A membrane glycoprotein from human neuroblastoma cells isolated with the use of a monoclonal antibody.
A membrane glycoprotein, Mr = 20,000, has been purified from human neuroblastoma cells (IMR-5) with the use of monoclonal antibody selected for binding capacity to human neuroblastoma cell lines. The antigen was extracted with 0.5% Nonidet P-40 from cells metabolically labeled with L-[3H]fucose or D-[3H]glucosamine. A double antibody affinity column was used to purify the membrane glycoprotein. Goat anti-mouse IgM was coupled to cyanogen bromide-activated Sepharose 4B. The absorption of the monoclonal antibody contained in ascites fluid completed the affinity column. Appropriate controls of similar material from other cell types and another monoclonal antibody demonstrated the specificity of the affinity column. Glycopeptides from the surface of human neuroblastoma cells, IMR-5 and CHP-134, had antigenic activity, as radioactive pronase-digested material bound to the affinity column and inhibited complement-mediated cytolysis. Glycolipids extracted from the cells had no antigenic activity. It was concluded that the carbohydrate residues of the glycoprotein conferred the antigenic specificity. Three methods were devised to aid in detection and purification of the antigen. These were: 1) an assay for the detection of complement-mediated cytolysis by measuring the enzyme creatine phosphokinase in the nonlysed target cells; 2) precipitation of the antigen . antibody complex with 4% polyethylene glycol; and 3) removal of the antibody by a wheat germ agglutinin-agarose column.
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface / Q000302:isolation & purification; D002460:Cell Line; D002846:Chromatography, Affinity; D002999:Clone Cells; D000939:Epitopes; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D009447:Neuroblastoma / Q000032:analysis*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D000939:Epitopes; D006023:Glycoproteins; D008565:Membrane Proteins