Ouchterlony Double Diffusion Teaching Kit  

Cat# BB-ITK020 (10 Reactions)

Objective: To study the reaction pattern of an antigen with a set of antibodies by Ouchterlony Double Diffusion method.

Principle: Interaction between antigen (Ag) and antibody (Ab) at the molecular level forms the basis for several techniques that are useful in modern day scientific studies and in routine clinical diagnosis. These techniques are either based on the use of labeled reagents, a tracer or immunoprecipitation. When soluble antigen and antibody samples are placed in adjacent wells in agarose gel, they diffuse radially into the agarose gel and set up two opposing concentration gradients between the wells. Once the gradients reach to an optimal proportion, interactions of the corresponding molecules occur and a line of precipitation will form. Using such a technique, the antigenic relationship between two antigens can be analyzed. Ouchterlony double diffusion (ODD) or double immunodiffusion technique is one of the simplest techniques extensively used to check antisera for the presence of antibodies for a particular Ag and to determine its titre.

In ODD assays, solutions of Ag and Ab are placed in adjacent wells cut in agarose gel and are allowed to diffuse radially. The Ag and Ab concentrations are relatively higher near their respective wells. As they diffuse farther from the wells, their concentration decreases. An antigen will react with its specific antibody to form an Ag-Ab complex. At one point their concentrations become equivalent and the Ag-Ab complex precipitates to form a precipitin line.

Duration of experiment: Experiment is carried out over a span of 2 days, approximate time taken on each day is indicated below:

Day 1: 1 hour (Preparation of gel & loading of antigen and antiserum)

Day 2: 20 minutes (Observation and Interpretation)

Materials Required:

Glassware: Conical flask, Measuring cylinder, Test tubes.

Reagent: Distilled water.

Other Requirements: Micropipette, Tips, Moist chamber (box with wet cotton).

Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival.

Note: Dilute the required amount of 10X assay buffer to 1X with distilled water. Wipe the glass plates with cotton, make it grease free for even spreading of agarose.

Procedure:

1. Boil to dissolve 60mg of agarose in 6ml of 1X assay buffer. Cool down to 55°C.
2. Pour 6ml of the gel solution onto a clean glass plate placed on a horizontal surface. Allow the gel to solidify; it takes approximately 20-30 minutes.
3. Place the gel plate on the template provided. Punch wells in the gel with the help of a gel puncher corresponding to the markings on the template. Use gentle suction to avoid forming rugged wells.
4. Serially dilute the test antiserum up to 1:32 dilution as follows

• Take 20μl of 1X assay buffer in each of the five vials.
• Add 20μl of test antiserum into the first vial and mix well. The dilution of antiserum in this vial is 1:2.
• Transfer 20μl of 1:2 diluted antiserum from the first vial into the second vial. The dilution in this vial is 1:4.
• Repeat the dilutions up to fifth vial as shown in figure below

5. Add 20 μl of the antigen to the center well and 20 μl each of neat (undiluted), 1:2, 1:4, 1:8, 1:16, 1:32 dilutions of antiserum into the surrounding wells as shown in figure below.

Fig 3: Pattern of addition of antigen and antiserum to the wells

6. Place the plate in a moist chamber and incubate at room temperature, overnight.
7. After incubation, observe for opaque precipitin line between the antigen and antisera wells.
8. Note down the highest dilution at which the precipitin line is formed. This is the titre value of the antiserum. A typical ODD pattern of high titre antisera is given below.

Antisera dilutions in wells 1-6:

      The center well contains antigen

Titre Value:     A: 1:8      B: 1:4

Result:

Observe for presence of precipitin lines and report the titer value of the test antiserum.

   Adapted from internet