One-step procedure for the rapid isolation of mouse monoclonal antibodies and their antigen binding fragments by fast protein liquid chromatography on a mono Q anion-exchange column.
A one-step chromatographic procedure was used to isolate rapidly mouse IgG monoclonal antibodies (mAbs) (IgG1, IgG2a, IgG2b) contained in ascites fluids and Fab fragments contained in papain-treated mAb suspensions. Chromatographic separations were performed on an anion-exchange Mono Q column connected to a fast protein liquid chromatographic (FPLC) system. Detection of mAb or their antigen binding fragments (Fab) in eluted peaks was performed using sodium dodecyl sulphate-polyacrylamide gel electrophoresis together with a silver or a Coomassie Brillant Blue R 250 staining technique and solid phase radioimmunoassay with 125I-labelled sheep anti-mouse antibodies directed against total immunoglobulins. Rapid assessment of the purity of isolated mAbs and their Fab fragments was performed by gel permeation chromatography on a TSK G 3000 SW column. Mouse mAbs and their Fab fragments were rapidly isolated (25 min), in a functionally active state, to a high degree of purity on the FPLC-Mono Q system compared to the time taken by other techniques.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001666:Binding Sites, Antibody; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D051379:Mice; D011863:Radioimmunoassay; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D012967:Sodium Dodecyl Sulfate