Immunodiffusion Plates

Immunodiffusion (ID) is a classic technique for detecting antibody-antigen interactions based on the theory of double diffusion originally described by Oudin 1 and Ouchterlony. 2 Antigens and antibodies are placed into separate wells that are cut into a gel matrix and allowed to diffuse towards each other. If the reaction is positive, a precipitate forms that appears as an opaque line. The precipitation reaction occurs when the antigen and antibody concentrations are combined at near equivalent proportions.

When multivalent antigens combine with divalent antibodies in solution, three-dimensional lattices are formed that aggregate and precipitate. The amount of precipitate varies in proportion to the concentration of the antigen and antibody. At equivalent or optimal proportions almost all the antigen and antibody will precipitate. If there is an excess of antibody, the complexes formed with the antigen are insoluble. When there is an excess of antigen, the precipitate has a tendency to dissolve caused by the formation of soluble complexes.

Advantages

• Gelling agent contains precipitin brighteners for easy visualization
• Immunodiffusion plates contain diffusion enhancers to help speed the interaction process
• Excellent precipitin bands with antibodies from all species (including rabbit antibodies)
• Gels can be washed, dried and stained for a permanent record

References

1. Oudin, J.; L’analyse, (1948). Immunochemique qualitative. Methode par diffusion des antigens au sein de l’immunoserum precipitant gelose. Premiere Parte. Inst. Pasteur 75: 30-52.
2. Ouchterlony, O. (1949). Antigen-antibody reactions in gels and the practical applications of this phenomenon in laboratory diagnosis of diphtheria. Med. Diss. Stockholm.