Precision silver carp allergy profiling and rapid IgE epitope mapping by a microfluidic chip-MALDI-TOF MS system and prediction models.

Food allergies are increasing at an alarming rate, and it has become necessary to identify patient-specific allergens for personalized allergy immunotherapy treatments. Herein, a precision fish allergy test was established for patient-specific allergen identification and IgE epitope mapping in silver carp by employing an integrated microfluidic chip-MALDI-TOF MS system. Tosyl-activated magnetic beads (MGBs) were functionalized with mouse monoclonal anti-human immunoglobulin E (IgE) antibodies and patient serum to form IgE-MGBs. For patient-specific allergen identification, silver carp (Hypophthalmichthys molitrix) muscle protein extracts were mixed with IgE-MGBs and injected into a microfluidic channel to capture allergenic proteins. Captured allergens were eluted, trypsin-digested, and characterized by MALDI-TOF MS and peptide mass fingerprinting. To map IgE epitopes, tryptic peptides from silver carp proteins were similarly captured by IgE-MGBs and characterized by MALDI-TOF MS and peptide mass fingerprinting. Ten potential allergens were identified from silver carp, nine of which are novel to this fish based on the use of fish allergic serum samples. The allergenicity of identified proteins was predicted using AlgPred and ALLERDET tools, while IgE epitopes were predicted by Bepipred Linear Epitope Prediction 2.0 and experimentally validated via the microfluidic chip-MALDI-TOF MS system. ELISA confirmed the protocol, showing a parvalbumin content of 458.2 µg g-1 in fish muscle. Comparison of fish allergy diagnosis methods further demonstrates the potential of the microfluidic chip-MALDI-TOF MS system to personalize fish allergy detection and aid in identifying patient-specific allergens and IgE epitopes for immunotherapy development.