Part III: Laboratory methods for detection and identification of infectious agents
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Enzyme-linked immunosorbent assay (ELISA)
Immunosorbent electron microscopy (ISEM) and antibody coating
Precipitation or precipitin reactions derive their name from the visible precipitate formed when adequate quantities of antigen and antibodies are allowed to interact.
When two reactants (antigen and antibody) capable of recognizing each other come into contact, a complex develops in the form of a lattice of antigen and antibody molecules. This results from the establishment of bonds between epitopes (i.e. antigenic determinants at the surface of the antigen molecule) and the corresponding antigen-combining sites located at the extremity of the arms of the antibody molecule (IgG, IgM). The antigen-antibody complex becomes insoluble and precipitates.
Precipitin reactions can take place in a liquid medium (liquid precipitin) when the reactants are mixed together or in an agarized medium (immunodiffusion in gel) when originally separated reactants are allowed to diffuse into one another through the pores of the agar.
Liquid precipitin is used for viruses whose particles are too long (500 nm and above) to diffuse in agar. Gel diffusion is used for viruses with particles up to 300 nm long (i.e. Tobraviruses Tobamoviruses, Furoviruses). Viruses with longer particles can be made to react in gel diffusion, provided that their particles are previously dissociated by SDS (sodium dodecyl sulphate) or another strong denaturing agent.
The microprecipitin test is the same as tube precipitin but is more economical, as it requires less antiserum. It is also more sensitive, as small precipitates not detected by the naked eye become visible under a dissecting microscope.
IMMUNODIFFUSION (GEL DOUBLE DIFFUSION TEST)
The advantage of the gel double diffusion test is that antigens and antibodies, while diffusing into one another, form a gradient that allows the precipitin reaction to occur where the ratio between the reactants reaches the right proportion. Furthermore, since different antigens diffuse at different rates and precipitate at different sites, gel diffusion tests reveal the presence of mixed antigens and allow their separation and the assessment of relationships between them.
Materials (Figure 260)
Procedure (Figure 261 )
FIGURE 260 Materials and tools needed for gel double diffusion tests. Background: bacteriological agar, buffered solution (PO`) or physiological solution (saline), glass or plastic (55 or 100 mm diameter) Petri dishes. Foreground: Pasteur pipettes, a preservative (sodium azide), antiserum and cork borers to be used as gel cutters
FIGURE 261 Procedure for gel diffusion tests (from left to right and top to bottom): leaf samples to be tested serologically in a porcelain mortar prior to grinding; leaf tissues ground preferably without addition of extraction media; a Pasteur pipette containing crude sap to be loaded in the wells made with a cork borer in the agarized medium contained in the plastic Petri dishes
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