergen Ara h 1 and the soybean allergen Gly m 5 using SPOT membrane technology and microarray slides . They found a 98% reproducibility between these techniques, although some additional IgE‐binding peptides were identified using microarray.…”

“…We found that only six studies addressed this question. Two studies analyzed technical sensitivity and specificity and concluded that microarray is a robust technology that can be applied for epitope mapping of food allergens with results that are comparable to previous SPOT membrane technique. Four studies analyzed the diagnostic sensitivity and specificity.…”

Pea (Pisum sativum) allergy in children: Pis s 1 is an immunodominant major pea allergen and presents IgE binding sites with potential diagnostic value

“…101 Celluspot™ peptides (15 amino acids, 4 amino acids offset) covering the entire primary structure of Pis s 1.0101 (Uniprot number Q702P1) were synthesized, as previously described, 29 on cellulose support, and spotted in quadruplicate onto microscope slides (Intavis AG). DMSO solvent was multiply spotted as a control for background signal.…”

Recombinant Allergens in Structural Biology, Diagnosis, and Immunotherapy

“…Indoor Biotechnology produces an IgE quantitative binding array for the simultaneous detection and quantitative determination of total and allergen-specific human IgE against 11 allergens. Kühne et al [47 ]demonstrated, in a proof-of-concept study, that multipeptide microarrays of the peanut allergen Ara h 1 and all 3 subunits of the soybean allergen Gly m 5 could be used for large-scale screening of IgE epitopes of food allergens.…”