TECHNIQUES IN PLANT VIROLOGYCIP Training Manual2.3 DETECTION/Serology

Section 2.3.4Double Diffusion Test InGels (Ouchterlony)

The gel double diffusion test (Ouchterlony) uses agar gels in whichpreparations of viral antigens and specific antibodies are placed inadjacent wells in a plate containing agar gel. The serological relationshipbetween viruses can be more precisely determined with this than with anyother method, except immuno-electrophoresis. In Ouchterlony, thenumber of precipitation lines between reacting substances in opposingwells corresponds to the number of antigenic components in a givenantigen-antibody system. However, this is one of the least sensitiveserological tests because the formation of precipitation lines depends onhigh and equivalent concentrations of specific antigens and antibodies.

Materials and Methods

The following materials are necessary:

− Agarose

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− Sodium azide (NaN3)− Sodium chloride (NaCl)− Distilled water− Plastic petri dishes (100 x 15 mm)− Cork borer punch (5 mm diameter)− Test tubes− Rubber bulb− Pasteur pipettes− Gauze pads (2.5 cm2)− Felt tipped pen or crayon− Erlenmeyer flasks (125 ml and 250 ml)− Test tube (100 ml)− Burner− Vacuum pump− Kitasato flask (125 ml)

1. Gel preparation

Gel mixture

Put agarose and water in an Erlenmeyer flask stopped with cotton. Heatthe mixture in a water bath until the agarose dissolves. Cool toapproximately 40oC. Add sodium azide. Mix carefully to prevent theformation of air bubbles. Dispense 15 ml of the solution on each petridish and partially cover until gel solidifies. Remove condensed water fromlids.

2. Sample preparation

Grind leaves in a mortar and using the tip of a pestle push sap to oneside. Cover macerated tissue with a damp gauze pad. Remove the juicewith a Pasteur pipette. Transfer to a labeled test tube.

3. Preparation of antiserum

Saline solution

Prepare the recommended antiserum dilution in saline solution. If theoptimum dilution is not known, try several titers (dilutions at 1/2, 1/4, 1/16,1/32, etc.).

4. Preparation of the test plate

a) Use cork borer punch to make wells in the gel according to adesired pattern. A 5-mm cork borer is normally used with 3 mmspacing between wells. The agarose disks are removed by using avacuum system consisting of a Pasteur pipette connected through

0.8 g agarose0.2 g NaN3 +100 ml distilled water

0.85 g NaCl100 ml distilled waterMix well

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a hose to a Kitasato flask, and a vacuum pump. If this equipmentis not available, use a dissection needle.

b) First, place the samples in the peripheral wells. Next, place theantiserum in the center well. Use a clean Pasteur pipette for eachsample and antisera.

5. Incubation of the test plate

Place plates in a wet chamber. Use a hermetically sealed plasticcontainer with a piece of damp paper inside and a support to prevent theplace from getting wet.

6. Readings

The first reading is made after 2 hours and the second reading at 18hours. For easier reading, use a dark box consisting of a wooden supportwith an opening at the top big enough to support the dish firmly in place.Place a fluorescent light inside the box.

Figure 1. M: test sample; V: positive control; As-V: antiserum against virus.A: Reaction of antiserum and the corresponding control but notwith sample. B: Identity reaction between sample and control. C:Partial identity reaction. D: Non-identity reaction.

Recommended LiteratureBall, E. 1974. Serological Test for the Identification of Plant Viruses.

Published by the American Phytopathological Society Inc. PlantVirology Committee, USA.