Inhibition of mannosidase in hybridomas yields monoclonal antibodies with greater capacity for carbohydrate labeling
Simonson, R.B.; Ultee, M.E.; Long, C.G.; Gillette, R.W.; McKearn, T.J.; Rodwell, J.D.
Clinical Chemistry 34(9): 1713-1716
1988
ISSN/ISBN: 0009-9147 PMID: 2970898 Document Number: 322489
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.