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Microarray immunoassay (MIA) for the parallel IgE and IgG4 epitope mapping of milk allergens
S238 Abstracts
J ALLERGY CLIN IMMUNOL FEBRUARY 2004
849
Microarray Immunoassay (MIA) for the Parallel IgE and IgG4 Epitope Mapping of Milk Allergens
W. G. Shreffler, L. Bardina, D. Lencer, K. Beyer, H. A. Sampson; Jaffe Food Allergy Institute, Department of Pediatrics, Division of Allergy & Immunology, Mount Sinai Medical Center, New York, NY. RATIONALE: The natural history of young patients with milk allergy is variable. Characteristics such as IgE/IgG4 ratio and specific epitope recognition patterns are being investigated for prognostic utility. Prospective, comprehensive analysis of patient epitope-specific antibody response is impractical with commonly used assays. We sought to develop a microarray-based immunoassay for parallel detection of whole allergen and peptide specific IgE and IgG4. METHODS: Sets of 15-residue peptides, each with biotin-SGSG linkers, were designed corresponding to milk allergen sequences. These were printed together with biotinylated beta- and kappa-caseins to avidinderivatized glass slides. Diluted human sera from milk-allergic patients and non-allergic controls were incubated over the arrayed peptides in a humidified chamber. Specific IgE and IgG4 were simultaneously detected by incubation with a cocktail of monoclonal secondary antibodies labeled with distinct fluorochromes. Slides were read with a microarray fluorescence scanner. RESULTS: Compared to normal sera, IgE signal [binding] with patient sera was 1.5-2 log higher to beta- and kappa-caseins respectively (p<0.01). IgG4 signal was also greater with patient sera (beta-casein, 1.5 log; kappa-casein 1 log; p<0.05), although IgG4 signal was clearly detectable in normals. Inter-assay CVs were less than 10%. For 80% of patients, kappa-casein was the dominant allergen with higher absolute IgE and IgG4 signals and higher IgE/IgG4 ratios. Individual and pooled patient sera bound specific peptides. CONCLUSION: MIA is a reproducible and specific assay for the investigation of allergen-specific antibody. Funding: NIH, FAAI, Mount Sinai Medical Center
MONDAY
J ALLERGY CLIN IMMUNOL FEBRUARY 2004
849
Microarray Immunoassay (MIA) for the Parallel IgE and IgG4 Epitope Mapping of Milk Allergens
W. G. Shreffler, L. Bardina, D. Lencer, K. Beyer, H. A. Sampson; Jaffe Food Allergy Institute, Department of Pediatrics, Division of Allergy & Immunology, Mount Sinai Medical Center, New York, NY. RATIONALE: The natural history of young patients with milk allergy is variable. Characteristics such as IgE/IgG4 ratio and specific epitope recognition patterns are being investigated for prognostic utility. Prospective, comprehensive analysis of patient epitope-specific antibody response is impractical with commonly used assays. We sought to develop a microarray-based immunoassay for parallel detection of whole allergen and peptide specific IgE and IgG4. METHODS: Sets of 15-residue peptides, each with biotin-SGSG linkers, were designed corresponding to milk allergen sequences. These were printed together with biotinylated beta- and kappa-caseins to avidinderivatized glass slides. Diluted human sera from milk-allergic patients and non-allergic controls were incubated over the arrayed peptides in a humidified chamber. Specific IgE and IgG4 were simultaneously detected by incubation with a cocktail of monoclonal secondary antibodies labeled with distinct fluorochromes. Slides were read with a microarray fluorescence scanner. RESULTS: Compared to normal sera, IgE signal [binding] with patient sera was 1.5-2 log higher to beta- and kappa-caseins respectively (p<0.01). IgG4 signal was also greater with patient sera (beta-casein, 1.5 log; kappa-casein 1 log; p<0.05), although IgG4 signal was clearly detectable in normals. Inter-assay CVs were less than 10%. For 80% of patients, kappa-casein was the dominant allergen with higher absolute IgE and IgG4 signals and higher IgE/IgG4 ratios. Individual and pooled patient sera bound specific peptides. CONCLUSION: MIA is a reproducible and specific assay for the investigation of allergen-specific antibody. Funding: NIH, FAAI, Mount Sinai Medical Center
MONDAY