[PRACTICAL 3] MTEB2404

OUCHTERLONY DOUBLE DIFFUSION (FOR ANTIBODY

TITRATION)

Objectives

To learn the technique of ouchterlony 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.
Ouchterlony double diffusion (ODD) or 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 titer.

Procedure
1.100mg of agarose was boiled to dissolve in 10ml of 1X assay
buffer. Cool to 55°C.
2.5ml of the gel solution was poured onto a clean glass plate placed
on a horizontal surface. The gel was allowed to set it took
approximately 20-30 minutes.
3.The gel plate was placed on the template provided. Wells in the gel
was punched with the help of a gel punch corresponding to the
markings on the template. Gentle suction was used to avoid forming
rugged wells.
4.The test antiserum was serially diluted up to 1:32 dilution as
follows:
•Take 20ul of 1x assay buffer in each of the five vials.
•Add 20ul of test antiserum into the first vial and mix well. The
dilution of antiserum in this vial is 1:2.
•Transfer 20ul of 1:2 diluted antiserums from the first vial into the
second vial. The dilution in this vial is 1:4.
•The dilution was repeated up to fifth vial.
5.10ul of the antigen was added to the center well and 10pl each of
neat (undiluted), 1:2, 1:4, 1:16, 1:32 dilutions of antiserum into the
surrounding wells as shown in figure 3.
6.The plate was placed in a moist chamber and incubated at room
temperature, overnight.
7.After incubation, opaque precipitin line between the antigen and
antisera wells was observed.
8.The observation was recorded.

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1:2
Ag

Ne
at 1:4

1:3
2 1:8

1:1
6

Figure 3: Pattern of addition of antigen and antiserum to the

wells.

Result
Precipitin line was formed at the well of 1:16 antiserum.

1:2
Ag

Ne 1:4
at

1:8
1:3
2
1:1
6

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Discussion

The Ouchterlony was

a test used for detecting
antigens and specific
antibodies which it placed in
adjacent wells in a plate
that containing agar gel.
The Anti-sera will be placed
in the central well, and
antigens will be added into
the wells around the central
well. Then, the antibody
and antigen molecules will
diffuse through the agarose
lawn. When antibody meets
with its specific antigen at
their equivalent zone, the
precipitation reaction
occurs. Antibody-antigen precipitates in agarose appear as a light
white band between the antibody and the antigen wells.

The serological relationship between antigens can be more

precisely determined with this method. However, this test was less
sensitive serology because the formation of precipitation lines is
depends on high and equivalent concentrations of specific antigens
and antibodies.

The Ouchterlony test also can be used to estimate the relative

concentration of antigens. When an antigen has a relatively higher
concentration, the equivalent zone will be formed a little bit away from
the antigen well. When an antigen has a relatively lower
concentration, the equivalent zone will be formed a little bit closer the
antigen well.

There are some possible error could be encounter during this

experiment done, which are:
1. Contaminating of antibodies which may develop a precipitin
lines between antiserum wells on double diffusion.
2. Sample that contains lipimic which produces non-specific
precipitin of protein gel.
3. Hemolysed sample were also given similar characteristic to
lipimic sample.
4. Others non-antibody such as C-Reactive protein,
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polysaccharides, immunoglobulin, DNA and others.

In the cases of sensitivity, the double immunodiffusion tests

were depending on the distant between the wells and reactant of
concentration. This means, more closer wells, the smaller amount
reactant will be needed.

Furthermore the concentration, thickness and viscosity were also

affected the sensitivity of the test. However, as to prevent these
problems, serial dilution can be beneficial.

Conclusion:

The precipitin line of the sample showed at the titer of 1:16.

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