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.

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 avidin-derivatized 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.

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.

MIA is a reproducible and specific assay for the investigation of allergen-specific antibody.