6.
OUCHTERLONY DOUBLE IMMUNODIFFUSION TEST
Aim:
To study the reaction pattern of an antigen with a set of antibodies by Ouchterlony Double
Diffusion method.
Introduction:
Immunodiffusion in gels encompasses a variety of techniques, which are useful for the analysis
of antigens and antibodies. Gel immunodiffusion can be classified into two groups:
1. Single Immunodiffusion
2. Double Immunodiffusion
In the Ouchterlony double diffusion, both the antigen and the antibody diffuse toward each
other in a semisolid medium to a point till their optimum concentration is reached. A band of
precipitation occurs at this point. The qualitative Ouchterlony Test can simultaneously monitor
multiple Antibody-Antigen system and can be used to identify particular antigens in a
preparation. This procedure was developed by Örjan Ouchterlony.
Principle:
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. Distinct precipitation line patterns are
formed against the same anti-sera depending on whether two antigens share all
antigenic epitopes or partially share their antigenic epitopes or do not share their antigenic
epitopes at all. 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. The pattern
of lines that form can be interpreted to determine the relationship between the antigens and
antibodies.
X Y Z
Pattern of Identity Pattern of Partial Identity Pattern of Non Identity
Fig 1: Antigen- Antibody Patterns formed in Ouchterlony Double Diffusion
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Pattern of Identity: X
Pattern of identity occurs when the antigens in the two wells are identical and specific for the
antibody in the antiserum present in the third well. The concentration of the two antigens been
the same, they will diffuse at the same rate resulting in a smooth line of precipitate. The
antibodies cannot distinguish between the two antigens i.e. the two antigens are
immunologically identical as shown in Fig 1.
Pattern of Partial Identity: Y
Pattern of partial identity occurs when the antigens in the two wells share some epitopes which
are same for both, yet each of the two antigens also have unique epitopes. In this case antiserum
contains polyclonal antibodies specific for each epitope. When one of the antigens has some of
the same epitopes compared to other, the polyclonal antibody population will respond
differently to the two antigens and the precipitin line formed for each antigen will be different.
The ‘spur’ is thought to result from the determinants present in one antigen but lacking in the
other antigen (refer to Fig 1). A similar pattern of partial identity is observed if the antibodies
are cross reactive with an epitope on one of the antigen that is similar, but not identical to that
present on the other antigen.
Pattern of Non-Identity: Z
Pattern of non-identity occurs when the antigens in the two wells are totally different. They are
neither cross reactive, nor do they have any epitopes which are same. In this case the antiserum
containing the antibodies is heterogeneous as some of the antibodies react with antigen in one
well while some react with antigen present in the other well. So the two antigens are
immunologically unrelated as far as that antiserum is concerned (refer to Fig 1).
Materials required:
Sl No. Materials Remarks
1 Agarose All the materials were
2 Assay buffer provided in HiPer®
3 Antiserum X Ouchterlony Double
4 Antiserum Y Diffusion Testing Kit
5 Antiserum Z
6 Antigen X1
7 Antigen X2
8 Antigen Y1
9 Antigen Y2
10 Antigen Z1
11 Antigen Z2
12 Glass plate
13 Gel puncher
14 Template
15 Measuring cylinder
16 Beaker
17 Alcohol
18 Incubator (37⁰C)
19 Micropipettes and Tips
20 Moist chamber
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Procedure:
1. Prepare 10 ml of 1% agarose (as given in important instructions).
2. Cool the solution to 55-60oC and pour 5 ml/plate on to grease free glass plates placed on
a horizontal surface. Allow the gel to set for 30 minutes.
3. Place the glass plate on the template provided.
4. Punch wells with the help of the gel puncher corresponding to the markings on the
template. Use gentle suction to avoid forming of rugged wells.
5. Add 10 ml each of the antiserum and the corresponding antigens to the wells as shown
in fig 2.
6. Keep the glass plate in a moist chamber overnight at 37oC.
7. After incubation, observe for opaque precipitin lines between the antigen and antiserum
wells.
Fig 2: Template for addition of antiserum and antigen to their respective wells
Observation and Result:
Observe for presence of precipitin lines between antigen and antisera wells. Note the pattern of
precipitin line observed in each case.
X Y Z
Pattern of Identity Pattern of Partial Identity Pattern of Non Identity
Fig 3: Diagram showing pattern of precipitin lines
Interpretation:
When antigen and antibody meet in optimal proportions a precipitation line is formed. In
Ouchterlony Double Diffusion (Antigen Antibody Pattern), three patterns of precipitin lines can
be observed.
1. Pattern X or pattern of identity is observed between the antigens and the antiserum, it
indicates that the antigens are immunologically identical.
2. Pattern Y or pattern of partial identity is observed, it indicates that the antigens are
partially similar or cross-reactive.
3. Pattern Z or pattern of non-identity is observed, it indicates that there is no cross-
reaction between the antigens. i.e. the two antigens are immunologically unrelated.
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Observation and Result:
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