Agriculture Reference
In-Depth Information
the wells. The highest dilution of antiserum that displays an arc represents the titre
of the antiserum. The
double diffusion exoantigen
technique is a variation of this
method in which the samples of different antigens surround a well filled with
antiserum. The antigens are deemed identical if the resultant arcs link; but if they
cross, the antigens are considered to be different. In other tests, if the antigens to be
compared are positioned parallel to the antiserum, the antigen-antibody zones of
precipitation also link if they match.
Gel electrophoresis
can be used to separate mixtures of antigens prior to
immunodiffusion. A narrow trough is cut in a thin gel parallel to an electric current
which passes close to the antigens along its length. Depending on its distinctive
charge, each antigen moves in a separate wave at a characteristic rate so the proteins
separate into bands. The current is then switched off and antiserum is added to the
trough cut in the gel so that precipitin arcs composed of complexes of antibodies and
antigens form at the individual bands of antigens in characteristic shapes and
patterns. Cross-reactions with other antigens can be reduced by initially absorbing
the antibody with the interfering antigen. The gel can also be split up after
electrophoresis so that and one half can be treated with antiserum and the other
stained to reveal the bands of antigen. Bands of pure antigen can be cut out from
such gels.
1.4.3 ELISA
Since the introduction of enzyme-linked immunosorbent assays (ELISA) for plant
virus detection (Clark and Adams, 1977), they have increasingly become the means
to rogue out infected plants from plant certification schemes. Only polyclonal anti-
serum was available at first but problems arose as carbohydrates from the host plants
can induce non-specific antibodies. These may impede ELISA tests, even when
double gel diffusion and other serological tests are largely unaffected. With viruses,
this can be partially overcome by cross-absorbing with healthy sap beforehand, but
there is no necessity for this extra stage if a specific monoclonal antibody is
substituted for the mixture of polyclonal antibodies. However, many tests still rely
on polyclonal antibodies because they give a clearer result as they can bind to
several characteristic epitopes. In conventional or 'direct' ELISA tests using 96-well
immunoplates, the end point is when the colourless substrate is converted to a
coloured product by an appropriate enzyme in proportion to the concentration of
antigen in the original sample. This obvious colour change can either be scanned
electronically with a spectrophotometer or assessed with the naked eye. The specific
antibody/antigen complex is detected by using a conjugated second antibody that
recognizes the non-specific antibody in 'indirect' ELISA. Morley and Jones (1980)
describe an interesting modification of the ELISA technique in which a poorly
antigenic microorganism can be more easily detected by incubating the substrate to
increase its fluorescence.
In addition to the relatively inconvenient conventional 96-well immunoplates,
cheaper and more portable assay systems have been developed using flexible
nitrocellulose dip-sticks surface-coated with a capture antibody. Antigens of the
