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.