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Double Diffusion test in gels (Ouchterlony)

Double Diffusion test in gels (Ouchterlony)

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Published by: Leidy Yudith Angarita Bautista on Jun 10, 2008
Copyright:Attribution Non-commercial

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02/18/2013

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TECHNIQUES IN PLANT VIROLOGYCIP Training Manual2.3 DETECTION/Serology
Section 2.3.4 
Double 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
 
P.V.
• 
Sec 2.3.4 – 99
 
 
Page 2 -
 
INTERNATIONAL POTATO CENTER
Sodium azide (NaN
3
)
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 cm
2
)
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 mixturePut agarose and water in an Erlenmeyer flask stopped with cotton. Heatthe mixture in a water bath until the agarose dissolves. Cool toapproximately 40
o
C. 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 preparationGrind 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 antiserumSaline solutionPrepare 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 NaN
3
+100 ml distilled water0.85 g NaCl100 ml distilled waterMix well

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