Agriculture Reference
In-Depth Information
Immunosorbent Dilution Plating.
Immunosorbent dilution plating procedure can be used to detect
bacteria. An inoculation stick is coated with antibody (Fig. 10.16). This is placed in a seed extract contain-
ing the target pathogen. Antibodies bind with the antigenic component of the target pathogen and thus select
out only the pathogen. The complex is then streaked onto a culture medium. The complex is recognized by
growth of a colony of the target pathogen.
Figure 10.16. Schematic diagram of the immunosorbent dilution plating procedure.
ELISA
. The introduction of enzyme-linked immunosorbent assay (ELISA) to plant pathology in 1976
(Voller et al., 1976) stimulated rapid advances in the use of serological assays for seedborne pathogens.
Various versions of ELISA are available, but the most common application is double-sandwich ELISA (Fig.
10.17A). In this technique a seed extract is added to wells in a polystyrene plate, already coated with the
capture antibody (Fig. 10.17B). If the target pathogen is present in the seed extract, the capture antibody
binds with the antigen. Non-bound material is washed off. The signal antibody is now added. This time the
antibody is conjugated to an enzyme, thus forming an antibody-antigen-antibody + enzyme complex. Non-
bound antibody + enzyme is washed off. A substrate with which the enzyme reacts is added and a reaction
which causes a color change is allowed to proceed for a set time period. An inhibitor is then added to stop
the reaction. The degree of color change is quantiied in a spectrophotometer. The degree of color change
relects the amount of enzyme present, which, in turn, is proportional to the amount of antigen present in
the seed tissues.
Advantages and limitations of serological methods.
Advantages of serological seed health tests are
that they can be very sensitive. They are effective in detecting seedborne bacteria, viruses, and fungi, They
also can be designed to detect strains and races of a pathogen. With diagnostic kits now available commer-
cially for ELISA and its variants, serology has become economical and practical for detection of seedborne
pathogens throughout the world. Limitations of this procedure are that viable and nonviable propagules
of the pathogen are not distinguished. This is a real problem in high-value crops such as hybrid vegetable
crops. One solution to this problem is to include a culture phase in the test protocol as in the immunosorbent
dilution plating technique described above (Fig. 10.16).
Successful serological testing relies on a source of good quality antiserum. Two types of antibodies
are available, polyclonal and monoclonal. Polyclonal antisera is drawn from the blood fraction of animals
