ANTIGEN ANTIBODY TITRATION.immunology experiment

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OUCHTERLONY  DOUBLE  DIFFUSION

[ANTIGEN ANTIBODY TITRATION]

OBJECTIVE

       To learn the technique of ouchterlong double diffusion.

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. 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.

      

KIT DISCRIPTION

        In this kit, an antigen and a test antiserum are supplied. Students will perform the ODD assay to determine the titre of the antiserum, by using serially diluted antiserum against the antigen and observing the formation of precipitin line. The highest dilution at which the precipitin line is formed is considered as the titre value of the antiserum.

      

              

               KT09S : The kit is designed to carry out 15 ODD experiments.

 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 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.

MATERIALS	QUANTITY	STORE
	KT09S (15 EXPTS)
Agarose 2 g	2 gm	4°C
10X Assay buffer	20 ml	4°C
Antigen	0.2 ml	4°C
Test antiserum	0.5 ml	4°C
Glass plate	5 Nos	4°C
Gel punch with syringe	I Nos	4°C
Template	2 Nos	4°C

 MATERIALS REQUIRED

Glassware : Conical flask, Measuring cylinder, Test tubes.

Reagent : Distilled water.

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

Note:

Ø  Read the entire procedure before starting the experiment.

Ø  Dilute the required amount of 10X assay buffer to 1X with distilled water.

Ø  Reconstitute the antigen vial with 0.2 ml of 1X assay buffer. Mix well, store at 4°C and use  within 3 months.

Ø  Reconstitute the antiserum vial with 0.5 ml of 1X assay buffer. Mix well, store at 4°C and us  within 3 months.

Ø  Wipe the glass plates with cotton, make it grease free for even spreading of agarose.

Ø  Cut the wells neatly without rugged margins.

Ø  Ensure that the moist chamber has enough wet cotton to keep the atmosphere humid.

PROTOCOL

Ø  Boil to dissolve 100 mg of agarose in 10 ml of 1X assay buffer. Cool to 55°C.

Ø  Pour 5 ml of the gel solution onto a clean glass plate placed on a horizontal surface. Allow the  gel to set, it  takes approximately 20 - 30 minutes.

Ø  Place the gel plate on the template provided. Punch wells in the gel with the help of a gel

punch corresponding to the markings on the template. Use gentle suction to avoid forming  rugged wells.

Ø  Serially dilute the test antiserum up to 1:32 dilution as follows:

o   Take 5 μl of 1X assay buffer in each of the five vials.

o   Add 5 μl of test antiserum into the first vial and mix well. The dilution of antiserum in this vial is 1:2.

o   Transfer 5 μl of 1:2 diluted antiserum from the first vial into the second vial. The dilution in this vial is 1:4.

o   Repeat the dilutions up to fifth vial

  

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

Ø  Place the plate in a moist chamber and incubate at room temperature, overnight.

Ø  After incubation, observe for opaque precipitin line between the antigen and antisera wells.

Ø  Note down the highest dilution at which the precipitin line is formed. This is the titre value of the antiserum.

OBSERVATION

We can see the presence of precipitin line in 1:16 dilution. So the titre value is 1:16.

 

                                                                                                                        

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