{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:61372"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:61372","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Herstellung, Expression und Charakterisierung von rekombinanten Antikörpern und Immuntoxinen gegen Phoma lingam, Sclerotinia sclerotiorum und Verticillium dahliae","abstract":"Rapeseed (Brassic napus) is growing importance as a crop used in the production of oil and animal feed. Increased production of the crop has been accompanied by the increased incidence of fungal pathogens that attack rapeseed and other crop species. These include Phoma lingam, Sclerotinia sclerotiorum and Verticillium dahliae. Current methods for controlling these pathogens are limited to prophylactic use of fungicides. Current fungicides are ineffective and naturally occurring resistance has yet to be identified. Transgenic plant technology offers the opportunity to overcome these limitations through the development of novel, inherently resistant rapeseed lines. The rationale of this thesis was to create resistant plant lines through the expression of antibody fusion proteins, so-called immunotoxins, that were specific for the pathogens and that carried fusion proteins that were toxic to the fungal pathogens. Thus, a prerequisite was the generation of pathogen specific antibodies. Further, these antibodies should maintain their specificity when expressed in transgenic plants. The first task of the work presented was the isolation of different, complex and to a large extent undefined fungal antigens for the subsequent immunological research. After the initial purification of polyclonal IgY antibodies from the egg yolk of immunized chicken, the separate immunisation of BALB/c mice by injection of a mycelium/secreted protein mixture took place. The spleen of the sacrificed animals was then utilised for the classic antibody generation by hybridoma technology as well as for the generation of single-chain (scFv) presenting phage libraries. Recombinant scFv could be subsequently selected by a „Bio-panning” in the scope of the phage display technology. Additionally the bivalent binding-characteristics of the parental monoclonal hybridoma cellines were transferred onto the monovalent scFv by hybridoma rescue. A total of seven pathogen specific antibodies and antibody fragments were generated in the course of this thesis: four specific for V. dahliae, two for P. lingam and one for S. sclerotiorum. Single chain antibodies were isolated that were specific for secreted protein, cell wall fragments, mycelium and spores. The binding characteristics of the antibodies to the pathogen were defined and were generally species specific and of high affinity. All of the scFvs were expressed as functional antibody fragments in bacteria and plant leaves. The antibodies were characterized in vitro (ELISA and Immunoblot) and in vivo (Immunofluorescence microscopy) for their capacity to bind antigen and no significant differences between native and heterologous expressed antibodies could be observed. The scFvs were expressed as fusion proteins to anti-fungal peptides, anti-microbial peptides, chitinase and glucanase and stably integrated into the genome of B. napus. In order to assess if the expressed antibodies could create fungal resistance, the T2 generation of transgenic B. napus plant lines were challenged by infection with a P. lingam spore suspension. Line were identified that were resistant to infection through the expression of pathogen specific single chain antibodies fused to anti-fungal peptides. These lines will now be crossed to establish a homozygous T3 generation, which will be evaluated for an improved acquired resistance against P. lingam, S. sclerotiorum and V. dahliae. The work presented here describes the in vitro selection of fungus specific antibodies against complex and undefined antigens of the phytopathogenic fungi P. lingam, S. sclerotinia and V. dahliae. These antibodies are able to detect their antigen in vivo and are therefore potentionally applicable in serological detection assays or in engineering antibody- or immunotoxin-based plant resistance.","abstract_html":"Rapeseed (Brassic napus) is growing importance as a crop used in the production of oil and animal feed. Increased production of the crop has been accompanied by the increased incidence of fungal pathogens that attack rapeseed and other crop species. These include Phoma lingam, Sclerotinia sclerotiorum and Verticillium dahliae. Current methods for controlling these pathogens are limited to prophylactic use of fungicides. Current fungicides are ineffective and naturally occurring resistance has yet to be identified. Transgenic plant technology offers the opportunity to overcome these limitations through the development of novel, inherently resistant rapeseed lines. The rationale of this thesis was to create resistant plant lines through the expression of antibody fusion proteins, so-called immunotoxins, that were specific for the pathogens and that carried fusion proteins that were toxic to the fungal pathogens. Thus, a prerequisite was the generation of pathogen specific antibodies. Further, these antibodies should maintain their specificity when expressed in transgenic plants. The first task of the work presented was the isolation of different, complex and to a large extent undefined fungal antigens for the subsequent immunological research. After the initial purification of polyclonal IgY antibodies from the egg yolk of immunized chicken, the separate immunisation of BALB/c mice by injection of a mycelium/secreted protein mixture took place. The spleen of the sacrificed animals was then utilised for the classic antibody generation by hybridoma technology as well as for the generation of single-chain (scFv) presenting phage libraries. Recombinant scFv could be subsequently selected by a „Bio-panning” in the scope of the phage display technology. Additionally the bivalent binding-characteristics of the parental monoclonal hybridoma cellines were transferred onto