PRODUCTION OF MONOCLONAL-ANTIBODIES BY TOBACCO HAIRY ROOTS

Hairy roots of tobacco (Nicotiana tabacum) were used to produce full-l ength murine IgG, monoclonal antibody. The presence of heavy (gamma) a nd light (kappa) chains and fully assembled antibody was verified by W estern blot analysis of root extracts. Antibody levels;in the biomass and medium were quantified by ELISA based on detection of gamma-kappa complexes. Antibody produced by hairy roots was fully functional as de monstrated in bacterial aggregation assays which confirmed bivalent an tigen-binding capacity. Eight antibody-producing hairy root clones ret ained their ability to produce mouse immunoglobulin over a period of 1 9 months after transformation with Agrobacterium rhizogenes. For hairy roots grown in Gamborg's B5 medium, the maximum level of assembled an tibody after 21-day culture in shake flasks was 18 mg L-1 or 1.8% tota l soluble protein; up to 14% of the antibody was secreted into the med ium. Antibody production by transgenic hairy roots had a negligible ef fect on growth compared with hairy roots of wild-type tobacco. Antibod y accumulation was growth associated with constant specific accumulati on rate at the beginning of the culture; however, degradation of antib ody was significant after 14 days and the amount of assembled antibody declined. Unlike hybridoma cultures, the time course of antibody accu mulation by hairy roots showed a distinctive maximum very soon after t he end of exponential growth. Total antibody levels were increased by addition of nitrate, polyvinylpyrrolidone, or gelatin to the medium. P olyvinylpyrrolidone and gelatin also markedly improved extracellular a ntibody concentrations; with these treatments, up to 43% of the antibo dy present was secreted into the medium. Antibody production was teste d using hairy roots grown in an air-driven bioreactor. The intracellul ar antibody content after 30-day bioreactor culture was similar to tha t measured in shake flasks; however, the final extracellular antibody level was 1.7 times higher than the maximum measured in shake flasks. (C) 1997 John Wiley & Sons, Inc.