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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.
