Shreffler WG, Beyer K, Chu TH, Burks AW, Sampson HA. J Allergy Clin Immunol. 2004;113:776–782
Purpose of the Study.
To develop a peptide microarray-based immunoassay to map IgE-binding segments (epitopes) of peanut allergens by using microliter quantities of serum.
Sera from 77 peanut-allergic patients and 15 non–peanut-allergic control patients were analyzed.
A set of 213 overlapping 20-residue peptides was synthesized corresponding to the primary sequences of the major peanut allergens, Ara h1, Ara h2, and Ara h3. These were arrayed in triplicate along with the corresponding recombinant proteins onto glass slides and used for immunolabeling.
The majority of patients (97%) had specific IgE to at least 1 of the recombinant allergens, and 87% had detectable IgE to sequential epitopes. Microarray mapping correlated well with previous studies. However, the analysis of individual patients revealed remarkable heterogeneity in the number and patterns of epitope recognition. High epitope diversity was found in patients with a history of more severe allergic reactions. Also, sensitization of effector cells with more diverse IgE antibodies conferred greater reactivity to specific allergens.
The protein microarray immunoassay confirmed that Ara h1, Ara h2, and Ara h3 are major peanut allergens and allows for parallel epitope analysis. This has led to the discovery of an additional important epitope of Ara h1 and the recognition of a high degree of patient heterogeneity. This qualitative difference in epitope diversity might provide prognostic information about the patient.
Current techniques for mapping large numbers of epitopes by using individual patient sera are relatively time consuming, labor intensive, expensive, and prone to error. However, such studies have been useful, because identification of certain IgE-binding segments correlates with clinical outcomes such as likelihood for an allergy to resolve. Peptide microarray technology is a novel assay that allows characterization of large numbers of individual patient samples simultaneously with minimal amounts of blood. Microarray technology may be a useful diagnostic tool to assess differences in epitope recognition among patients and may provide more prognostic information regarding patients’ peanut allergies. In addition, these assessments of allergens may speed the production of allergy vaccines engineered in the future.