Inhibition of mannosidase in hybridomas yields monoclonal antibodies with greater capacity for carbohydrate labeling.
Labeling an antibody site specifically through its carbohydrate residues preserves more of its antigen-binding activity than does labeling through protein moieties. To boost the amount of immunoglobulin G carbohydrate capable of being labeled, we treated hybridoma cells with a mannosidase inhibitor, deoxymannojirimycin (dMM). Polyacrylamide gel electrophoresis showed formation of a glycoprotein with high mannose content, in that endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96) could digest the antibody from the dMM-treated cells, but not from control cultures. Carbohydrate analysis confirmed this conclusion, indicating that the antibody from the dMM-treated cells had twice as much mannose as did the control antibody. The glucosamine content of the treated-cells' antibodies was half that of the control, and no additional carbohydrate residues were detectable in the antibodies secreted by the dMM-treated cells. We conjugated both the dMM and control antibodies through their carbohydrate to a chelator. In labeling, the dMM antibody conjugate incorporated approximately threefold as much 111In isotope as the control conjugate. The two labeled antibodies were injected into mice and showed similar organ distributions.
Simonson
R B
RB
Cytogen Corp., Princeton, NJ 08540.
Ultee
M E
ME
Long
C G
CG
Gillette
R W
RW
McKearn
T J
TJ
Rodwell
J D
JD
D017485:1-Deoxynojirimycin; D000117:Acetylglucosamine / Q000032:analysis; D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002241:Carbohydrates / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005944:Glucosamine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006825:Hybridomas / Q000201:enzymology* / Q000276:immunology; D007205:Indium Radioisotopes; D008358:Mannose / Q000032:analysis; D008361:Mannosidases / Q000037:antagonists & inhibitors*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D009374:Neoplasms, Experimental / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D000941:Antigens; D002241:Carbohydrates; D007205:Indium Radioisotopes; D017485:1-Deoxynojirimycin; D008361:Mannosidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D005944:Glucosamine; D008358:Mannose; D000117:Acetylglucosamine