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.

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.

Figure 1

Time course experiment for optimization of serum incubation. 3 replicate arrays were immunolabeled with 1/500 diluted serum pool or 1/5 diluted negative control serum for each condition. Data presented are averaged Z scores of the replicates. Reactions to the pool and negative control serum are showed as ◆ and □, respectively. The percentage of positive peptides (Z score>3) are indicated.

Figure 2

Scatter plots showing the effects of different variables (printing lot, immunolabeling day and serum pool batch) on reproducibility of the peptide microarray. A, data from two replicate arrays with the same 3 variables; B-D, data from two replicate arrays differing in one of the 3 variables: immunolabeling day (B), serum pool batch (C) or printing lot (D). Coefficient of correlation was calculated (p<0.01) and shown in each scatter plot.

Figure 3

Results from serum dilution experiment (A) and peptide inhibition assay (B). Data are presented as Z scores using TMeV. Areas showing non-specific binding are indicated with black arrows. Targeted peptides, which are the peptides pre-incubated with the serum pool are indicated with red arrows for each inhibition group. Areas with possible cross-inhibition based on sequence alignment are indicated with blue arrows.

Figure 4

Comparison between the epitopes identified from SPOT membrane immunoassays and peptide microarray. Peptides with amino acid sequences of at least 90% overlapped with the major epitopes and minor epitopes identified using SPOT membrane technology are shown in and , respectively, while peptides with positive reactions (z score >3) to positive serum pool in peptide microarray are shown in .