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

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-reactiv...

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Autores principales: Bublin, Merima, Kostadinova, Maria, Fuchs, Julian E., Ackerbauer, Daniela, Moraes, Adolfo H., Almeida, Fabio C. L., Lengger, Nina, Hafner, Christine, Ebner, Christof, Radauer, Christian, Liedl, Klaus R., Valente, Ana Paula, Breiteneder, Heimo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651496/
https://www.ncbi.nlm.nih.gov/pubmed/26579717
http://dx.doi.org/10.1371/journal.pone.0142625
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author Bublin, Merima
Kostadinova, Maria
Fuchs, Julian E.
Ackerbauer, Daniela
Moraes, Adolfo H.
Almeida, Fabio C. L.
Lengger, Nina
Hafner, Christine
Ebner, Christof
Radauer, Christian
Liedl, Klaus R.
Valente, Ana Paula
Breiteneder, Heimo
author_facet Bublin, Merima
Kostadinova, Maria
Fuchs, Julian E.
Ackerbauer, Daniela
Moraes, Adolfo H.
Almeida, Fabio C. L.
Lengger, Nina
Hafner, Christine
Ebner, Christof
Radauer, Christian
Liedl, Klaus R.
Valente, Ana Paula
Breiteneder, Heimo
author_sort Bublin, Merima
collection PubMed
description 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%. (1)H/(15)N 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.
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spelling pubmed-46514962015-11-25 A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope Bublin, Merima Kostadinova, Maria Fuchs, Julian E. Ackerbauer, Daniela Moraes, Adolfo H. Almeida, Fabio C. L. Lengger, Nina Hafner, Christine Ebner, Christof Radauer, Christian Liedl, Klaus R. Valente, Ana Paula Breiteneder, Heimo PLoS One Research Article 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%. (1)H/(15)N 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. Public Library of Science 2015-11-18 /pmc/articles/PMC4651496/ /pubmed/26579717 http://dx.doi.org/10.1371/journal.pone.0142625 Text en © 2015 Bublin et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Bublin, Merima
Kostadinova, Maria
Fuchs, Julian E.
Ackerbauer, Daniela
Moraes, Adolfo H.
Almeida, Fabio C. L.
Lengger, Nina
Hafner, Christine
Ebner, Christof
Radauer, Christian
Liedl, Klaus R.
Valente, Ana Paula
Breiteneder, Heimo
A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope
title A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope
title_full A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope
title_fullStr A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope
title_full_unstemmed A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope
title_short A Cross-Reactive Human Single-Chain Antibody for Detection of Major Fish Allergens, Parvalbumins, and Identification of a Major IgE-Binding Epitope
title_sort cross-reactive human single-chain antibody for detection of major fish allergens, parvalbumins, and identification of a major ige-binding epitope
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4651496/
https://www.ncbi.nlm.nih.gov/pubmed/26579717
http://dx.doi.org/10.1371/journal.pone.0142625
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