• The Precipitation Tests

Precipitation tests involve the reaction of soluble antigens with specific antibodies (in general, Ig G or Ig M) to convert the soluble antigen to a solid precipitate called lattice. Using the precipitation test (PT), one can demonstrate the antigen in such tiny amounts which can not be detected by chemical tests. The use of the PT for sanitary and hygienic control of foodstuffs makes it possible to uncover adulteration of meat, fish, and flour products, as well as admixtures in milk, etc.

Precipitation tests occur in two distinct stages. First, there is the rapid interaction between antigen and antibody to form small antigen-antibody complexes. This interaction occurs within seconds and is followed by a slower reaction, which may take minutes to hours, in which the antigen-antibody complexes form lattices that precipitate from solution.

P recipitation reactions normally occur only when there is an optimal ratio of antigen to antibody. The optimal ratio is produced when separate solutions of antigen and antibody are placed adjacent to each other and allowed to diffuse together. In a precipitin ring test (see PRACTICAL WORK), a cloudy line of precipitation (ring) appears in the area in which the optimal ratio has been reached (the zone of equivalence) -Fig. 6 and 7.

Immunodiffusion tests are precipitation reactions carried out in an agar or gel medium. In one such test, the Ouchterlony test, wells are cut into a purified agar gel in a Petri plate. A serum containing antibodies (antiserum) is added to one well, usually centrally located and soluble test antigens are added to each surrounding well. A line of visible precipitate develops between the wells at the point where the optimal antigen-antibody ratio is reached. The Ouchterlony test is most useful in determining whether the antigens are identical, partially identical, or totally different.

The Ouchterlony test (the gel-diffusion plate technique) is used to demonstrate toxigenicity of diphtheria bacilli. A filter paper strip saturated with antitoxin- containing antiserum (antitoxins as an antibodies) is placed on the culture medium, which favors the production of diphtheria toxin (antigen). Several strains of Corynebacterium diphtheriae are then seeded by streak technique. The toxigenic strains (the diphtheria toxin-producing strains) develop V-shaped lines of precipitate in the zone of optimal antigen-antibody proportion.

The Mantchini test is commonly used to measure the concentration of immunoglobulins in patient’s serum. The monoclonal or animal antibodies (“known antibodies”) against corresponding class of human immunoglobulins are mixed with melted purifies agar and poured into Petri plate. Wells are cut in the agar and the patient’s serum (certain class of immunoglobulins as “unknown antigens”) is added to one well. Two-fold dilutions of standard immunoglobulins are prepared and added to other wells. A zone of precipitation surrounds each well due to the antigen-antibodies complexes production. The diameter of zone precipitation depends on the concentration of immunoglobulins in the patient’s serum: the lager zone demonstrates the lager concentration. To determine the immunoglobulins concentration the diameter of precipitation zone around the test well is compared with precipitation zone around the standard wells.

O ther precipitation tests do not depend entirely on the passive diffusion of antigen and antibody in a gel but instead use electrophoresis to speed up their movement. Protein mixtures can be separated rapidly, sometimes in less than an hour, with this method. A modification of the precipitation reaction combines the techniques of immunodiffusion and electrophoresis in a procedure called immunoelectrophoresis. This procedure is used in research to separate proteins in human serum and is the basis of certain diagnostic tests (for example, countercurrent immunoelectrophoresis - CIE). CIE is based on the fact that some antigens and antibodies have opposite charges when they are placed in buffers of correction strength and pH. When an electrical current is applied the antigens and antibodies move toward the pole with the electrical charge opposite to the one they carry. They pass through each other to do so, and precipitation line appears within an hour if a reaction occurs (Fig. 8).