Microcheck 176

Fluorescent dyes are used to visualize antibodies under the fluorescence microscope, either directly or indirectly.

■ How do the direct and indirect immunofluorescent antibody tests differ?

■ Why are anti-human-gamma-globulin antibodies labeled with detectable substances used in many immunological tests, such as indirect fluorescent antibody tests?

■ How could different antigens in tissues be located?

adsorbs to the plastic surface, and the excess is washed away. The serum to be tested is added to the tube or plate and incubated for a short time. If specific antibodies are present, they bind to the antigen. To detect the antigen-antibody reaction, the ELISA uses anti-HGG chemically coupled with an enzyme label such as peroxidase. This binds to the test antibody, excess labeled anti-HGG is washed away, and chromogen is added. Chromogen is a colorless substrate that produces a colored end product when acted on by an enzyme such as peroxidase. This color change can be measured and is directly related to the amount of antibody bound (figure 17.9).

Direct ELISA tests are also useful in studying infectious diseases. Modifications of ELISA tests are available commer-

17.7 Radioimmunoassay (RIA), Enzyme-Linked Immunosorbent Assay (ELISA), and Western Blot

RIA and ELISA are widely used, because these extremely sensitive tests can be employed to analyze a large number of samples quickly, using very little sample and reagents. For example, the ELISA technique is now routinely used to test donated blood for antibodies against the human immunodeficiency virus, or HIV (the virus that causes AIDS), before the blood is used for transfusion. The presence of specific antibodies implies that the virus is present. The Western blot combines electrophoresis with ELISA to separate and then detect specific proteins.

Radioimmunoassay (RIA)

RIA is a competitive inhibition assay used to measure antigens or antibodies that can be radioactively labeled. For example, to test for antigen in a sample, a fixed amount of unlabeled antibody is adsorbed onto plastic wells, and then the ability of a test sample to inhibit the binding of a labeled specific antigen is measured. Antigen binding is measured by the amount of radioactivity retained in the well, determined by using a gamma counter. The test is based on competition for specific antibody between known amounts of radioactively labeled antigen and unknown amounts of unlabeled antigen in the sample being tested. The concentration of the unknown is determined by comparison with a standard curve prepared using several concentrations of a known standard unlabeled antigen. This highly sensitive method is used to measure extremely small amounts of hormones or drugs in a clinical sample. A special RIA called radioallergosorbent test (RAST) is used to measure the very minute amounts of IgE antibody that react with a specific antigen. This method can detect as little as 1 to 50 ng/ml of serum antibody.

Enzyme-Linked Immunosorbent Assay (ELISA)

In the indirect ELISA, the known antigen is put into a plastic test tube or on a plastic microtiter plate. Some of the antigen

Microtiter well

Known antigen adsorbed to plastic well

Serum to be tested added to well

Antibody in test serum (human gamma globulin, HGG)

If serum contains specific antibody, it will bind to the antigen

Microtiter well

Known antigen adsorbed to plastic well

Serum to be tested added to well

Antibody in test serum (human gamma globulin, HGG)

If serum contains specific antibody, it will bind to the antigen

Wash and add antibody (anti-HGG) that has been coupled to the enzyme peroxidase

- Enzyme peroxidase linked to anti-HGG (antibody that reacts with any human immunoglobulin)

Colored end product

Colorless chromogen interacts with enzyme to produce colored end product

The amount of colored end product can be measured optically

Figure 17.9 Enzyme-Linked Immunosorbent Assay (ELISA)

Wash and add chromogen (a colorless substrate which when acted upon produces a colored end product)

Colored end product

Colorless chromogen interacts with enzyme to produce colored end product

The amount of colored end product can be measured optically

Figure 17.9 Enzyme-Linked Immunosorbent Assay (ELISA)

17.8 The Complement Fixation Test and Neutralization Tests

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