Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens

Henrik Ditzel*, Karin Erb, Graham Leslie, Jens Chr Jensenius

*Kontaktforfatter for dette arbejde

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    Resumé

    The large size of human IgM monoclonal antibodies (MAbs) may impede the tumor-localizing capacity. A procedure is described for the preparation of antigen-binding monomeric (IgMm) and half-monomeric (IgMI½m fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiotreitol, 2-mercaptoethanol, 2-mercaptoethylamine, L-cysteine, metabisulphite, ascorbic acid) were investigated over a range of concentrations, pHs, and incubation periods. The reduced IgM preparations were alkylated with iodoacetamide and fractionated by high-performance gel permeation chromatography. The fractions were directly collected on ELISA plates coated with extracts of colon cancer cells. Antigen-binding IgMm and IgMI½m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgMI½m fragments were also obtained after dithiotreitol treatment. These fragments were, however, nonreactive. The pH during the reduction was important for optimal yields of the fragments. The fragments obtained with 2-mercaptoethanol and mercaptoethylamine were most effective in binding to the cancer cell extract. The association constants per binding site for intact, monomeric, and half-monomeric COU-1 were by competitive inhibition assays estimated at 1.5 × 108 M−13.1 × 108 M−l and 4.0 × 106 M−1respectively. The reduction of human IgM MAbs to IgMm and IgMI½m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.

    OriginalsprogEngelsk
    TidsskriftHuman Antibodies
    Vol/bind4
    Udgave nummer2
    Sider (fra-til)86-93
    ISSN1093-2607
    DOI
    StatusUdgivet - 1. jan. 1993

    Fingeraftryk

    Cysteamine
    Mercaptoethanol
    Colorectal Neoplasms
    Neoplasms
    Iodoacetamide
    Cell Extracts
    Colonic Neoplasms
    Gel Chromatography
    Colon
    metabisulfite

    Citer dette

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    title = "Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens",
    abstract = "The large size of human IgM monoclonal antibodies (MAbs) may impede the tumor-localizing capacity. A procedure is described for the preparation of antigen-binding monomeric (IgMm) and half-monomeric (IgMI½m fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiotreitol, 2-mercaptoethanol, 2-mercaptoethylamine, L-cysteine, metabisulphite, ascorbic acid) were investigated over a range of concentrations, pHs, and incubation periods. The reduced IgM preparations were alkylated with iodoacetamide and fractionated by high-performance gel permeation chromatography. The fractions were directly collected on ELISA plates coated with extracts of colon cancer cells. Antigen-binding IgMm and IgMI½m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgMI½m fragments were also obtained after dithiotreitol treatment. These fragments were, however, nonreactive. The pH during the reduction was important for optimal yields of the fragments. The fragments obtained with 2-mercaptoethanol and mercaptoethylamine were most effective in binding to the cancer cell extract. The association constants per binding site for intact, monomeric, and half-monomeric COU-1 were by competitive inhibition assays estimated at 1.5 × 108 M−13.1 × 108 M−l and 4.0 × 106 M−1respectively. The reduction of human IgM MAbs to IgMm and IgMI½m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.",
    keywords = "Antigen-binding, Colorectal cancer, Fragment, Half-monomeric, Human monoclonal antibody, Monomeric",
    author = "Henrik Ditzel and Karin Erb and Graham Leslie and Jensenius, {Jens Chr}",
    year = "1993",
    month = "1",
    day = "1",
    doi = "10.3233/HAB-1993-4207",
    language = "English",
    volume = "4",
    pages = "86--93",
    journal = "Human Antibodies",
    issn = "1093-2607",
    publisher = "I O S Press",
    number = "2",

    }

    Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens. / Ditzel, Henrik; Erb, Karin; Leslie, Graham; Jensenius, Jens Chr.

    I: Human Antibodies, Bind 4, Nr. 2, 01.01.1993, s. 86-93.

    Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

    TY - JOUR

    T1 - Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens

    AU - Ditzel, Henrik

    AU - Erb, Karin

    AU - Leslie, Graham

    AU - Jensenius, Jens Chr

    PY - 1993/1/1

    Y1 - 1993/1/1

    N2 - The large size of human IgM monoclonal antibodies (MAbs) may impede the tumor-localizing capacity. A procedure is described for the preparation of antigen-binding monomeric (IgMm) and half-monomeric (IgMI½m fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiotreitol, 2-mercaptoethanol, 2-mercaptoethylamine, L-cysteine, metabisulphite, ascorbic acid) were investigated over a range of concentrations, pHs, and incubation periods. The reduced IgM preparations were alkylated with iodoacetamide and fractionated by high-performance gel permeation chromatography. The fractions were directly collected on ELISA plates coated with extracts of colon cancer cells. Antigen-binding IgMm and IgMI½m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgMI½m fragments were also obtained after dithiotreitol treatment. These fragments were, however, nonreactive. The pH during the reduction was important for optimal yields of the fragments. The fragments obtained with 2-mercaptoethanol and mercaptoethylamine were most effective in binding to the cancer cell extract. The association constants per binding site for intact, monomeric, and half-monomeric COU-1 were by competitive inhibition assays estimated at 1.5 × 108 M−13.1 × 108 M−l and 4.0 × 106 M−1respectively. The reduction of human IgM MAbs to IgMm and IgMI½m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.

    AB - The large size of human IgM monoclonal antibodies (MAbs) may impede the tumor-localizing capacity. A procedure is described for the preparation of antigen-binding monomeric (IgMm) and half-monomeric (IgMI½m fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiotreitol, 2-mercaptoethanol, 2-mercaptoethylamine, L-cysteine, metabisulphite, ascorbic acid) were investigated over a range of concentrations, pHs, and incubation periods. The reduced IgM preparations were alkylated with iodoacetamide and fractionated by high-performance gel permeation chromatography. The fractions were directly collected on ELISA plates coated with extracts of colon cancer cells. Antigen-binding IgMm and IgMI½m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgMI½m fragments were also obtained after dithiotreitol treatment. These fragments were, however, nonreactive. The pH during the reduction was important for optimal yields of the fragments. The fragments obtained with 2-mercaptoethanol and mercaptoethylamine were most effective in binding to the cancer cell extract. The association constants per binding site for intact, monomeric, and half-monomeric COU-1 were by competitive inhibition assays estimated at 1.5 × 108 M−13.1 × 108 M−l and 4.0 × 106 M−1respectively. The reduction of human IgM MAbs to IgMm and IgMI½m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.

    KW - Antigen-binding

    KW - Colorectal cancer

    KW - Fragment

    KW - Half-monomeric

    KW - Human monoclonal antibody

    KW - Monomeric

    U2 - 10.3233/HAB-1993-4207

    DO - 10.3233/HAB-1993-4207

    M3 - Journal article

    C2 - 8518368

    AN - SCOPUS:0027309549

    VL - 4

    SP - 86

    EP - 93

    JO - Human Antibodies

    JF - Human Antibodies

    SN - 1093-2607

    IS - 2

    ER -