Gel Diffusion

1. Gel Diffusion Period:3 Melisa Blazevic, Rene Boyas, Benjamin Vega, Thomas Zemen, Mario

2. Background/ Facts • Örjan Ouchterlony, is the Swedish physician who invented the test in 1948. • Useful because it can help determine if the blood being tested belongs to a human. • Electrical fields can be used and its called the electrophoretic method. With this method an electrical potential is applied to an antigen or anti body. The reaction is noted by a line of precipitation formed between the blood extract and the hole containing the human antiserum. •  It can be used in particular for detecting the presence of specific antibodies in a serum. • The tests  sensitive for detecting antigens. Percent Error can be high due to the many affecting variables such as lighting and temperature. Gel Diffusion is widely used to determine antigenic relationships; the bands of precipitate that form where the reactants meet in optimal concentration are of three patterns, referred to as reaction of identity, reaction of partial identity, and reaction of nonidentity.

3. Procedure and Function of Gel Diffusion • -A gel plate is cut to form wells in the gel.-The unknown sample is put into one well and the purified antibodies are placed into another well. -The plate is left untouched for 48 hours to form and the antibodies in each sample diffuse out of their wells.-When the two meet, if any antibodies recognize each other, they bind and form an immune complex.-A line of precipitation forms when the immune complex precipitates in the form of a thin, white line.

4. Method • The method of gel diffusion is based on the diffusion of antigens and antibodies in solid medium from wells placed opposite. When antibody molecules encounter antigen molecules, the antigen-antibody binding results in the precipitation of immune complexes in the region of meeting if the antibody recognizes antigen. The precipitate forms in the area where the concentrations of both solutions that are optimal for the amount of antibody saturated the antigenic sites, ie the zone of equivalence. The precipitates are in the form of an arc whitish visible to the naked eye.   •  The position of the precipitate depends on the relative concentration of antigens and antibodies, it is quantitative.  • After a few hours of diffusion, precipitin arcs are examined ,but they can also be colored to enhance their visibility.  • Mainly used to detect antibody/antigen interaction • used to compare the similarities of antigens with the use of these three rules identity, reaction of partial identity, and reaction of nonidentity.

5. Case/Extras • The gel double diffusion (Gdd) test using a lysate antigen of M. smegmatis was used to test the serums of 5 experimentally infected cattle. Four animals had been inoculated with M. bovis and one had been previously exposed to donor animals infected with M. bovis. Two unexposed non-tuberculous animals were used as controls. All 5 tuberculosis animals showed positive results to the test. In the case of 1 animal, a positive reaction to the test was observed throughout the study for a total of 63 weeks. Control animals remained negative to the test. When evaluated with serums of tuberculosis and non-tuberculous cattle in the field, the technique showed poor results. In many instances non-tuberculous herds were classified positive due to the fact that at least one animal in the herd was positive to the Gdd test. The detection of tuberculous animals did verify the existence of cross reaction between antibodies of M. bovis and antigens of M. smegmatis, however, this phenomenon was not considered reliable as the basis for a test to diagnose tuberculosis. • Double diffusion gel precipitation tests are very sensitive fordetecting antigens. They are most suitable for demonstrationand identification of an antigen in an antigenic mixture provided that a proper antiserum reagent is available.

6. Strength and weaknesses • The tests are good because they can detect antigens and different types of blood at the smallest levels. • The tests may show poor results which may may sometimes be unreliable. • Lighting and temprature can have a big affect on the results. • Contamination has been a factor in results so some people may be sceptical on using the tests.

7. Photos/Video http://www.youtube.com/watch?v=hmK7yYr2T54

8. Questions • 1.Who invented the gel diffusion tests? • 2.What can affect the tests? • 3.During the procedure what is bonding? • 4.After the procedure the precipitates are in the form of what? • 5.When electric fields are used for diffusion what is the method called?

9. Citations • "Definition: 'Gel Diffusion Precipitin Tests In Two Dimensions'" Gel Diffusion Precipitin Tests In Two Dimensions. N.p., n.d. Web. 04 Apr. 2013. • Richards, W. D. "Evaluation of the Gel Double Diffusion Test Using A Phage Lysate Antigen of Mycobacterium Smegmatis to Diagnose Tuberculosis of Cattle." Ncbi. N.p., n.d. Web. 3 Apr. 2013. • http://triscience.com/Animals/Cattle/evaluation-of-the-gel-double-diffusion-test-using-a-phage-lysate-antigen-of-mycobacterium-smegmatis-to-diagnose-tuberculosis-of-cattle/doculite_view