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

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

Research output: Journal article (peer-reviewed)Journal article

9 Citations (Scopus)

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.

Original languageEnglish
Article number0142625
Pages (from-to)e0142625
JournalPLoS ONE
Volume10
Issue number11
DOIs
Publication statusPublished - 1 Nov 2015

Keywords

  • Allergens/immunology
  • Animals
  • Cross Reactions/immunology
  • Epitope Mapping
  • Epitopes/immunology
  • Fishes/immunology
  • Food Hypersensitivity/immunology
  • Humans
  • Immunoglobulin E/immunology
  • Parvalbumins/immunology
  • Single-Chain Antibodies/immunology

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