Tandem purification of mouse IgM monoclonal antibodies produced in vitro using anion-exchange and gel fast protein liquid chromatography.
A tandem chromatographic procedure was used to isolate rapidly mouse IgM monoclonal antibodies produced by cultivation of hybridomas in vitro. Hybridoma culture supernatants containing mouse IgM monoclonal antibodies were first chromatographed on an anion-exchange Mono Q column connected to a fast protein liquid chromatography system. This anion-exchange step offers the advantage of obtaining IgM antibodies in a concentrated form. The IgM-rich fractions from the Mono Q column were then injected on a gel filtration Superose 6 column equilibrated with a low-ionic strength buffer and eluted with a high-ionic strength buffer. Assessment of the purity of isolated IgM monoclonal antibodies was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis together with a Coomassie Brillant Blue R 250 staining technique. Assessment of the immunoreactivity of isolated IgM monoclonal antibodies was performed by an enzyme linked immunosorbent assay using a solid phase adsorbed antigen against which IgM monoclonal antibodies were directed. The chromatographic procedures described allows the rapid isolation of mouse IgM monoclonal antibodies produced in vitro at a high degree of purity and in an immunoreactive state.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas; D007075:Immunoglobulin M / Q000302:isolation & purification*; D051379:Mice; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D012967:Sodium Dodecyl Sulfate