Precipitation shows the following features:
- It is a type of antigen–antibody reaction, in which the antigen occurs in a soluble form.
- It is a test in which antibody interacts with the soluble antigen in the presence of electrolyte at a specified pH and temperature to produce a precipitate.
- A lattice is formed between the antigens and antibodies; in certain cases, it is visible as an insoluble precipitate.
- Antibodies that aggregate soluble antigens are called precipitins.
- Formation of an antigen–antibody lattice depends on the valency of both the antibody and antigen.
- The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments.
- The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same epitope, or have different epitopes that react with different antibodies present in polyclonal antisera.
Types of precipitation reactions:
Precipitation reactions can be broadly 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.
- In this test, antigen solution is layered over antiserum in a test tube.
- Precipitation between antigen and antibodies in antiserum solution is marked by the appearance of a ring of precipitation at the junction of two liquid layers.
- Flocculation test may be performed in a slide or tube.
- VDRL test for detection of antibodies in syphilis is an example of a slide flocculation test.
- In this test, a drop of VDRL antigen suspension is added to a drop of patients’ serum on a cavity slide, and the result is recorded after shaking the slide on a VDRL shaker.
- In a positive test the floccules appear, which can be demonstrated well under a microscope.
Precipitation in agar:
- The precipitation test in agar gel is termed as immunodiffusion test.
- In this test, 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.
Types of immunodiffusion reactions:
Single diffusion in one dimension:
- Single diffusion in one dimension, as the name suggests, is the single diffusion of antigen in agar in one dimension.
- It is otherwise called Oudin procedure because this technique was pioneered by Oudin who for the first time used gels for precipitation reactions.
- In this method, antibody is incorporated into agar gel in a test tube and the antigen solution is poured over it.
- During the course of time, the antigen diffuses downward toward the antibody in agar gel and a line of precipitation is formed.
- The number of precipitate bands shows the number of different antigens present in the antigen solution.Single diffusion in two dimensions:
Single diffusion in two dimensions or radial immunodiffusion:
- Single diffusion in two dimensions is also called radial immunodiffusion.
- In this method, antiserum solution containing antibody is incorporated in agar gel on a slide or Petri dish.
- The wells are cut on the surface of gel.
- The antigen is then applied to a well cut into the gel.
- When antibody already present in the gel reacts with the antigen, which diffuses out of the well, a ring of precipitationis formed around the wells.
- The diameter of the ring is directly proportional to the concentration of antigen.
- However, the test has recently been replaced by more sensitive and automated methods, such as nephelometry and enzyme-linked immunosorbent assays (ELISAs).
Double diffusion in one dimension:
- This method is also called Oakley–Fulthrope procedure.
- In this method, the 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.
- In this zone of plain agar, both antigen and antibody react with each other to form a band of precipitation at their optimum concentration.
Double diffusion in two dimensions or Ouchterlony Double diffusion:
- This method is also called the Ouchterlony Double diffusion.
- In this method, both the antigen and antibody diffuse independently through agar gel in two dimensions, horizontally and vertically.
- The test is performed by cutting wells in the agar gel poured on a glass slide or in a Petri dish.
- The antiserum consisting of antibodies is placed in the central well, and different antigens are added to the wells surrounding the center well.
- After an incubation period of 12–48 hours in a moist chamber, the lines of precipitins are formed at the sites of combination of antigens and antibodies.
- Three types of reactions can be demonstrated as follows:
- Line of precipitation at their junction forming an arc represents serologic identity or the presence of a common epitope in antigens.
- A pattern of crossed lines demonstrates two separate reactions and indicates that the compared antigens are unrelated and share no common epitopes thus non-identity.
- Fusion of two lines indicates cross-reaction or partial identity. In this last case, the two antigens share a common epitope, but some antibody molecules are not captured by the antigen and traverse through the initial precipitin line to combine with additional epitopes found in the more complex antigen.
Precipitation in agar with an electric field:
- Immunoelectrophoresis is a process of combination of immunodiffusion and electrophoresis.
- It is a method in which different antigens in serum are separated according to their charge under an electric field.
- In this method, a drop of antigen is placed into a well in agar on a glass slide.
- An electric current is then passed through the agar.
- During electrophoresis, antigens move in the electric field according to their charge and size.
- after electrophoresis a trough is cut into the agar and is filled with the antibody and diffusion is allowed to occur.
- As the antigen and antibody diffuse toward each other, they form a series of lines of precipitation.
- The main advantage of immunoelectrophoresis is that a number of antigens can be identified in serum.
- The method is used to detect normal as well as abnormal proteins, such as myeloma proteins in human serum.
- Counter-current immunoelectrophoresis depends on movement of antigen towards the anode and of antibody towards the cathode through the agar under electric field.
- The test is performed on a glass slide with agarose in which a pair of wells is punched out.
- One well is filled with antigen and the other with antibody.
- Electric current is then passed through the gel.
- The migration of antigen and antibody is greatly facilitated under electric field, and the line of precipitation is made visible in 30–60 minutes.
- This technique is an adaptation of radial immunodiffusion developed by Laurell.
- It is called so due to the appearance of the precipitin bands in the shape of cone-like structures (rocket appearance) at the end of the reaction.
- In this method, antibody is incorporated in the gel and antigen is placed in wells cut in the gel.
- Electric current is then passed through the gel, which facilitates the migration of antigen into the agar.
- This results in formation of a precipitin line that is conical in shape, resembling a rocket.
- The height of the rocket, measured from the well to the apex, is directly in proportion to the amount of antigen in the sample.
- Rocket electrophoresis is used mainly for quantitative estimation of antigen in the serum.