Antigen Detection Assays

The use of an antibody to detect the presence of a specific microbial antigen is

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Through developments in immunology, a more specific type of antibody known as a monoclonal antibody can now be produced in large quantities. In contrast to the previously used polyclonal antibodies, monoclonal antibodies react with antigenic determinants that are unique to one microorganism and not shared by others. As a result, false-positive test reactions are greatly reduced and a wide variety of antigen-detection tests can now be performed in the clinical microbiology laboratory.

Many immunoassays are available, but three major types are in common use: immunofluorescence, latex agglutination, and enzyme immunoassay or EIA. For these immunoassays, monoclonal antibodies are labeled with (attached to) a "marker" molecule that provides a means of detecting whether an antigen-antibody reaction has taken place. Polyclonal antibody preparations are sometimes used for special purposes. The principles of these three assays are described briefly.

Immunofluorescence

In immunofluorescence assays, antibodies are labeled with a fluorescent dye called fluores-cein. When the antibodies combine with their specific antigen in a preparation, the bright fluorescence can be visualized with a fluorescence microscope fitted with an ultraviolet illuminator and special filters. The test is performed by placing a smear of a clinical sample on a microscope slide and fixing it with a suitable reagent. In the simplest method, known as a direct fluorescent antibody (DFA) test, the fluorescein-labeled antibody preparation is applied directly to the specimen slide, which is then incubated, washed, and viewed under the fluorescence microscope. A positive test is indicated by the presence of brightly fluorescing organisms in the preparation (see colorplates 23, 40, and 53).

For the indirect fluorescent antibody (IFA) test, two antibody preparations are needed. The first, which is not labeled with the fluorescent dye, contains antibodies against the mi-crobial agent we wish to detect. If the agent is present in the specimen smear, an antigen-antibody reaction occurs. To detect this combination, the second antibody, labeled with fluorescein, is applied to the preparation. This second antibody has been prepared to react with the first, unlabeled antibody. Again, a positive result is indicated by bright fluorescence under the microscope.

Figure 19.1 illustrates the principle of direct and indirect fluorescence assays. Fluorescent antibody tests are in widespread use to diagnose infections caused by a variety of microbial agents including bacteria, viruses, and protozoa.

Latex Agglutination

In latex agglutination assays, antibodies are attached to latex (polystyrene) beads that serve as the marker for detecting the antigen-antibody interaction. Each latex particle is about 1 ^m in diameter and can be charged with thousands of antibody molecules. Antibody-coated latex particles form a milky suspension, but when they are mixed with a preparation containing specific antigen, the resulting antigen-antibody complex results in visible clumping. Figure 19.2 illustrates the events associated with the latex particle agglutination reaction.

Latex agglutination tests are usually performed on a glass slide or a specially treated cardboard surface using small volumes of latex particles and liquid clinical sample. The reagent is mixed with the clinical sample using a stirrer, and the slide is rocked by hand or rotated with a mechanical device for several minutes before being examined visually for clumping of the latex particles. Colorplate 24 illustrates the appearance of positive and negative latex agglutination slide tests.

In clinical laboratories, latex agglutination tests are used to detect soluble micro-bial antigens directly in serum or cerebrospinal fluid specimens, or for identifying various types of bacteria recovered from culture plates.

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Figure 19.1 (a) In the direct fluorescent antibody (DFA) test, antibody specific for the microorganism sought is conjugated with the dye fluorescein. The antibody preparation is added to a specimen fixed to a slide. If the specific microorganism is present, the preparation will fluoresce when viewed under a fluorescence microscope. (b) In the indirect fluorescent antibody (IFA) test, the antibody specific for the microorganism is not conjugated with the dye, but will bind to the specific microorganism on the slide. A second antibody preparation, labeled with fluorescein, has been prepared to react with the first, unlabeled antibody and will fluoresce when viewed microscopically. (Modified from

L. M. Prescott et al. Microbiology, 4th ed., WCB/McGraw Hill).

Mcgraw Hill Steps Antibody Production
(a)
Fluorescent Enzyme Immunoassay Principle

Enzyme Immunoassay (EIA)

As in fluorescent antibody tests, the antibody in EIAs is conjugated with a marker that can be detected when an antigen-antibody reaction has taken place. In EIAs, the marker is an enzyme, typically alkaline phosphatase or horseradish peroxidase. These enzymes catalyze the breakdown of a colorless substrate to a colored end-product. To visualize the binding of antigen and the enzyme-linked antibody, the appropriate substrate for the enzyme must be added. A positive reaction results in the production of a colored end-product that can be detected visually or measured quantitatively in a spectrophotometer.

