title: A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope.
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.
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Merima
authors.initials: M
authors.lastname: Bublin
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Maria
authors.initials: M
authors.lastname: Kostadinova
authors.affiliation: Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Julian E
authors.initials: JE
authors.lastname: Fuchs
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Daniela
authors.initials: D
authors.lastname: Ackerbauer
authors.affiliation: Centro Nacional de Ressonância Magnética, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Adolfo H
authors.initials: AH
authors.lastname: Moraes
authors.affiliation: Centro Nacional de Ressonância Magnética, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Fabio C L
authors.initials: FC
authors.lastname: Almeida
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Nina
authors.initials: N
authors.lastname: Lengger
authors.affiliation: Karl Landsteiner Institute for Dermatological Research, St. Pölten, Austria, Department of Dermatology, Karl Landsteiner University for Medical Sciences, St.Pölten, Austria.
authors.forename: Christine
authors.initials: C
authors.lastname: Hafner
authors.affiliation: Allergy Clinic Reumannplatz, Vienna, Austria.
authors.forename: Christof
authors.initials: C
authors.lastname: Ebner
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Radauer
authors.affiliation: Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.
authors.forename: Klaus R
authors.initials: KR
authors.lastname: Liedl
authors.affiliation: Centro Nacional de Ressonância Magnética, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Valente
authors.affiliation: Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria.
authors.forename: Heimo
authors.initials: H
authors.lastname: Breiteneder
mesh_terms: D000485:Allergens / Q000276:immunology* / Q000302:isolation & purification; D000818:Animals; D003429:Cross Reactions / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005399:Fishes / Q000276:immunology; D005512:Food Hypersensitivity / Q000276:immunology*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology* / Q000302:isolation & purification; D010320:Parvalbumins / Q000276:immunology* / Q000302:isolation & purification; D057127:Single-Chain Antibodies / Q000276:immunology
chemical_list: D000485:Allergens; D000939:Epitopes; D010320:Parvalbumins; D057127:Single-Chain Antibodies; D007073:Immunoglobulin E