Precipitation reactions can be broad of three types:
- Precipitation in solution.
- Precipitation in agar.
- Precipitation in agar with an electric field.
Precipitation in solution
Ring test and flocculation test are examples of precipitation in solution. It is also called simple precipitation.
- Ring test: In this test, a clear solution of the test antigen is layered slowly over the clear solution of antiserum in narrow test tube. Following a period of incubation, precipitation between antigen and antibodies in the antiserum solution is marked by the appearance of a white ring at the junction of two liquid layers. C-reactive protein (CRP), Lancefield grouping of β-haemolytic streptococci, Ascoli’s thermoprecipitin test are the examples of the ring test.
- Flocculation test: Flocculation test may be performed in a slide or tube. In the slide test, a drop of antigen solution is added to drop of serum solution in the slide and mixed well. Visible clumps appear in positive cases. VDRL test for the detection of reaginic antibodies in syphilis is an example of a slide flocculation test. Tube flocculation test for standardization of toxins and toxoids, Kahn test for syphilis is an example of a tube flocculation test.
Precipitation in agar
The precipitation test in agar gel is termed as an immunodiffusion test. Here, reactants are added to the gel and antigen-antibody combination occurs by means of diffusion. The rate of diffusion is affected by the size of the particles, temperature, gel viscosity, amount of hydration, and interactions between the matrix and reactants. An agar concentration of 0.3–1.5% allows for the diffusion of most reactants.
- The reaction is visible in the form of a distinct band of precipitation.
- When only antigen or antibody diffuse with a corresponding antibody or antigen being incorporated on agar gel, this is called single diffusion.
- When both antigen and antibody diffuse in agar gel, it is double diffusion.
- Oudin immunodiffusion: It is single diffusion in one direction (single diffusion of antigen in agar in one dimension). Here, the antibody is incorporated in agar gel in the test tube and the antigen is layered over it. Antigen diffuses and forms the line of a precipitate.
- Oakley Fulthorpe immunodiffusion: It is double diffusion in one dimension. Here antibody is incorporated in agar gel in a test tube, above which a layer of plain agar is placed. The antigen is then layered on top of this plain agar. During the course of time, the antigen and antibody move toward each other through the intervening layer of plain agar and form band of the precipitate.
- Radial immunodiffusion: Single diffusion in two dimensions is called radial immunodiffusion. Here antiserum solution containing antibody is incorporated in agar gel on a slide or Petri dish. The antigen is then applied to a wells cut into the gel. When the antibody present in the gel reacts with the antigen, which diffuses out of the well, a ring of precipitation is formed around the wells. The diameter of the precipitin ring formed is directly proportional to the concentration of the antigen.
- Ouchterlony immunodiffusion: It is double diffusion in two dimensions. Both antigen and antibody diffuse independently through agar gel in two dimensions, horizontally and vertically. In this method, wells are cut in agar gel prepared in the slide. The antibody is placed in the central well and the antigen is placed on wells surrounding the central well. Antigen and antibody diffuse and precipitation bands are formed where they meet in optimal conditions.
Types of Precipitation Reactions in agar with an electric field
Also called immunoelectrophoresis as it combines electrophoresis with immunodiffusion.
Various concentrations of antigens are loaded side by side in small circular wells along the edge of agarose gel incorporated with the specific antibody. On electrophoresis, antigens begin to migrate towards the anode and interact with antibodies to form the complex. As electrophoresis continues more antigen moves toward the anode and at the equivalence point, antigen-antibody complex precipitates in the form of a rocket. The height of the rocket is proportional to the amount of antigen-loaded.
Antigen and antibody are placed in separate wells (opposite directions) cut in agar gel as the test relies on the movement of antigen and antibody in the opposite direction. On electrophoresis, antigen (Negatively charged) moves towards positive pole and antibody (Positively charged) moves towards negative pole. Line of precipitation appears at the point where the two meet.