<< Chapter < Page Chapter >> Page >

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 ( [link] ).

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.

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)

Radial immunodiffusion assay

The radial immunodiffusion (RID) assay is similar to the Ouchterlony assay but is used to precisely quantify antigen concentration rather than to compare different antigens. In this assay, the antiserum is added to tempered agar (liquid agar at slightly above 45 °C), which is poured into a small petri dish or onto a glass slide and allowed to cool. Wells are cut in the cooled agar, and antigen is then added to the wells and allowed to diffuse. As the antigen and antibody interact, they form a zone of precipitation. The square of the diameter of the zone of precipitation is directly proportional to the concentration of antigen. By measuring the zones of precipitation produced by samples of known concentration (see the outer ring of samples in [link] ), we can prepare a standard curve for determining the concentration of an unknown solution. The RID assay is a also useful test for determining the concentration of many serum proteins such as the C3 and C4 complement proteins, among others.

At the top is a photograph of 4 clear dots in a row. Dot 1 has a small ring around it, dot 2 has a larger ring, dot 3 has a larger ring, and dot 4 has an even larger ring. These have arrows leading to a graph that shows that the size of the ring (zone of precipitation diameter)  relates to the concentration of antigen. The lower antigen concentration results in a smaller ring.  Another dot (#5) off to the side contains an unknown antigen concentration. The size of the ring is measured and used to find the concentration of antigen. This is done by finding the ring size on the line from the graph and connecting that to the X-axis to find the concentration of antigen.
In this radial immunodiffusion (RID) assay, an antiserum is mixed with the agar before it is cooled, and solutions containing antigen are added to each well in increasing concentrations (wells 1–4). An antigen solution of an unknown concentration is added to well 5. The zones of precipitation are measured and plotted against a standard curve to determine the antigen concentration of the unknown sample. (credit circles: modification of work by Kangwa M, Yelemane V, Polat AN, Gorrepati KD, Grasselli M, Fernández-Lahore M)
  • Why does a precipitin ring form in a precipitin ring test, and what are some reasons why a ring might not form?
  • Compare and contrast the techniques used in an Ouchterlony assay and a radial immunodiffusion assay.

Get Jobilize Job Search Mobile App in your pocket Now!

Get it on Google Play Download on the App Store Now




Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
Google Play and the Google Play logo are trademarks of Google Inc.

Notification Switch

Would you like to follow the 'Microbiology' conversation and receive update notifications?

Ask