IgE and IgG4 epitope mapping by microarray immunoassay reveals the diversity of immune response to the peanut allergen, Ara h 2.
BACKGROUND
Detailed assessment of antibody responses to allergens reveals clinically relevant information about both host response and antigen structure. Microarray technology offers advantages of scale and parallel design over previous methods of epitope mapping.


OBJECTIVE
We designed a redundant peptide microarray for IgE and IgG4 epitope mapping of the previously characterized peanut allergen, Ara h 2.


METHODS
Six complete sets of overlapping peptides were commercially synthesized and site-specifically bound to epoxy-derivatized glass slides in triplicate. Peptides were 10, 15, or 20 amino acids in length with an offset of either 2 or 3 amino acids. A total of 10 control and 45 peanut-allergic sera were assayed. Specific IgE and IgG4 were detected by using fluorochrome-labeled monoclonal secondary antibodies.


RESULTS
By using 15-mer and 20-mer peptides, we could define 11 antigenic regions, whereas only 5 were identifiable using 10-mers. Controls and patients produced IgG4 recognizing a comparable number of Ara h 2 peptides, although the dominant epitopes were distinct. As expected, patient IgE bound a larger number of Ara h 2 peptides (9.4% vs 0.9%). IgE and IgG4 epitopes recognized by patients were largely the same, and there was a positive association between IgE and IgG(4) signal, suggesting coordinate regulation. Cluster analysis of peptide binding patterns confirmed the specificity of antibody-peptide interactions and was used to define 9 core epitopes ranging from 6 to 16 residues in length-7 of which (78%) agreed with previous mapping.


CONCLUSIONS
Epitope mapping by microarray peptide immunoassay and cluster analysis reveals interpatient heterogeneity and a more detailed map.
Shreffler
Wayne G
WG
Jaffe Food Allergy Institute, Department of Pediatrics, Division of Allergy and Immunology, Mount Sinai School of Medicine, New York, NY 10029, USA. wayne.shreffler@mssm.edu
Lencer
Doerthe A
DA
Bardina
Ludmilla
L
Sampson
Hugh A
HA
D055516:2S Albumins, Plant; D000485:Allergens / Q000235:genetics* / Q000276:immunology*; D000595:Amino Acid Sequence; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D052179:Antigens, Plant; D010367:Arachis / Q000235:genetics* / Q000276:immunology*; D016022:Case-Control Studies; D016000:Cluster Analysis; D018604:Epitope Mapping / Q000379:methods* / Q000706:statistics & numerical data; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D021183:Peanut Hypersensitivity / Q000235:genetics* / Q000276:immunology*; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology*; D040081:Protein Array Analysis
D055516:2S Albumins, Plant; D000485:Allergens; D052179:Antigens, Plant; C500890:Ara h 2 allergen, Arachis hypogaea; D006023:Glycoproteins; D007074:Immunoglobulin G; D010940:Plant Proteins; D007073:Immunoglobulin E