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Fish allergy is associated with moderate to severe IgE-mediated reactions to the calcium binding parvalbumins present in fish muscle. Allergy to multiple fish species is caused by parvalbumin-specific cross-reactive IgE recognizing conserved epitopes. In this study, we aimed to produce cross-reactive single chain variable fragment (scFv) antibodies for the detection of parvalbumins in fish extracts and the identification of IgE epitopes. Parvalbumin-specific phage clones were isolated from the human ETH-2 phage display library by three rounds of biopanning either against cod parvalbumin or by sequential biopanning against cod (Gad m 1), carp (Cyp c 1) and rainbow trout (Onc m 1) parvalbumins. While biopanning against Gad m 1 resulted in the selection of clones specific exclusively for Gad m 1, the second approach resulted in the selection of clones cross-reacting with all three parvalbumins. Two clones, scFv-gco9 recognizing all three parvalbumins, and scFv-goo8 recognizing only Gad m 1 were expressed in the E. coli non-suppressor strain HB2151 and purified from the periplasm. scFv-gco9 showed highly selective binding to parvalbumins in processed fish products such as breaded cod sticks, fried carp and smoked trout in Western blots. In addition, the scFv-gco9-AP produced as alkaline phosphatase fusion protein, allowed a single-step detection of the parvalbumins. In competitive ELISA, scFv-gco9 was able to inhibit binding of IgE from fish allergic patients' sera to all three β-parvalbumins by up to 80%, whereas inhibition by scFv-goo8 was up to 20%. 1H/15N HSQC NMR analysis of the rGad m 1:scFv-gco9 complex showed participation of amino acid residues conserved among these three parvalbumins explaining their cross-reactivity on a molecular level. In this study, we have demonstrated an approach for the selection of cross-reactive parvalbumin-specific antibodies that can be used for allergen detection and for mapping of conserved epitopes.

Beta-parvalbumins from different fish species have been identified as the main elicitors of IgE-mediated reactions in fish-allergic individuals. Here, we report for the first time the NMR determination of the structure and dynamics of the major Atlantic cod (Gadus morhua) allergen Gad m 1 and compare them with other known parvalbumins. Although the Gad m 1 structure and accessibility of putative IgE epitopes are similar to parvalbumins in mackerel and carp, the charge distribution at the putative epitopes is different. The determination of the Gad m 1 structure contributes to a better understanding of cross-reactivity among fish parvalbumins. In addition, the high-pressure NMR and temperature variation experiments revealed the important contribution of the AB motif and other regions to the protein folding. This structural information could assist the future identification of hot spots for targeted mutations to develop hypoallergenic Ca(2+) -free forms for potential use in immunotherapy., (© 2014 Wiley Periodicals, Inc.)