Ring Precipitation Test

Ring Precipitation Test Protocol

Two test tubes were prepared, one with a 5 mm depth of undiluted, normal rabbit serum (control) and another tube contained a 5 mm depth of un-diluted anti-BSA. Addition of BSA solution to the test tubes resulted in the subsequent formation of an uniform ring precipitate (white) in the tube containing the anit-BSA at the boundary, but not in the rabbit serum control. Shaking the two tubes and inspecting after half an hour later, displayed that the precipitate had redissolved (no sharp boundary). Some precipitate did remain at the bottom of the tube, but was very transparent.

Ring Precipitation Test Background Information

The Ring Precipitation Test is the simplest way to depict the presence of precipitating antibodies in a serum, through the use of the precipitin reaction. Even though this assay is insensitive and may produce negative results for a serum with antibodies present, it provides a simple and fairly quick way of detecting antigen-specific antibodies. The ring test works on the principle that layering an antigen solution over top an antiserum solution results in a ring precipitation formation at the boundary between the two solutions. This ring of precipitation only occurs if the serum (antiserum) contains antibodies specific to the antigen. (2,3)

Proceeding throught the mentioned protocol, a bovine serum albumin (BSA) solution was placed over top a solution of undiluted anti-BSA. A control reaction, in which BSA was placed over top a solution of normal rabbit serum (no antibodies specific for BSA present). A ring precipiate will form in the solution of BSA/anto-BSA tube, because of the presence of antibodies specific against the antigen, BSA. No ring precipitate will form in the control reaction tube. Subsequently shaking the two tubes, with a one and a half hour room incubation period, redissolved the precipitate and failed to reform any ring precipitate with a sharp boundary, but rather an opaque solution in only the anti-BSA tube bottom. Mixing causes the loss of any sharp boundary ring precipitate, as now the higher gravity fluid has become mixed throughout with the lower gravity fluid, and there is no diffusion, from the heavy molecules, antigen (higher gravity) to the less dense molecules (lower gravity) antibodies, from the top of the tube to the bottom, in order for an interaction to occur. The antigen would slowly proceed to the bottom of the tube and meet the less dense antibodies on the way and subsequently react and crosslink. All the antigen would be proceeding downwards and therefore could be caught by the antibodies and precipitated in the ring as large aggregates, as antibody met antigen.

However, upon mixing, the majority of antigen becomes situated at the bottom, with the majority of the less dense antibodies higher up, with no future probable encounter. The reaction of antigen and antibody is a reversible interaction, in which the non-covalent and weak van der Waals forces, hydrogen bonds, hydrophobic bonds and electrostatic forces are broken and the antigen and antibody are redistributed throughout. Now, there is no separation and antibody is binding to antigen throughout the mixture, without forming large visible aggregates as a ring and to trap incoming antigen, because there is no area of high enough concentration of both antibody and antigen to form a zone of equivalence. Therefore, there is no lattice formation. The optimal ration of antigen and antibody that was obtained at the equivalence zone, the precipitation ring, was disrupted by the mixing and subsequently dispersed. The mixed solution now contains either a probable excess of antigen or antibody, with no optimal antigen-antibody ratio binding to form a precipitate. (2,3)

References:

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA.

2. Delves, P., and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA.

3. Cruse, J. and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA.

4. Bryant, N. 1986. Laboratory Immunology and Serology. B. Venable, W.B. Saunders Company, 1st ed., Philadelphia, USA.