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

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    Abstrakt

    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

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