Development of a novel peptide microarray for large-scale epitope mapping of food allergens - 20/08/11
, Ludmilla Bardina, MSc a, Wayne G. Shreffler, MD, PhD a, Doerthe A. Andreae, MD a, Yongchao Ge, PhD b, Julie Wang, MD a, Francesca M. Bruni, MD c, Zhiyan Fu, PhD a, Youngshin Han, PhD d, Hugh A. Sampson, MD a
Reprint requests: Jing Lin, PhD, Pediatric Allergy and Immunology, Mount Sinai School of Medicine, Box 1198, New York, NY 10029-6574.Abstract |
Background |
The peptide microarray is a novel assay that facilitates high-throughput screening of peptides with a small quantity of sample.
Objective |
We sought to use overlapping peptides of milk allergenic proteins as a model system to establish a reliable and sensitive peptide microarray-based immunoassay for large-scale epitope mapping of food allergens.
Methods |
A milk peptide microarray was developed by using commercially synthesized peptides (20-mers, 3 offset) covering the primary sequences of ⍺s1-casein, ⍺s2-casein, β-casein, κ-casein, and β-lactoglobulin. Conditions for printing and immunolabeling were optimized using a serum pool of 5 patients with milk allergy. Reproducibility of the milk peptide microarray was evaluated using replicate arrays immunolabeled with the serum pool, whereas specificity and sensitivity were assessed by using serial dilution of the serum pool and a peptide inhibition assay.
Results |
Our results show that epitopes identified by the peptide microarray were mostly consistent with those identified previously by SPOT membrane technology, but with specific binding to a few newly identified epitopes of milk allergens. Data from replicate arrays were reproducible (r ≥ 0.92) regardless of printing lots, immunolabeling, and serum pool batches. Using the serially diluted serum pool, we confirmed that IgE antibody binding detected in the array was specific. Peptide inhibition of IgE binding to the same peptide and overlapping peptides further confirmed the specificity of the array.
Conclusion |
A reliable peptide microarray was established for large-scale IgE epitope mapping of milk allergens, and this robust technology could be applied for epitope mapping of other food allergens.
Le texte complet de cet article est disponible en PDF.Key words : Epitope mapping, peptide microarray, allergy, milk allergen
Abbreviations used : DFU, DMSO, PPB, MAD, PBST, TMeV
Plan
| Y.H. was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD; KRF-2006-214-C00099). H.A.S. and J.L. were supported in part by a grant from National Institute of Allergy and Infectious Diseases (AI-44236). Studies were supported in part by grants from the National Institute of Allergy and Infectious Diseases (AI-44236) and the National Center for Research Resources (MO1-RR-00071). |
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| Disclosure of potential conflict of interest: W. G. Shreffler receives grant support from the Food Allergy and Anaphylaxis Network. Y. Han receives grant support from the Korean Research Foundation. H. A. Sampson is a consultant for and 4% shareholder in Allertein Pharmaceuticals, LLC; is on the advisory Board for Schering-Plough; receives grant support from the Food Allergy Initiative and the National Institute of Allergy and Infectious Diseases/National Institutes of Health; is a consultant/scientific advisor for the Food Allergy Initiative; is president of the American Academy of Allergy, Asthma & Immunology; and is 45% owner of Herbal Springs, LLC. The rest of the authors have declared that they have no conflict of interest. |
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| This is the first article describing the optimization and validation of the peptide microarray-based immunoassay. It provides solid tools for current and future research for mapping of allergenic epitopes. |
Vol 124 - N° 2
P. 315 - août 2009 Retour au numéro
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