Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens
Ditzel, H.; Erb, K.; Leslie, G.; Jensenius, J.C.
Human Antibodies and Hybridomas 4(2): 86-93
1993
ISSN/ISBN: 0956-960X PMID: 8518368 Document Number: 413172
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 (IgM-m) and half-monomeric (IgM-1/2m) 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 IgM-m and IgM-1/2m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgM-m and IgM-1/2m 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 times 10-8 M-1, 3.1 times 10-8 M-1 and 4.0 times 10-6 M-1, respectively. The reduction of human IgM Mabs to IgM-m and IgM-1/2m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.