Cross-reactive monoclonal antibodies against fish parvalbumins as a tool for studying antigenic similarity of different parvalbumins and analysis of fish extracts

Abstract

Fish parvalbumins are heat-stable calcium-binding proteins that are highly cross-reactive in causing allergy symptoms in fish-sensitized patients. The reactivities of parvalbumin-specific monoclonal or polyclonal antibodies with parvalbumins of different fish species allowed their application for development of various immunoassays for allergen identification in fish samples. In this study, monoclonal antibodies (MAbs) were generated against two parvalbumins - natural Atlantic cod parvalbumin and recombinant common carp β-parvalbumin expressed in E. coli. Large collections of recombinant parvalbumins and natural allergen extracts of different fish species and other animals were used to identify the specificities of these MAbs using ELISA, Western blot, and dot blot. MAbs demonstrated different patterns of cross-reactivities with recombinant parvalbumins. Their binding affinities were affected by the addition and removal of Ca²⁺ ions. Moreover, all MAbs showed a broad reactivity with the target antigens in natural fish, chicken, and pork extracts. The ability of two MAbs (clones 7B2 and 3F6) to identify and isolate native parvalbumins from allergen extracts was confirmed by Western blot. Epitope mapping using recombinant fragments of Atlantic cod parvalbumin (Gad m 1) and common carp parvalbumin (Cyp c 1) revealed that 4 out of 5 MAbs recognize parvalbumin regions that contain calcium binding sites. In conclusion, the generated broadly reactive well-characterized MAbs against fish β-parvalbumins could be applied for investigation of parvalbumins of fish and other animals and their detection in allergen extracts.

Keywords: Allergen extracts; Cross-reactivity; Fish allergy; Monoclonal antibodies; Parvalbumin.