Agriculture Reference
In-Depth Information
1.4.1 Principles of immunoassay
There are numerous different diagnostic immunoassay procedures, but all ultimately
depend on the efficacy of a biochemical response of living animals, particularly
mammals. Animals manufacture antibodies that react to some of the characteristic
substances that comprise pathogens and other environmental agents that potentially
challenge their integrity. Antibodies that otherwise share a basic common structure
(two heavy and two light chains held together by disulphide bonds) differ in the
ability of their tips to bind to sites on different 'foreign' protein, glycoprotein,
lipoprotein, lipopolysaccharide and carbohydrate molecules. Provided such
antigenic substances are not already present in the body of that animal, they
stimulate its immune system to produce an assortment of specific antibodies, each of
which specifically recognizes and will link almost exclusively to its complementary
epitope on the antigen. This interaction between an antibody and an antigen depends
on an extraordinary pairing of an individual epitope of the latter in three dimensions
between the contours of the tips of the side chains of a particular antibody, since the
molecular configuration of each of these binding site regions varies subtly in both
affinity and avidity. While the overall capacity of the system is truly astounding,
nearly all of this enormous potential is normally latent. Nonetheless, once an animal
has been stimulated by inoculation, in theory a specific antibody can be generated
that possesses the ability to bind with any of a virtually unlimited variety of different
antigens. Afterwards, the immune system of the animal can produce more copies of
an antibody from 'memory'. In practice, as well as several constituents of the walls
of pathogens, the antigens recognized by antibodies include a diversity of other
innocuous substances. Unfortunately, many fungi and other microorganisms consist
of many non-specific antigenic substances that are immunodominant. As a result,
polyclonal serum may be insufficiently reliable for certain diagnoses so monoclonal
antibodies are necessary (Dewey, 1992). A uniform population of a particular
monoclonal antibody is formed by each hybridoma (Fox, 1993a). Although each
monoclonal antibody generally reacts with its own particular antigenic determinant
(epitope), this site could be restricted to a particular strain or it may be common to
an entire species, genus or even more widely (Hardham
et al.,
1985). Barring
mishaps in culture due to contamination by noxious yeasts and mycoplasmas or
unexpected thawing, a hybridoma chosen for its valuable properties can be kept
almost indefinitely at low temperature. This makes it possible to maintain a plentiful
supply of a highly specific monoclonal antibody (MCA). Monoclonal antibodies are
valuable for numerous applications such as increasing the specificity of detection of
the antigen - for example, after electrophoresis (Western blotting), and to purify yet
more antigen (affinity chromatography).
1.4.2 Classical immunoassay tests
Several immunoidentification techniques, including agglutination and precipitation
tests, have a long and successful history and have proved very useful in the
epidemiology of viruses as they are portable and the results are clearly visible to the
eye. These simple serological tests depend on two similar properties: agglutination
