J. Biol. Chem., Vol. 255, Issue 24, 11914-11921, Dec, 1980
A membrane glycoprotein from human neuroblastoma cells isolated with the use of a monoclonal antibody
M Momoi, RH Kennett and MC Glick
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.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
