Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody.
In vitro and in vivo experiments to explain the function of natural polyreactive antibodies, usually of the IgM isotype, require large amounts of purified antibodies. We have developed a two-step purification procedure using a human natural polyreactive monoclonal IgM antibody (CB03). This combines hydrophobic interaction chromatography on phenyl-Superose and gel filtration over Superose 12 and readily permits scaling-up to isolate mg to g amounts of antibody. Retention of the CB03 antibody during gel filtration by precipitation and interaction with the gel matrix was overcome by the addition of 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. The yield of purified antibody was 34% and Fab fragments were obtained from the purified CB03 antibody by hot tryptic digestion (yield, 68% of theoretical amount). In an enzyme-linked immunosorbent assay, Fab and complete antibody had similar reaction patterns with different antigens.
Roggenbuck
D
D
Department of Medical Immunology, Medical School (Charité), Humboldt University of Berlin, Germany.
Marx
U
U
Kiessig
S T
ST
Schoenherr
G
G
Jahn
S
S
Porstmann
T
T
D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D002845:Chromatography; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000276:immunology* / Q000302:isolation & purification; D008214:Lymphocytes / Q000276:immunology; D013154:Spleen / Q000166:cytology; D013921:Thrombocytopenia / Q000276:immunology
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007075:Immunoglobulin M