A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.

Merima Bublin,Daniela Ackerbauer,Christine Hafner,Fabio C L Almeida,Nina Lengger,Klaus R Liedl,Heimo Breiteneder,Christof Ebner,Ana Paula Valente,Christian Radauer,Julian E Fuchs,Maria Kostadinova,Adolfo H Moraes,Zhaozhong Han

doi:10.1371/journal.pone.0142625

Abstract

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.

Highlights

Fish is one of the eight most important food allergen sources which cause the majority of foodinduced IgE-mediated allergic reactions [1,2,3]
To isolate cross-reactive single chain variable fragment (scFv) antibodies from the ETH-2 phage display library sequential antigen biopanning against purified Gad m 1, Cyp c 1 and Onc m 1 was performed (Table 1)
Parvalbumin-specific scFv enrichment was confirmed by polyclonal phage-scFv Enzyme Linked Immunosorbent Assay (ELISA) using the phage libraries obtained from each biopanning

Summary

Introduction

Fish is one of the eight most important food allergen sources which cause the majority of foodinduced IgE-mediated allergic reactions [1,2,3]. In the past two decades, fish consumption has undergone major changes due to the globalization of the food industry and to innovations and improvement in processing, transportation and distribution. The consumption of fish and processed fish products has steadily increased due to the recognition of their high nutritional value [5]. Fish allergy often persists throughout life and in allergic individuals consumption, inhalation or contact with fish and fish containing products can lead to mild local symptoms to severe systemic anaphylactic reactions [4]

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