Efficient purification of mouse anti-FGF receptor IgM monoclonal antibody by magnetic beads.
Affinity chromatography has been widely used for the purification of monoclonal antibodies (MAb). Traditionally, activated agarose beads conjugated with specific antisera have been used as a solid support in chromatographic protein purification. Magnetic beads conjugated with various antibodies have recently become an alternative method for the isolation of diverse proteins, nucleic acids, and cell types. In this study, murine anti-fibroblast growth factor receptor 1 (FGFR1) immunoglobulin M (IgM) was isolated from protein solutions to compare immunoaffinity column chromatography and magnetic bead IgM purification methods. Using immobilized rat anti-mouse IgM MAb, an UltraLink 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC)/diaminodipropylamine (DADPA) immunoaffinity column and polystyrene-coated magnetic beads were used for the purification of mouse IgM from bovine serum albumin/phosphate-buffered saline (BSA/PBS) as well as from crude ascites. Protein quantitation and percent IgM yield were determined by reducing SDS-PAGE electrophoresis followed by silver staining, then IgM and protein contaminants were quantitated using densitometry analysis. IgM anti-FGFR1 binding specificity and immunologic activity were determined by enzyme-linked immunosorbant assay (ELISA). This study demonstrates that magnetic bead isolation of IgM from ascites is more effective than traditional affinity chromatography purification as determined by greater IgM yield, purity, and immunologic activity.
Quitadamo
I J
IJ
Department of Genetics and Cell Biology, Washington State University, Pullman 99164-4234, USA.
Schelling
M E
ME
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000918:Antibody Specificity; D001201:Ascites / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006825:Hybridomas / Q000276:immunology; D007075:Immunoglobulin M / Q000302:isolation & purification*; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017468:Receptors, Fibroblast Growth Factor / Q000032:analysis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D017468:Receptors, Fibroblast Growth Factor