Production of monoclonal antibodies by tobacco hairy roots

Abstract

Hairy roots of tobacco (Nicotiana tabacum) were used to produce full-length murine lgG(1) monoclonal antibody. The presence of heavy (gamma) and light (kappa) chains and fully assembled antibody was verified by Western 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 demonstrated in bacterial aggregation assays which confirmed bivalent antigen-binding capacity. Eight antibody-producing hairy root clones retained their ability to produce mouse immunoglobulin over a period of 19 months after transformation with Agrobacterium rhizogenes. For hairy roots grown in Gamborg's B5 medium, the maximum level of assembled antibody after 21-day culture in shake flasks was 18 mg L(-1) or 1.8% total soluble protein; up to 14% of the antibody was secreted into the medium. Antibody production by transgenic hairy roots had a negligible effect on growth compared with hairy roots of wild-type tobacco. Antibody accumulation was growth associated with constant specific accumulation rate at the beginning of the culture; however, degradation of antibody was significant after 14 days and the amount of assembled antibody declined. Unlike hybridoma cultures, the time course of antibody accumulation by hairy roots showed a distinctive maximum very soon after the end of exponential growth. Total antibody levels were increased by addition of nitrate, polyvinylpyrrolidone, or gelatin to the medium. Polyvinylpyrrolidone and gelatin also markedly improved extracellular antibody concentrations; with these treatments, up to 43% of the antibody present was secreted into the medium. Antibody production was tested using hairy roots grown in an air-driven bioreactor. The intracellular antibody content after 30-day bioreactor culture was similar to that measured in shake flasks; however, the final extracellular antibody level was 1.7 times higher than the maximum measured in shake flasks.