the monovalent scFv by hybridoma rescue. A total of seven pathogen specific antibodies and antibody fragments were generated in the course of this thesis: four specific for V. dahliae, two for P. lingam and one for S. sclerotiorum. Single chain antibodies were isolated that were specific for secreted protein, cell wall fragments, mycelium and spores. The binding characteristics of the antibodies to the pathogen were defined and were generally species specific and of high affinity. All of the scFvs were expressed as functional antibody fragments in bacteria and plant leaves. The antibodies were characterized in vitro (ELISA and Immunoblot) and in vivo (Immunofluorescence microscopy) for their capacity to bind antigen and no significant differences between native and heterologous expressed antibodies could be observed. The scFvs were expressed as fusion proteins to anti-fungal peptides, anti-microbial peptides, chitinase and glucanase and stably integrated into the genome of B. napus. In order to assess if the expressed antibodies could create fungal resistance, the T2 generation of transgenic B. napus plant lines were challenged by infection with a P. lingam spore suspension. Line were identified that were resistant to infection through the expression of pathogen specific single chain antibodies fused to anti-fungal peptides. These lines will now be crossed to establish a homozygous T3 generation, which will be evaluated for an improved acquired resistance against P. lingam, S. sclerotiorum and V. dahliae. The work presented here describes the in vitro selection of fungus specific antibodies against complex and undefined antigens of the phytopathogenic fungi P. lingam, S. sclerotinia and V. dahliae. These antibodies are able to detect their antigen in vivo and are therefore potentionally applicable in serological detection assays or in engineering antibody- or immunotoxin-based plant resistance.","abstract_has_math":false,"creators":["Dorfmüller, Simone"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Fischer, Rainer"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2002,"date_issued":"2002","date_published":"2002","updated_at":"2026-07-30T19:43:10Z","subjects":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie","Immuntoxine","Antikörper","scFv","Pilze","Pathogene","Antikörper-vermittelte Resistenz","Genetic engineering","transgene Pflanzen","fungitoxische Proteine","Ph"],"languages":["ger"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123042%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123042%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123042%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/61372","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fischer, Rainer"]},{"key":"dc:creator","label":"Author","values":["Dorfmüller, Simone"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2002"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-5983","info:eu-repo/semantics/altIdentifier/doi/10.18154/RWTH-CONV-123042"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","Biowissenschaften, Biologie","Immuntoxine","Antikörper","scFv","Pilze","Pathogene","Antikörper-vermittelte Resistenz","Genetic engineering","transgene Pflanzen","fungitoxische Proteine","Ph"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["ger"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/61372","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123042%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Rapeseed (Brassic napus) is growing importance as a crop used in the production of oil and animal feed. Increased production of the crop has been accompanied by the increased incidence of fungal pathogens that attack rapeseed and other crop species. These include Phoma lingam, Sclerotinia sclerotiorum and Verticillium dahliae. Current methods for controlling these pathogens are limited to prophylactic use of fungicides. Current fungicides are ineffective and naturally occurring resistance has yet to be identified. Transgenic plant technology offers the opportunity to overcome these limitations through the development of novel, inherently resistant rapeseed lines. The rationale of this thesis was to create resistant plant lines through the expression of antibody fusion proteins, so-called immunotoxins, that were specific for the pathogens and that carried fusion proteins that were toxic to the fungal pathogens. Thus, a prerequisite was the generation of pathogen specific antibodies. Further, these antibodies should maintain their specificity when expressed in transgenic plants. The first task of the work presented was the isolation of different, complex and to a large extent undefined fungal antigens for the subsequent immunological research. After the initial purification of polyclonal IgY antibodies from the egg yolk of immunized chicken, the separate immunisation of BALB/c mice by injection of a mycelium/secreted protein mixture took place. The spleen of the sacrificed animals was then utilised for the classic antibody generation by hybridoma technology as well as for the generation of single-chain (scFv) presenting phage libraries. Recombinant scFv could be subsequently selected by a „Bio-panning” in the scope of the phage display technology. Additionally the bivalent binding-characteristics of the parental monoclonal hybridoma cellines were transferred onto the monovalent scFv by hybridoma rescue. A total of seven pathogen specific antibodies and antibody fragments were generated in the course of this thesis: four specific for V. dahliae, two for P. lingam and one for S. sclerotiorum. Single chain antibodies were isolated that were specific for secreted protein, cell wall fragments, mycelium and spores. The binding characteristics of the antibodies to the pathogen were defined and were generally species specific and of high affinity. All of the scFvs were expressed as functional antibody fragments in bacteria and plant leaves. The antibodies were characterized in vitro (ELISA and Immunoblot) and in vivo (Immunofluorescence microscopy) for