Monoclonal anti-glycoprotein antibody that blocks cell adhesion in Polysphondylium pallidum.
Polyclonal and monoclonal antibodies were prepared against a glycoprotein (gp 64) of Polysphondylium pallidum previously shown to act as a target site of adhesion-blocking Fab prepared from antisera against whole membranes of aggregation-competent cells. The purified glycoprotein, with a nominal Mr of 64000, could be fractionated into two subspecies, gp 64I and gp 64II, with apparent Mr of 66000 and 60000, as determined in 7.5% sodium dodecyl sulfate/polyacrylamide gels. Rabbit antibodies against purified gp 64 reacted not only with the two subspecies but also with many other membrane proteins. Almost all the cross-reactivity could be abolished by absorption of the antibodies with extensively purified gp 64. All monoclonal antibodies obtained by screening with gp 64 showed similar cross-reactivity. One monoclonal antibody specifically precipitating gp 64 was selected by screening with antigen that had been pretreated with anhydrous hydrogen fluoride for removal of carbohydrates. Fab from polyclonal anti-(gp 64) sera as well as one monoclonal Fab completely blocked cell adhesion of aggregation-competent P. pallidum cells. A carbohydrate fraction prepared by treatment of gp 64 with proteases and hydrazine completely neutralized the adhesion-blocking Fab. The product of hydrazinolysis contained less than 3% of the original peptide as based on the glucosamine recovered, but the specific neutralizing activity of the carbohydrate was essentially the same as that of the glycoprotein. In conclusion, monoclonal as well as polyclonal adhesion-blocking Fab reacted with carbohydrates; gp 64 shared the relevant carbohydrate moieties with other membrane proteins.
D000268:Adhesiveness; D000818:Animals; D000908:Antibodies, Fungal / Q000302:isolation & purification*; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D002241:Carbohydrates / Q000302:isolation & purification; D055598:Chemical Phenomena; D002621:Chemistry; D005656:Fungal Proteins / Q000276:immunology*; D006023:Glycoproteins / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments; D009235:Myxomycetes / Q000276:immunology*; D011817:Rabbits
D000908:Antibodies, Fungal; D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D005656:Fungal Proteins; D006023:Glycoproteins; D007140:Immunoglobulin Fab Fragments