Hairy roots of tobacco (Nicotiana tabacum) used in clinical diagnosis, in commercial analyses, as were used to produce full-length murine IgG 1 monoclonal therapeutic agents, and as binding moieties in affinity antibody. The presence of heavy (β₯) and light () chains separations. Large-scale production of monoclonal antiand fully assembled antibody was verified by Western bodies is carried out using hybridoma cells formed by blot analysis of root extracts. Antibody levels in the biofusion of animal lymphocytes with myeloma cells. One mass and medium were quantified by ELISA based on detection of β₯complexes. Antibody produced by hairy of the features of these production systems is the reroots was fully functional as demonstrated in bacterial quirement for nutrient media containing complex and aggregation assays which confirmed bivalent antigenexpensive protein additives which raise the cost of the binding capacity. Eight antibody-producing hairy root raw materials and interfere with purification of antibody clones retained their ability to produce mouse immunofrom the culture broth.
globulin over a period of 19 months after transformation with Agrobacterium rhizogenes. For hairy roots grown in
Several heterologous systems such as nonlymphoid
Gamborg's B5 medium, the maximum level of assembled mammalian cells (Piccioli et al., 1991;Weidle et al., 1987), antibody after 21-day culture in shake flasks was 18 mg insect cells (Hasemann and Capra, 1990), yeast (Horwitz L Οͺ1 or 1.8% total soluble protein; up to 14% of the antibody et al., 1988), and bacteria (Better et al., 1988; Carter et was secreted into the medium. Antibody production by al., 1992; Skerra and Plu Β¨ckthun, 1988) have been develtransgenic hairy roots had a negligible effect on growth compared with hairy roots of wild-type tobacco. Antibody oped in recent years for expression of antibody proteins. accumulation was growth associated with constant spe-Despite numerous investigations, complete antibodies cific accumulation rate at the beginning of the culture;
containing both constant and variable regions have so far however, degradation of antibody was significant after only been assembled and secreted from eukaryotic cells; 14 days and the amount of assembled antibody declined.
proper folding of protein molecules or fragments does
Unlike hybridoma cultures, the time course of antibody accumulation by hairy roots showed a distinctive maxinot occur in bacteria such as Escherichia coli as intracellumum very soon after the end of exponential growth. Total lar conditions are insufficiently oxidizing for formation antibody levels were increased by addition of nitrate, of all the disulfide bonds required (Carter et al., 1992).
polyvinylpyrrolidone, or gelatin to the medium. Polyvinyl-Recombinant antibodies from eukaryotes typically dispyrrolidone and gelatin also markedly improved extracellular antibody concentrations; with these treatments, up play good antigen-binding activity; however, because of to 43% of the antibody present was secreted into the the different glycosylation patterns produced in heterolmedium. Antibody production was tested using hairy ogous systems compared with mammalian cells, they do roots grown in an air-driven bioreactor. The intracellular not always retain antibody effector functions and may antibody content after 30-day bioreactor culture was simielicit immunogenic reactions in therapy. Strategies to lar to that measured in shake flasks; however, the final extracellular antibody level was 1.7 times higher than the overcome problems with immunogenicity and the poor maximum measured in shake flasks.