The Ouchterlony Assay


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A gel with a central circle labeled A and 5 outer circles numbered 1-5. Between A and 1 is a white band labeled precipitin band. This zone is magnified and a diagram shows that circle A contains antigens in a well. Circle 1 contains antibodies in a well. The antigens and antibodies diffuses outward and meet in the region of the precipitin band. Here they bind together.
The Ouchterlony test places antigen (well A) and antisera (wells 1 through 5) in a gel. The antibodies and antigen diffuse through the gel, causing a precipitin arc to form at the zone of equivalence. In this example, only the antiserum in well 1 contains antibodies to the antigen. The resulting precipitin arc is stable because the lattice is too large to diffuse through the gel. (credit left: modification of work by Higgins PJ, Tong C, Borenfreund E, Okin RS, Bendich A)

OpenStax Microbiology

While the precipitin ring test provides insights into antibody-antigen interactions, it also has some drawbacks. It requires the use of large amounts of serum, and great care must be taken to avoid mixing the solutions and disrupting the ring. Performing a similar test in an agar gel matrix can minimize these problems. This type of assay is variously called double immunodiffusion or the Ouchterlony assay for Orjan Ouchterlony, who first described the technique in 1948.

When agar is highly purified, it produces a clear, colorless gel. Holes are punched in the gel to form wells, and antigen and antisera are added to neighboring wells. Proteins are able to diffuse through the gel, and precipitin arcs form between the wells at the zone of equivalence. Because the precipitin lattice is too large to diffuse through the gel, the arcs are firmly locked in place and easy to see.

Although there are now more sensitive and quantitative methods of detecting antibody-antigen interactions, the Ouchterlony test provides a rapid and qualitative way of determining whether an antiserum has antibodies against a particular antigen. The Ouchterlony test is particularly useful when looking for cross-reactivity. We can check an antiserum against a group of closely related antigens and see which combinations form precipitin arcs.


Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: