The Protein Protocols Handbook

pp 749-752

Ouchterlony Double Immunodiffusion

  • Graham S. BaileyAffiliated withDepartment of Biological and Chemical Sciences, University of Essex

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Immunodiffusion in gels encompasses a variety of techniques that are useful for the analysis of antigens and antibodies (1). The fundamental immunochemical principles behind their use are exactly the same as those that apply to antigen-antibody interactions in the liquid state. Thus, an antigen will react with its specific antibody to form a complex, the composition of which will depend on the nature, concentrations, and proportions of the initial reactants. As increasing amounts of a multivalent antigen are allowed to react with a fixed amount of antibody, precipitation occurs, in part because of extensive crosslinking between the reactant molecules. Initially, the antibody is in excess, and all the added antigen is present in the form of soluble antigen-antibody adducts. Addition of more antigen leads to the formation of an immune precipitate (precipitin) where all of the antigen and antibody molecules are in an extensive lattice of antigen-antibody complex. Further addition of antigen produces an excess of antigen and leads to a reduction in the amount of the precipitate as soluble antigen-antibody adducts are formed again. The analysis of such interactions occurring in gels is of much higher sensitivity and resolution than that for the liquid state, thus explaining the extensive use of immunochemical gel techniques.