MadSci Network: Immunology

Re: What do white precipitate lines after you immunoelectrophorese show?

Date: Fri Jan 14 16:54:34 2000
Posted By: Richard Deem, Staff, Inflammatory Bowel Disease, Cedars-Sinai Medical Center
Area of science: Immunology
ID: 947200000.Im

Re: What do white precipitate lines after you immunoelectrophorese show?


I must say that your school is doing some fairly sophisticated experiments. It sounds like you have a good teacher. The kinds of experiments you mentioned are usually done in introductory immunology classes in college.

In the 2nd part, the white precipitate lines represent areas where antibodies ("cow antibodies") and antigen (the 6 sera) have reacted to form an insoluble precipitate. I assume that the "cow antibodies" were made by injecting the cow with some serum protein(s) from one of the 6 species you mentioned. Because of the similarity in the amino acid sequence and structure of mammalian proteins, you should have seen some cross-reactivity between the cow antibodies generated against serum proteins of mammal #1 with the other 5 mammalian sera. The test you describe is called "single radial immuno-diffusion," or the "Ouchterlony" test. In this test, antibody ("cow antibodies") is placed in the center well and antigen (the 6 sera) is placed in the surrounding wells. The antibody and the antigen diffuse through the agarose in all directions. Where the antibodies and antigen interact (in the area between the center well and the outside wells), you will get a precipitin line. Since antibodies are divalent (have two antigen-binding sites), they can bind two antigen molecules together. This is not sufficient to cause a precipitin line, since literally thousands are required to be linked together to form an insoluble complex. However, the "cow antibodies" you have are most likely made against several epitopes (specific areas of the antigen molecule), and so, multiple antibody molecules can bind to each antigen molecule, which will allow for the formation of very large complexes. These complexes are too large to remain in solution in the water within the gel, and so form a visible precipitate.

There are three different kinds of precipitin reactions that can occur (see below).

         O                 O                  O    /
	____		_______            _______/__
	    \  O            \                    /
	     \               \  O	        /   O
      Identity    Partial Identity     Non-Identity

This kind of test is rarely used today, since there are better ways to examine the questions you are asking. Yes, there is very little information available on the web. However, I found one link that you might want to look at for more information: t13.htm

What could have gone wrong in your experiment? The most likely problem would be something wrong with your antibodies. It would be unlikely that your serum samples would become completely non-reactive. Antibodies can become bad through improper storage temperature (most should be refrigerated or frozen for long-term stability). Also, antibodies can become contaminated with bacteria if there is no preservative (usually sodium azide). Your antibody solution should be clear. If it is turbid or cloudy, it is likely bad - order some new antibody.

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