500 1000 1500 2000 Antigen concentration (mg/100 ml)
A standard curve prepared using known concentrations of antigen shows a direct correlation between the diameter of the precipitin ring and the logarithm of the antigen concentration. Diameters of the test rings are compared with the standard curve to determine the concentration of antigen in each test well. Test sample A contains about 1600 mg/100 ml; test sample B contains about 1250 mg/100 ml.
Figure 17.4 Radial Immunodiffusion Is Used to Measure the Concentration of Antigen in a Sample
(a) Antigen diffusing from a well into antibody-containing gel forms a ring of precipitation in the zone of optimal proportions. (b) The amount of antigen in a sample is determined by comparing the diameter of the precipitin ring with a standard curve prepared using known concentrations of antigen.
Another immunodiffusion test, the double diffusion in gel, is a qualitative test. It involves diffusion of both antigen and antibody (figure 17.5). Antigen and antibody are placed in separate wells cut in the gel and are allowed to diffuse toward each other. When quantities of a specific antigen and its antibody meet at optimal proportions between the wells, a line of precipitate forms. Since there is often more than one antigen present in the sample and many antibodies in the serum, more than one line can form, each in its area of optimal proportions. This method is useful for identifying unknown substances and can be used to identify more than one antigen or antibody in a mixture.
Immunoelectrophoresis is a variation of the precipitation in gel technique, combining precipitation with electrophoresis. Electrophoresis is used to separate mixtures of protein antigens on the basis of their movement through an electrical field (figure 17.6). Once the proteins have been separated, the antibodies are placed in a trough and allowed to diffuse toward the separated protein antigens. A line of precipitate forms at the location of each antigen that is recognized by antibodies.
One of the many applications of immunoelectrophoresis is to determine whether people have normal immunoglobulins. The person's serum being tested for immunoglobulins, the
antigens in this case, is placed in the well; then, electrophore-sis is used to separate the proteins in the sample. Anti-immunoglobulin antibodies are placed in the trough and allowed to diffuse toward the antigen. Where each antibody meets an antigen in the area of optimal proportions, a line of precipitation is formed. In normal serum, lines will form for IgA, IgG, and IgM. IgD and IgE are normally present in such small quantities that they do not yield visible precipitates. Some patients lack immunoglobulins, and their sera will give no lines of precipitation with anti-human antibodies. Other patients have myeloma tumors, in which a single plasma cell has given rise to a tumor. All the cells of the tumor produce the same immunoglobulin that the original cell produced. In this case, the serum from the myeloma patient will give a heavy, thick line for the class of immunoglobulin being produced. Thus, abnormally high or low production of immunoglobulins is readily detected by immunoelectrophoresis. ■ electrophoresis, p. 236
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