their capacity to bind antigen and no significant differences between native and heterologous expressed antibodies could be observed. The scFvs were expressed as fusion proteins to anti-fungal peptides, anti-microbial peptides, chitinase and glucanase and stably integrated into the genome of B. napus. In order to assess if the expressed antibodies could create fungal resistance, the T2 generation of transgenic B. napus plant lines were challenged by infection with a P. lingam spore suspension. Line were identified that were resistant to infection through the expression of pathogen specific single chain antibodies fused to anti-fungal peptides. These lines will now be crossed to establish a homozygous T3 generation, which will be evaluated for an improved acquired resistance against P. lingam, S. sclerotiorum and V. dahliae. The work presented here describes the in vitro selection of fungus specific antibodies against complex and undefined antigens of the phytopathogenic fungi P. lingam, S. sclerotinia and V. dahliae. These antibodies are able to detect their antigen in vivo and are therefore potentionally applicable in serological detection assays or in engineering antibody- or immunotoxin-based plant resistance."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University II, 182 S. : Ill., graph. Darst. (2002). doi:10.18154/RWTH-CONV-123042 = Aachen, Techn. Hochsch., Diss., 2002"]},{"key":"dc:title","label":"Title","values":["Herstellung, Expression und Charakterisierung von rekombinanten Antikörpern und Immuntoxinen gegen Phoma lingam, Sclerotinia sclerotiorum und Verticillium dahliae"]}]}],"canonical_facts":{"dc:contributor":["Fischer, Rainer"],"dc:coverage":["DE"],"dc:creator":["Dorfmüller, Simone"],"dc:date":["2002"],"dc:description":["Rapeseed (Brassic napus) is growing importance as a crop used in the production of oil and animal feed. Increased production of the crop has been accompanied by the increased incidence of fungal pathogens that attack rapeseed and other crop species. These include Phoma lingam, Sclerotinia sclerotiorum and Verticillium dahliae. Current methods for controlling these pathogens are limited to prophylactic use of fungicides. Current fungicides are ineffective and naturally occurring resistance has yet to be identified. Transgenic plant technology offers the opportunity to overcome these limitations through the development of novel, inherently resistant rapeseed lines. The rationale of this thesis was to create resistant plant lines through the expression of antibody fusion proteins, so-called immunotoxins, that were specific for the pathogens and that carried fusion proteins that were toxic to the fungal pathogens. Thus, a prerequisite was the generation of pathogen specific antibodies. Further, these antibodies should maintain their specificity when expressed in transgenic plants. The first task of the work presented was the isolation of different, complex and to a large extent undefined fungal antigens for the subsequent immunological research. After the initial purification of polyclonal IgY antibodies from the egg yolk of immunized chicken, the separate immunisation of BALB/c mice by injection of a mycelium/secreted protein mixture took place. The spleen of the sacrificed animals was then utilised for the classic antibody generation by hybridoma technology as well as for the generation of single-chain (scFv) presenting phage libraries. Recombinant scFv could be subsequently selected by a „Bio-panning” in the scope of the phage display technology. Additionally the bivalent binding-characteristics of the parental monoclonal hybridoma cellines were transferred onto the monovalent scFv by hybridoma rescue. A total of seven pathogen specific antibodies and antibody fragments were generated in the course of this thesis: four specific for V. dahliae, two for P. lingam and one for S. sclerotiorum. Single chain antibodies were isolated that were specific for secreted protein, cell wall fragments, mycelium and spores. The binding characteristics of the antibodies to the pathogen were defined and were generally species specific and of high affinity. All of the scFvs were expressed as functional antibody fragments in bacteria and plant leaves. The antibodies were characterized in vitro (ELISA and Immunoblot) and in vivo (Immunofluorescence microscopy) for their capacity to bind antigen and no significant differences between native and heterologous expressed antibodies could be observed. The scFvs were expressed as fusion proteins to anti-fungal peptides, anti-microbial peptides, chitinase and glucanase and stably integrated into the genome of B. napus. In order to assess if the expressed antibodies could create fungal resistance, the T2 generation of transgenic B. napus plant lines were challenged by infection with a P. lingam spore suspension. Line were identified that were resistant to infection through the expression of pathogen specific single chain antibodies fused to anti-fungal peptides. These lines will now be crossed to establish a homozygous T3 generation, which will be evaluated for an improved acquired resistance against P. lingam, S. sclerotiorum and V. dahliae. The work presented here describes the in vitro selection of fungus specific antibodies against complex and undefined antigens of the phytopathogenic fungi P. lingam, S. sclerotinia and V. dahliae. These antibodies are able to detect their antigen in vivo and are therefore potentionally applicable in serological detection assays or in engineering antibody- or immunotoxin-based plant resistance."],"dc:identifier":["https://publications.rwth-aachen.de/record/61372","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123042%22"],"dc:language":["ger"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-5983","info:eu-repo/semantics/altIdentifier/doi/10.18154/RWTH-CONV-123042"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University II, 182 S. : Ill., graph. Darst. (2002). doi:10.18154/RWTH-CONV-123042 = Aachen, Techn. 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