Two basic formats for EIA testing are in use. In the first, monoclonal antibody specific for the antigen sought is bound to a solid surface such as plastic tubes, beads, or wells of a microtiter tray. The clinical sample is added to this solid surface followed by incubation and washing steps. If the antigen is present in the sample, it will bind to the antibody and unbound material is washed away. Now, the enzyme-labeled antibody is added to detect the antigen-antibody complex. This step is accomplished either in a direct or indirect manner. In the direct method, the second antibody, which is conjugated to the enzyme, reacts with antigen bound by the first antibody on the solid surface. In the indirect method, two additional antibodies are needed to develop the reaction. The first is unlabeled antibody specific for the bound antigen and the second is an enzyme-labeled antibody that reacts with the first antibody. In this way, the indirect EIA is similar to the IFA test. Finally, in both test methods the substrate for the enzyme is added. The amount of colored end-product that develops indicates the amount of antigen present in the clinical sample. Figure 19.3 illustrates direct and indirect EIA methods.

EIA kits for detecting certain parasites, rotavirus, and other enteric viruses, and toxins of diarrhea pathogens, are available in this format. Colorplate 25 illustrates such a kit.

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Figure 19.2 Diagram of a latex reaction. (a) When latex particles that are coated with an antibody (for example, group A

Streptococcus antibody) react with a specific antigen (group A Streptococcus antigen), the particles join together to form clumps that agglutinate on the test slide (lower left corner of (a)). In the control test that is always run in parallel on the same slide, the same antigen is mixed with latex particles that are not coated with antibody, therefore, the particles remain in suspension and do not agglutinate. In diagram section (b), the antibody-coated particles do not react with the nonspecific antigen (for example, group B Streptococcus antigen), therefore, no clumps are formed, and the test as well as the control suspension shows no agglutination (lower left corner of (b)).

Figure 19.2 Diagram of a latex reaction. (a) When latex particles that are coated with an antibody (for example, group A

Streptococcus antibody) react with a specific antigen (group A Streptococcus antigen), the particles join together to form clumps that agglutinate on the test slide (lower left corner of (a)). In the control test that is always run in parallel on the same slide, the same antigen is mixed with latex particles that are not coated with antibody, therefore, the particles remain in suspension and do not agglutinate. In diagram section (b), the antibody-coated particles do not react with the nonspecific antigen (for example, group B Streptococcus antigen), therefore, no clumps are formed, and the test as well as the control suspension shows no agglutination (lower left corner of (b)).

Latex Bead Agglutination

Positive Control test

Positive Control test

Direct And Indirect Immunoassays

Negative Control test

Negative Control test

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Figure 19.3 Direct and indirect enzyme immunoassays. (Modified from L. M. Prescott et al. Microbiology, 4th ed., WCB/McGraw Hill).

(a) Direct enzyme immunoassay

(b) Indirect enzyme immunoassay

Figure 19.3 Direct and indirect enzyme immunoassays. (Modified from L. M. Prescott et al. Microbiology, 4th ed., WCB/McGraw Hill).

(a) Direct enzyme immunoassay

(b) Indirect enzyme immunoassay

Antibody specific for the patient antigen sought is bound to a solid surface.

Antibody specific for the patient antigen sought is bound to a solid surface.

Antibody specific for the patient antigen sought is bound to a solid surface.

Antibody specific for the patient antigen sought is bound to a solid surface.

Clinical sample is added. If the specific antigen is present, it will bind to the

Clinical sample is added. If the specific antigen is present, it will bind to the

Clinical sample is added. If the specific antigen is present, it will bind to the

Unlabeled antibody specific for patient antigen then binds to antigen.

Enzyme-linked antibody specific for patient antigen then binds to antigen.

Unlabeled antibody specific for patient antigen then binds to antigen.

Enzyme-linked antibody specific for patient antigen then binds to antigen.

Enzyme's substrate ( ■ ) is added, and reaction produces a visible color change ( # ).

Enzyme-labeled antibody reacts with first antibody.

Wash

Enzyme's substrate ( ■ ) is added, and reaction produces a visible color change ( # ).

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Figure 19.4 An enzyme immunoassay kit for "strep" throat. The left side illustrates a negative test, with only the control (C) line positive. On the right, both the control (C) and patient specimen (T) are positive as illustrated by the appearance of two lines.

Figure 19.4 An enzyme immunoassay kit for "strep" throat. The left side illustrates a negative test, with only the control (C) line positive. On the right, both the control (C) and patient specimen (T) are positive as illustrated by the appearance of two lines.

Streptococcus Enzyme Immunoassay

In another format for performing EIA tests, the specific enzyme-labeled antibody is bound to a porous nitrocellulose or nylon membrane. The membrane is contained in a disposable plastic cassette with a small chamber to which the liquid clinical sample can be added (fig. 19.4). An absorbent material on the underside of the membrane serves to draw the liquid sample through the membrane. If antigen is present in the sample, it will bind to the membrane-bound antibody as the material passes through the membrane. After the colorless substrate is added and passed through the membrane filter, development of a colored area indicates a positive test (fig. 19.4).

Membrane-bound EIA tests are very popular because they are easy to perform and reliable results are usually available within 5 to 10 minutes. They are available for direct specimen detection of some bacteria and viruses.

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Responses

  • katri
    What type of immunoassay typically results in visible clumping of particles?
    6 years ago
  • amna
    What does a positive strep test look like?
    3 years ago

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