Serum Antibody Tests: Precipitation

On this page you will find the following topics: Precipitation in Solution Immunodiffusion in Agar Gels Radial Immunodiffusion (RID) Immunoelectrophoresis (IEP) A precipitation reaction occurs as a result of the combination of antibodies in solution with soluble substances with which the antibodies react. If such a precipitation reaction occurs in vivo in the joints or the kidney, inflammation results because the immune complexes are filtered out in those areas, causing irritation. In a serum precipitin test, dilutions of antigen and antibody in aqueous solution are combined until a precipitation reaction occurs and visible particles accumulate. The amount of "flocculation" can be measured by an instrument designed to measure the light scattering properties of particulate aggregations (nephelometer). The rapid plasma reagin test remains the "Gold Standard" as a screening test for syphilis and has an immunological flocculation reaction as its basis.		These immune complexes have fallen out of solution. The Ab at the bottom in the illustration at left is still in the soluble phase.

The precise "Zone of Equivalence" where there is a perfect concentration of both antibody and antigen in solution, is difficult to achieve. Many tubes of various antibody and antigen concentrations would need to be prepared in order to get just the right amount; the thin ring of precipitate visible only after centrifugation is much more likely than the tube full of precipitate shown at the top of the page. In practice, precipitation reactions are generally prepared as agar diffusion reactions, to make precipitates more visible.

Serum antibodies are not homogeneous (you have antibodies that recognize all kinds of infectious organisms and allergens). It is difficult to obtain a single antigen even in the purest preparations, so there are several antigen-antibody reactions possible in a single mixture. Gel diffusion permits the examination of such multiple systems since the technique allows the separation of the reactions. The individual components in a system react independently of each other so precipitation tests in agar are termed "double immunodiffusion" tests or simply immunodiffusion. (Ouchterlony is used in the older terminology). The hazy white background of the solid agar matrix allows visualization of the precipitation reaction. The method is used clinically in immunology/rheumatology evaluations.

In this picture, the Center contains the antiserum & wells 1 & 2 contain samples of homologous antigen that the serum recognizes. Note the band of identity between wells 1 & 2.

This technique has many different applications; examples include: Determining the homogeneity of antigen-antibody systems Diagnosing specific autoimmune disorders Following the purification of an antigenic mixture Elucidating the reactions among serologically related antigens

The precipitation appears as a continuous line in the form of an angle between those two wells and the C well. There are no spurs at the angle and this type of reaction is termed a band of identity.

FIGURE 2: If a solution with antigens X and Y is placed in well 1, a solution with antigen X only is placed in well 2, and antiserum containing antibodies specific for both X and Y is placed in well 3, a reaction similar to that appearing in Fig. 2 will occur. Notice that there is a spur reaction towards the XY well. This indicates that the two antigenic materials in wells 1 and 2 are related, but that the material in well 1 possesses an antigenic specificity not possessed by the material in well 2. Such a reaction with spur formation indicates partial identity.

If the material in wells 1 and 2 do not possess common antigens and the antiserum in well 3 possesses specificities for both materials, the reaction will appear as two crossed lines as in Fig. 3

Immunoelectrophoresis (IEP)
Immunoelectrophoresis (IEP) is a procedure combining separation of antigens by zone electrophoresis with diffusion of precipitating antibodies at right angles to the direction of antigen migration. The extent of migration of any protein during electrophoresis is dependent upon the electrophoresis buffer, time, voltage, and the isoelectric point of the protein. Particularly, pH determines the separation of one protein species from another. The net difference between the isoelectric point of a given protein and the pH of the electrophoresis buffer determines the extent of migration toward the cathode or the anode.
This immunological method is used to detect abnormalities in specific serum proteins and to identify specific immunoglobulins using anti-human antibody reagents. The anti-human antibody reagents have been prepared against purified fractions of human globulins and are commercially available for research or biomedical purposes.

THE SETUP Human serum proteins are electrophoretically separated in agar gels during the first part of the procedure. After the separation is completed, anti-human globulin sera are added to troughs cut in the agar and allowed to diffuse into the gel. Precipitation occurs within the agar matrix at the point where the diffusing anti-human globulin sera meet with their corresponding serum proteins.

 

ASSESSMENT OF RESULTS  View the stained IEP slides with an immunoviewer and identify the immuno-arcs relating to the globulin and albumin fractions. This is best accomplished by comparing the arcs produced with antisera to whole serum with those produced by anti-IgG, anti-IgM, and anti-IgA specific antisera.

Biomedical Laboratory Information Last modified 11-21-03.