The classic method of detecting soluble antigen (i.e., antigen in solution) is the Ouchterlony method, a double immunodiffusion precipitation method.
Double Immunodiffusion
In the double immunodiffusion method, small circular wells are cut in an agarose gel, a gelatin-like matrix derived from agar, which is a chemical purified from the cell walls of brown algae. The agarose forms a porous material through which molecules can readily diffuse. The patient specimen containing antigen is placed in a well, and antibody directed against the antigen is placed in the adjacent well. Over 18 to 24 hours, the antigen and antibody diffuse toward each other, producing a visible precipitin band (a lattice structure or visible band) at the point in the gel where the antigen and antibody are in equal proportion (zone of equivalence). If the concentration of antibody is significantly higher than that of the antigen, no lattice forms and no precipitation reaction occurs; this is known as prozone effect. Conversely, if excess antigen prevents lattice formation, resulting in no band formation, the effect is termed post zone. Immunodiffusion is currently used to detect exoantigens produced by the systemic fungi to confirm their presence in culture (Figure 1). However, the technique is extremely time-consuming and is no longer used regularly in the clinical laboratory for antigen detection in patient specimens.
Fig1. Exo-Antigen Identification System (Immuno-Mycologics, Inc., Norman, Okla.) The center well is filled with a 50× concentrate of an unknown mold. The arrow identifies well 1; wells 2 to 6 are shown clockwise. Wells 1, 3, and 5 are filled with anti-Histoplasma. anti-Blastomyces, and anti-Coccidioides reference antisera, respectively. Wells 2, 4, and 6 are filled with Histoplasma antigen, Blastomyces antigen, and Coccidioides antigen, respectively. The unknown organism can be identified as Histoplasma capsulatum based on the formation of line(s) of
