IMMUNOLOGY

Experiment 3

Aim of the Experiment

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 in 1948.

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.

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 being 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 antigen 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).

Fig 1: Antigen- Antibody Patterns formed in Ouchterlony Double Diffusion

Procedure

1. Prepare 10 ml of 1% agarose.

2. Cool the solution to 55-60^oC 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 μl 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 37^oC.

7. After incubation, observe for opaque precipitin lines between the antigen and antiserum 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.

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. If pattern X or pattern of identity is observed between the antigens and the antiserum, it indicates that the antigens are immunologically identical.

2. If pattern Y or pattern of partial identity is observed, it indicates that the antigens are partially similar or cross-reactive.

3. If 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.

Reference Material

1. Himedia Protocol

2. Amrita Lab animation

3. Double Immunodiffusion

Questions

1. What are the different antigen-antibody patterns observed in a typical Ouchterlony double diffusion experiment?

2. How Ouchterlony double diffusion helpful in evolutionary studies?

3. Why do polyclonal antisera produce a better precipitin reaction?

4. Both IgM and IgG antibodies can be used in precipitation reactions. However, one of these immunoglobulin classes will form precipitates at much lower concentrations than the other. Which class is this, and why is it so much more efficient in this regard?