Ouchterlony Double Diffusion (Molecular Biology)

A direct extension of the precipitin reaction to immunoassays is the Ouchterlony double diffusion technique (1). Here, two or more wells are cut in an agar slab and filled with either antigen or antibody. The two materials slowly diffuse throughout the slab. As the antibody and antigen molecules encounter each other and react, a precipitate forms in the gel. Because of the highly cooperative nature of immune complex lattice formation, the precipitate forms an extremely sharp line between the two wells. The position of the precipitin line, compared to lines generated from other dilutions of antigen or antibody, gives a semiquantitative measure of the relative concentration of the reactant. However, the true value of the Ouchterlony technique is the remarkable number of details that can be inferred from the precipitin patterns formed. From the presence of multiple lines, or complex precipitin patterns formed at the intersection with multiple antigen or antibody wells in the agar slab, conclusions can be drawn about such properties as antigen heterogeneity, antigen molecular weight, shared antigenic determinants, and cross-reactivity of different antisera (2). The Ouchterlony technique is now obsolete, but it is commonly described in older literature.