IMMUNODIFFUSION TECHNIQUE- OUCHTERLONY METHOD

 

 IMMUNODIFFUSION TECHNIQUE- OUCHTERLONY METHOD

 

AIM:

 

 Understand the antigen antibody interactions and familiar with different antigens nature in a mixture.

 

PRINCIPLE:

 

This method was first introduced by Orjan Ouchterlony. There are two types of antigens such as soluble antigen and surface antigen. Based on the physical and chemical nature of the antigen biological reaction agglutination and precipitation would occur. The soluble antigen and respective antibody in agarose gel well, they diffuse radially and result a precipitation band between the wells.

MATERIALS REQUIRED:

 

Antigens, antiserum, agarose, well template, well puncture, clean glass slide, petri plate,

 cotton

 PROCEDURE:

 

1.   In a beaker 10 ml of 1% agarose shall be prepared.

2.   Allow the solution to cool up to bearable heat and pour the agarose on to a clean grease free glass plate and keep it in a plane surface.

3.   Allow the gel to set for 30 minutes.

4.   A template is used to punch the well on gel and care must be taken to avoid forming of rugged well.

5.   Add 10 µl each of the antiserum and the corresponding antigens to the well

        6.   The glass plate is placed in a petriplate with moistened filter paper along with wet cotton over night at 370 C.

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

RESULT AND DISCUSSION:

 

Observe for presence of precipitin lines between antigen and antisera wells. Note the pattern of 

precipitin line observed in each case.

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.