title: Development of a novel peptide microarray for large-scale epitope mapping of food allergens.
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 alpha(s1)-casein, alpha(s2)-casein, beta-casein, kappa-casein, and beta-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 > or = 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.
  
  
  CONCLUSIONS
  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.
authors: Pediatric Allergy and Immunology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA. jing.s.lin@mssm.edu
authors: Jing
authors: J
authors: Lin
authors: Ludmilla
authors: L
authors: Bardina
authors: Wayne G
authors: WG
authors: Shreffler
authors: Doerthe A
authors: DA
authors: Andreae
authors: Yongchao
authors: Y
authors: Ge
authors: Julie
authors: J
authors: Wang
authors: Francesca M
authors: FM
authors: Bruni
authors: Zhiyan
authors: Z
authors: Fu
authors: Youngshin
authors: Y
authors: Han
authors: Hugh A
authors: HA
authors: Sampson
mesh_terms: D000485:Allergens / Q000276:immunology*; D000595:Amino Acid Sequence; D000818:Animals; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology*; D005512:Food Hypersensitivity / Q000175:diagnosis / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D008892:Milk / Q000276:immunology; D016269:Milk Hypersensitivity / Q000175:diagnosis / Q000276:immunology*; D008894:Milk Proteins / Q000276:immunology*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D040081:Protein Array Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000485:Allergens; D000939:Epitopes; D008894:Milk Proteins; D010455:Peptides; D007073:Immunoglobulin E