Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

Henriette T. Boesen, Tim Kåre Jensen, Gregers Jungersen, Ulla Riber, Mette Boye, Kristian Møller

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.
    Original languageEnglish
    JournalVeterinary Microbiology
    Volume105
    Issue number3-4
    Pages (from-to)199-206
    ISSN0378-1135
    DOIs
    Publication statusPublished - 2005

    Keywords

    • Lawsonia intracellularis
    • lipopolysaccharide
    • monoclonal antibodies

    Cite this

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    title = "Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen",
    abstract = "Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.",
    keywords = "Lawsonia intracellularis, lipopolysaccharide, monoclonal antibodies",
    author = "Boesen, {Henriette T.} and Jensen, {Tim K{\aa}re} and Gregers Jungersen and Ulla Riber and Mette Boye and Kristian M{\o}ller",
    year = "2005",
    doi = "10.1016/j.vetmic.2004.10.022",
    language = "English",
    volume = "105",
    pages = "199--206",
    journal = "Veterinary Microbiology",
    issn = "0378-1135",
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    }

    Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen. / Boesen, Henriette T.; Jensen, Tim Kåre; Jungersen, Gregers; Riber, Ulla; Boye, Mette; Møller, Kristian.

    In: Veterinary Microbiology, Vol. 105, No. 3-4, 2005, p. 199-206.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

    AU - Boesen, Henriette T.

    AU - Jensen, Tim Kåre

    AU - Jungersen, Gregers

    AU - Riber, Ulla

    AU - Boye, Mette

    AU - Møller, Kristian

    PY - 2005

    Y1 - 2005

    N2 - Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.

    AB - Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol-water extract. The inhibitory effect of periodate oxidation on the antigen-antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.

    KW - Lawsonia intracellularis

    KW - lipopolysaccharide

    KW - monoclonal antibodies

    U2 - 10.1016/j.vetmic.2004.10.022

    DO - 10.1016/j.vetmic.2004.10.022

    M3 - Journal article

    VL - 105

    SP - 199

    EP - 206

    JO - Veterinary Microbiology

    JF - Veterinary Microbiology

    SN - 0378-1135

    IS - 3-4

    ER -