Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates.
A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D-galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies.
D000373:Agglutinins / Q000302:isolation & purification; D000918:Antibody Specificity; D002241:Carbohydrates / Q000276:immunology*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology*; D002846:Chromatography, Affinity / Q000379:methods*; D003601:Cytotoxicity Tests, Immunologic; D006023:Glycoproteins / Q000276:immunology; D007074:Immunoglobulin G / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000302:isolation & purification; D007136:Immunoglobulins / Q000302:isolation & purification*
D000373:Agglutinins; D002241:Carbohydrates; D006023:Glycoproteins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007136:Immunoglobulins