PLANT DISEASE DIAGNOSIS - The Epidemiology of Plant Diseases - page 13

purified from crude immune serum or antiserum humanely collected from the blood
of living animals that have been injected with doses of antigenic material from the
pathogen; usually this must be done only in registered premises.
In this way, a variety of antigenic substances have been used as immunogens,
including peptides (e.g. Robard, 1987; Fox et al., 1989; Martin et al., 1992a) and
proteins (e.g. Benhamou et al., 1984; Groves et al., 1988). The sources of these
immunogens have included purified material, electrophoresis bands, crude mycelial
culture extracts (Ouellette and Benhamou, 1987) and freeze dried mycelium (Fox
and Hahne, 1988). Since many antigens in plants are shared by microorganisms,
polyclonal antibodies to fungi are often ineffective for diagnosing them in host
tissues, even when unwanted cross-reactions have been reduced by using pure
antigen. Nonetheless, antisera to some fungi have been used successfully (e.g.
Mohan, 1989).
One possible way around the problems caused by the mixed population of
antibodies present in antisera is to produce a uniform population of a single
monoclonal antibody (MCAs, mAbs or MAbs) that only reacts with a particular
individual antigenic determinant (epitope). Such a homogeneous population can be
produced because it is possible to select a particular stable hybridoma created by the
hybridization between the B lymphocyte spleen cell responsible for the production
of that particular antibody and a myeloma cell (Kohler and Milstein, 1975).
However, only a few per cent (at most) of these hybridomas usually produce a
useful antibody (Lin and Chen, 1985; Ouellette and Benhamou, 1987). Once a
hybridoma has been selected by monoclonal antibody enzyme-linked immuno-
sorbent assay (MCA, mAb or MAb ELISA) of its particular antibody, it can be
cloned in vitro .
Hybridomas are cheap to maintain and can be used to produce monoclonal
antibodies when required, so an ample and theoretically unending supply of a highly
specific monoclonal antibody can continue to be provided routinely by resuscitating
and growing subcultures of clones of a hybridoma stored at low temperature. This
technique has allowed adequate supplies of antibodies specific to immunological
markers to a particular strain, species, genus or any other taxon to be provided suffi-
ciently economically for commercial kits as well as research. Nonetheless, it should
also be understood that coupled with the low probability of obtaining an appropriate
antibody, it might not form precipitin bands in double gel diffusion tests or readily
conjugate to enzymes. The probability of success can be anticipated by obtaining
small amounts of polyclonal antiserum from some drops of blood squeezed from a
small nick in the tail of the immunized animal to confirm the presence of general
activity by carrying out polyclonal antibody ELISA. The veracity of this prediction
must be treated with caution since an intense colour reaction in indirect polyclonal
antibody ELISA may be largely due to non-specific binding. A hybridoma that
produces an antibody unique to any qualitative or quantitative immunogenic
difference can be selected by screening the latter for specificity, thus obviating the
need for preliminary purification of the immunogen (Goding, 1983).
Many ELISA protocols have been devised for particular purposes (Fox, 1993a).
In practice, polyclonal antibody ELISA is still predominant in these. Most MCA
ELISA have been used in academic research, for example, for taxonomy (Benhamou