Double Immunodiffusion


Assays involving the diffusion of soluble antigen and antibody include the precipitation reaction. When the concentrations reach a critical point, a visible precipitate is formed, which is made up of an insoluble complex of antigens and antibodies. In an agarose gel, precipitation reactions are the most stable. Double immunodiffusion, radial immunodiffusion, and flocculation tests are also precipitation techniques that can be used to diagnose infectious diseases.

Double Immunodiffusion

In the laboratory, double immunodiffusion or Ouchterlony gel diffusion is used to conduct antibody-antigen reactions in agarose. In this method, cylindrical holes or wells are cut out of an agarose gel and placed in a small Petri dish or glass top, spaced accordingly. A recognized crude or purified antigen extract of a microorganism is placed in one well of the agar plate for antibody detection, and a patient’s serum is placed in an adjacent well. The antigen and antibody molecules in solution diffuse into the porous agarose and out of the wells.

If antigen-specific antibody is present, the two components merge at a concentration point known as the zone of equivalence to form a visible precipitin band, or line of precipitation. The size and shape of the wells, the distance between them, the temperature, and the incubation time are all optimized. The band position is determined by the concentration and rate of diffusion of antigen and antibody. The excess amount of antibody is depicted by the band closest to the antigen. Antibodies related to autoimmune diseases such as Rheumatoid Arthritis and Systemic Lupus Erythematosus can be identified using the double diffusion process.

Double Immunodiffusion

Identity Reaction

When both antigens diffuse at the same rate and combine with antibody to create a single smooth band region or chevron, identity reactions are produced, meaning that the antigens in both wells are of the same type.

Non-Identity Reaction

Nonidentity reactions are those in which band lines cross each other, suggesting the non-identical presence of antigens found in two samples.

Partial Identity Reactions

When band lines converge to form a spur in partial identity reactions, it means two antigens are cross reactive but not similar. The development of a spur means that one of the antibodies does not react with the cross-reacting antigen and instead migrates past it until it comes across an antigen that contains the epitope for which it has specificity.

The antigen and antibody molecules in solution diffuse into the porous agarose and out of the wells. If antigen-specific antibody is present, the two components join at a point of maximum concentration, known as the zone of equivalence, to form a visible precipitation band or line. Diffusion is gradual and not suitable to quick diagnosis because the test depends on passive diffusion of molecules.


  1. Isenberg, H. D., & American Society for Microbiology. (1992). Clinical microbiology procedures handbook. Washington, D.C: American Society of Microbiology.
Default image

A tech enthusiast medical molecular technologist, biotechnologist and now also a blogger.

Articles: 66

Leave a Reply