Ch14

G.P. Martelli

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.

MICROPRECIPITIN TEST

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.

Materials

glass or plastic Petri dishes 100 mm in diameter. If glass plates are used, their bottoms
must be given a coat of silicone paste to be made hydrophobic
micropipettes or Pasteur pipettes
paraffin oil
reactants: antiserum and virus-containing plant extract (usually clarified plant sap)

Procedure

Put a series of drops of the infected extract in the bottom of the Petri dish.
Add to each drop an equal amount of antiserum at increasing dilution.
Mix drops thoroughly.
Cover with paraffin oil.
Incubate for 1 hour at 37°C.
Read reaction under a dissecting microscope. Drops in which a precipitate has formed are scored positive.

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)

glass or plastic Petri dishes 55 or 100 mm in diameter. Glass plates need to be made hydrophobic with a coat of silicone paste
Pasteur pipettes
gel cutters (cork borers) or preformed templates for well patterns
gel medium composed of 7 to 15 g bacteriological agar or agarose, to be dissolved by heating in 1 litre of 0.2 M
buffer (phosphate, citrate) or saline (0.85 percent sodium chloride), with the addition of a preservative (e.g. 0.02 percent sodium azide)
reactants: antiserum and infected crude or clarified plant sap

Procedure (Figure 261 )

Place Petri dishes on a flat surface. Pour warm medium to form a layer about 3 mm thick and leave to solidify at room temperature. Plates can be stored in a plastic bag in a refrigerator (4°C) until needed.
Choose the desired well pattern. A very common pattern consists of a central well 2 to 3 mm in diameter for the antiserum, surrounded by eight peripheral wells 4 mm in diameter for the antigens, each 4 to 5 mm from the edge of the central well.
Cut cylinders of agar with the gel cutter, following the chosen pattern, and remove agar plugs with a needle or by suction.
Place reactants in the wells.
Cover Petri dish and leave to stand at room temperature or at 4°C until precipitin lines are formed. Although the reaction may begin to appear as soon as 3 hours after filling the wells with reactants, precipitin lines are fully developed after 12 to 14 hours.

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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