Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens.
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 (IgM1/2m) fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiothreitol, 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 IgM1/2m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgM1/2m fragments were also obtained after dithiothreitol 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 x 10(8) M-1, 3.1 x 10(8) M-1 and 4.0 x 10(6) M-1, respectively. The reduction of human IgM MAbs to IgMm and IgM1/2m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.
Ditzel
H
H
Department of Medical Microbiology, University of Odense, Denmark.
Erb
K
K
Leslie
G
G
Jensenius
J C
JC
D000478:Alkylation; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D000912:Antibodies, Neoplasm / Q000276:immunology* / Q000378:metabolism; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D015179:Colorectal Neoplasms / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D007075:Immunoglobulin M / Q000276:immunology* / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D007128:Immunoglobulin Fragments; D007075:Immunoglobulin M