Highly-sensitive peptide array using peptides immobilized on microbeads: Application to cow's milk allergy analysis

with peptide microarrays containing linear epitopes of allergenic proteins is expected to provide information on the clinical status of the patient, but peptide arrays are still limited to research use. We thus aimed at developing a simple and sensitive peptide array that operates with more cost-effective ECL detection, so that it can be routinely used in clinical practice. For this purpose, instead of directly immobilizing the peptides onto the microarray surface as in the previous reports, we developed a two-step immobilization technique using microbeads. Peptides biotinylated at the N-terminal are first bound to microbeads with streptavidin (sAV) on the surface, followed by immobilization of the peptide-bound beads onto the microarray substrate using the photoreactive crosslinker. In this way, we were able to overcome the limitations of direct immobilization in increasing the amount and accessibility of peptides and greatly enhance the sensitivity so that ECL detection became possible. In the present study, we analyzed sera from cow's milk allergy (CMA) patients with our peptide array containing 20 peptides from αS1-casein. The results showed that IgE epitope patterns of patients could be visualized individually, and confirmed that the pattern is unique to each patient.