Double Diffusion In Gel
Utilizing the same principle of diffusion, precipitation reactions can also develop in an agar gel through which contains soluble antigen and their corresponding antibodies within adjacent wells. Antigen and its' corresponding antibody solutions are placed within adjacent but separate wells. The antigen and antibody will diffuse through time through the gel medium, and a line of precipitation will form at any point of interaction between antigen and antibody. The visible bands, which are precipitin reaction precipitates, will form at any points of equivalence, where antibodies and antigens will interact, bind, crosslink and form a lattice, and also possibly a precipitation spectrum. The precipitates are dependent on the degree of diffusability (e.g. agar concentration), but more greatly dependent on the concentration of the reactants, antigen and antibody, at their initial sources. Any precipitation band formed and stabilized becomes a specific barrier for further diffusion for the integrated antigen and its' antibody, but not for reactants with differeing serology. In the Ouchterlony comparative plate technique, this mechanism is taken advantage of, where sources of diffusion are distributed in wells in a pattern possible of forming patterns representative of certain possible interactions. For antigen and immune serum comparisons, there can be three outcomes and also no outcome in an absence of interaction, whcih are mentioned below.
Reaction of Identity - is depicted with the formation of precipitate as a continuous, fused arc, that displays that the diffusing antibody is precipitating the two antigens with identical specificities. This reveals the identity of the antigens (shared epitopes recognized by antibody). This occurs when there are no shared epitopes.
Reaction of Non-Identity - is depicted with the formation of precipitate as independent lines to one another, that intersect and resemble crossed swords, displaying that parts of the antiserum (antibodies) interact independently to each antigen, resulting in the antigens being a mixture of different specificities, epitopes, that do not shar any antigenic determinants. Therefore, no epitopes shared between antigens.
Reaction of Partial Identity - is depicted with the formation of a precipitate that resembles a spur. Antigenic determinants common to both antigens give the continuous arc, but any unique determinants found in a single antigen produce an overshooting of the precipitate, seen as a spur. This demonstrates that the two antigens, share some epitopes, displayed by the fused arc, however, not others, as seen by the spur. (2,3)
Double diffusion, however, can only be used as a qualitative assay, unlike the radial immunodiffusion assay, even though the thickness of bands may indicate antibody concentration levels and as the position of the precipitin line is dependent on the concentrations of both the antigen and antibody. In addition, this technique gives numerous cross reactions with antibodies of high affinity, does not reveal low affinity antibodies, requires high concentrations of both antigen and antibody and also is incapable of revealing any epitopes not reacted with the antisera. Moreover, this test is mainly used to reveal the specificities antisera, as in clinical uses for detection of antibodies in systemic lupus. (3,4)
Setting up various antibody-antigen diffusion systems to display the three reaction types mentioned above can easily be done.
The absence of precipitation in a reaction of double diffusion in a gel, could be due to inexperience in laboratory work, as in making adequate wells and filling them appropriately, poor incubation and trasport of the agar plates that could of disturbed the diffusing molecules into a mixture, as seen in the ring precipitation reaction. Such poor handling could have disturbed the main principles that this test works on. The band sizes are greatly dependent on the degree of diffusibility and more importantly, the concentration of the reactants at their 'initial sources.' Any movement or spill could have destroyed any consistency in the initial source concentration and therefore disrupted any entrance into the zone of equivalence for precipitation to occur. In addition, the double diffusion test works on the ability of the precipitation band to act as a specific barrier for further diffusion for the integrated specific antigen and antibody. Without a formation of bands, there could have been no build up of visibility.
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.