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Clinical Chemistry 34: 1713-1716, 1988;
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Clinical Chemistry, Vol 34, 1713-1716, Copyright © 1988 by American Association for Clinical Chemistry

Inhibition of mannosidase in hybridomas yields monoclonal antibodies with greater capacity for carbohydrate labeling

RB Simonson, ME Ultee, CG Long, RW Gillette, TJ McKearn and JD Rodwell
Cytogen Corp., Princeton, NJ 08540.

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.




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Copyright © 1988 by the American Association for Clinical Chemistry.