OUCHTERLONY DOUBLE IMMUNODIFFUSION TECHNIQUE.pptx

The Ouchterlony double diffusion (ODD) 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. This method has been widely used for detection and qualitative diagnostic procedures.

1. OUCHTERLONY DOUBLE IMMUNODIFFUSION TECHNIQUE PRESENTED BY, BP241502 KESAVARDHINI K 1 ST M.SC MICROBIOLOGY

2. INTRODUCTION Immuno -diffusion is a technique for the detection or measurement of antibodies and antigens by their precipitation which involves diffusion through a substance such as agar or gel agarose . Simply, it denotes precipitation in gel. Interaction between antigen and antibody at the molecular level

3. Immunodiffusion reactions classified based on the Number of reactants diffusing (Single diffusion/Double diffusion) Direction of diffusion (One dimension/Two dimension)  They thus may be of the following types: Single diffusion in one dimension Single diffusion in two dimensions Double diffusion in one dimension Double diffusion in two dimensions

4. Objectives The Ouchterlony double immunodiffusion test may be carried out with one or more of the following objectives: To detect antigen-antibody complexes. Describe the circumstances under which antigen-antibody complexes precipitate out. Detect the presence of an antigen-specific antibody. To test the similarity between antigens.

5. Materials Required Antigen solution Test serum Assay buffer Glass plate Agarose Micropipette and Tip Distilled water Gel puncture pattern Petriplate

6. Procedure : Dissolve 100 mg of agarose in 10 ml of the buffer by boiling to completely dissolve the agarose . Cool solution to 55 °C and pour agarose solution to a depth of 1 – 2 mm on a clean glass plate ( petri dish or rectangular plate) placed on a horizontal surface. Allow the gel to set for 30 minutes. Wells are punched into the gel using a gel borer corresponding to the marks on the template if used. Fill wells with solutions of antigen and antiserum (of same or different dilutions).Antiserum is usually placed in the central well and different antigens are added to the wells surrounding the center well. Incubate the glass plate in a moist chamber overnight at 37 °C.

7. Results : The presence of an opaque precipitant line between the antiserum and antigen wells indicates antigen-antibody interaction. Absence of precipitant line suggests the absence of reaction. When more than one well is used there are many possible outcomes based on the reactivity of the antigen and antibody selected.

8. The results may be either of the following: A full identity (i.e. a continuous line):  Line of precipitation at their junction forming an arc represents serologic identity or the presence of a common epitope in antigens. Non-identity (i.e. the two lines cross completely):  A pattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens are unrelated and share no common epitopes . Partial identity (i.e. a continuous line with a spur at one end):  The two antigens share a common epitope , but some antibody molecules are not captured by the antigen . The pattern of the lines that form can determine whether the antigens are the same.

9. Applications It is useful for the analysis of antigens and antibodies. It is used in the detection, identification, and quantification of antibodies and antigens, such as immunoglobulins and extractable nuclear antigens. Agar gel immunodiffusions are used as serologic tests that historically have been reported to identify antibodies to various pathogenic organisms such as  Blastomyces . Demonstration of antibodies in serodiagnosis of smallpox. Identification of fungal antigens.