Double diffusion (DD) is based on the principle that antigen and antibody diffuse
through a semisolid medium and form a precipitin line. In Ouchterlony's method, a
layer of agar gel is deposited in a petri dish and circular wells are punched out
near one another in the gel. Antibody is then added to one well while antigen is
added to the other. These materials are allowed to diffuse radially from their
respective wells. As the perimeter of the diffusing substance increases, the
concentration of that substance within the perimeter continually decreases. When
the optimal concentration of antigen and antibody is reached, a line of precipitation
is formed in the gel. The precipitin line is relatively straight and is perpendicular to
the axis line between the two wells. The immunologic reactions in double gel
diffusion are of three types.
Consider this: A solution of the same antigen is placed in two wells and its
corresponding antibody is placed in the center well. Two precipitin bands form,
joining at their contiguous ends and fusing. This is a reaction of identity.
To produce a reaction or partial identity, the antigen in one well and the antibody in
the central well are homologous, and the antigen in the other well is a cross-
reacting antigen. The precipitin bands fuse, but in addition form a spike that
extends toward the cross-reacting antigen. This is a reaction of partial identity.
When two unrelated antigens are placed in adjacent wells and diffuse toward a
central well that contains antibodies for each, two precipitin bands form
independently of each other and cross. This is a reaction of non-identity.
Currently, double diffusion is utilized in the clinical laboratory for immune complex
studies and comparing antigens or antibodies for the presence of identical or
A screening procedure utilized to detect abnormalities of the various protein
fractions is serum protein electrophoresis. The patient's serum is applied to a
support medium along with a normal control. The agarose gel with the serum
applied is placed in a barbital buffer (pH 8.6) and then subjected to an electrical
charge. Due to the pH of the barbital buffer, the serum proteins will assume a net
negative charge. The serum protein components migrate to one of the five
possible characteristic zones (albumin, alpha-1, alpha-2, beta, and gamma) based
upon their net negative electrical charge, size, and molecular weight. Following a
staining procedure using a protein stain, the serum protein components are
compared to those of the normal control.