Microarray immunoassay: association of clinical history, in vitro IgE function, and heterogeneity of allergenic peanut epitopes.
BACKGROUND
IgE epitope mapping of food allergens is a prerequisite for engineering hypoallergenic immunotherapeutic agents and might reveal basic information regarding a patient's immune response. Mapping of large numbers of epitopes by using individual patient sera has been impractical with current techniques.


OBJECTIVE
We sought to develop a peptide microarray-based immunoassay to map peanut epitopes by using microliter quantities of serum.


METHODS
A set of 213 overlapping 20-residue peptides was synthesized corresponding to the primary sequences of Ara h 1, Ara h 2, and Ara h 3. These were arrayed in triplicate along with the corresponding recombinant proteins onto glass slides and used for immunolabeling.


RESULTS
Seventy-seven patient and 15 control sera were analyzed. The majority of patients (97%) had specific IgE to at least one 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 allergen.


CONCLUSIONS
The protein microarray immunoassay confirmed that Ara h 1, Ara h 2, and Ara h 3 are major peanut allergens and allows for parallel epitope analysis. This has led to the discovery of an additional important epitope of Ara h 1 and the recognition of a high degree of patient heterogeneity. This qualitative difference in epitope diversity might provide prognostic information about the patient.
Shreffler
Wayne G
WG
Jaffe Food Allergy Institute, Division of Allergy and Immunology, Department of Pediatrics, Mount Sinai Medical Center, Box 1198, One Gustave L. Levy Place, New York, NY 10029, USA.
Beyer
Kirsten
K
Chu
Te-Hua Tearina
TH
Burks
A Wesley
AW
Sampson
Hugh A
HA
D055516:2S Albumins, Plant; D000293:Adolescent; D000328:Adult; D000485:Allergens / Q000276:immunology*; D052179:Antigens, Plant; D002648:Child; D002675:Child, Preschool; D000939:Epitopes; D006023:Glycoproteins / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D007073:Immunoglobulin E / Q000276:immunology*; D066298:In Vitro Techniques; D007223:Infant; D008565:Membrane Proteins; D020411:Oligonucleotide Array Sequence Analysis; D021183:Peanut Hypersensitivity / Q000276:immunology* / Q000503:physiopathology; D010940:Plant Proteins / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D055314:Seed Storage Proteins; D012720:Severity of Illness Index
D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500889:Ara h 1 protein, Arachis hypogaea; C078642:Ara h II protein, Arachis hypogaea; D000939:Epitopes; D006023:Glycoproteins; D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D055314:Seed Storage Proteins; C118207:allergen Ara h3; D007073:Immunoglobulin E