The Precipitin Ring Test


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A diagram with multiple test tube dilutions labeled: 1, ½, ¼, 1/8, 1/16, 1/32. The bottom of each test tube contains a standard antigen solution. The top contains serial dilutions of antiserum. Tubes 1, ½, and 1/32 have no band at the interface between these two zones. Tubes ¼, and 1/6 have a thin band. Tube 1/8 has a thick band labeled zone of equivalence – visible precipitation.
A precipitin ring test is performed using a standard antigen solution in the bottom of the tube and a serial dilution of antiserum in the top of the tube. Glycerol prevents the two solutions from mixing so that precipitation only occurs at the interface. A visible ring of precipitation is seen in the 1/4, 1/8, and 1/16 dilutions, indicating that these concentrations are within the equivalence zone. Since 1/16 is the highest dilution in which a precipitin is observed, the titer is the reciprocal, or 16.

Source: OpenStax Microbiology

OpenStax Microbiology

A variety of techniques allow us to use precipitin formation to quantify either antigen concentration or the amount of antibody present in an antiserum. One such technique is the precipitin ring test, which is used to determine the relative amount of antigen-specific antibody in a sample of serum. To perform this test, a set of test tubes is prepared by adding an antigen solution to the bottom of each tube. Each tube receives the same volume of solution, and the concentration of antigens is constant (e.g., 1 mg/mL). Next, glycerol is added to the antigen solution in each test tube, followed by a serial dilution of the antiserum. The glycerol prevents mixing of the antiserum with the antigen solution, allowing antigen-antibody binding to take place only at the interface of the two solutions. The result is a visible ring of precipitin in the tubes that have an antigen-antibody ratio within the equivalence zone. This highest dilution with a visible ring is used to determine the titer of the antibodies. The titer is the reciprocal of the highest dilution showing a positive result, expressed as a whole number. In the image above, the titer is 16.

While a measurement of titer does not tell us in absolute terms how much antibody is present, it does give a measure of biological activity, which is often more important than absolute amount. In this example, it would not be useful to know what mass of IgG were present in the antiserum, because there are many different specificities of antibody present; but it is important for us to know how much of the antibody activity in a patient’s serum is directed against the antigen of interest (e.g., a particular pathogen or allergen).


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