Principles of Immunological Testing

The specificity of immunological reactions is useful in many diagnostic tests. During most infectious diseases, antibodies and immune cells specific for the infecting agent are produced. In a number of conditions other than infections, antibodies or immune cells are also formed. These can be detected and measured to aid in diagnosis and management of the condition. Specific antibodies are found in many locations; they are plentiful in the blood and are easily obtained from serum, the fluid from clotted blood. Serology refers to the use of serum antibodies to detect and measure antigens, or conversely, the use of antigens to detect serum antibodies. Immunoassay is another term used to describe assays, or tests, using immunological reagents such as antigens and antibodies. Monoclonal antibodies are often used in immunoassays (see Perspective 17.1).

Quantifying Antigen-Antibody Reactions

Individuals exposed to infectious agents for the first time usually do not have detectable specific antibodies in the blood serum until about a week or 10 days after infection. The change from negative serum without specific antibodies to serum positive for specific antibodies is called seroconversion. As the infection progresses, more and more antibodies are formed and the amount of antibody in the blood, the titer, increases. Small but steady amounts of antibody could result from infection at a previous time or for other reasons, but a rise in titer of antibodies is characteristic of an active infection.

Specimens that can be tested for the presence of antigens or antibodies include blood serum (the fluid portion remaining after blood clots) and plasma (the fluid portion of blood, treated with an anticoagulant to prevent clotting). In addition, urine, cerebrospinal fluid, sputum, or other body constituents may contain antigens or antibodies. Also, antigens and antibodies can be detected in solid tissues in thin sections examined with tagged reagents, such as fluorescent-labeled antibodies. Blood serum is probably the specimen most often used. A single sample of serum or other specimen can be used for many different tests, due to the availability of micromethods and automated techniques.

The amount of antibodies or the titer of a serum sample is usually determined in serial dilutions. Serum is diluted in either 2-fold or 10-fold dilutions, antigen is added to each dilution, and the titer is assigned to the last dilution that gives a detectable antigen-antibody reaction. For example, undiluted serum is diluted with a saline solution half and half, to give a 1:2 dilution; this dilution is again mixed with an equal volume of saline to give a 1:4 dilution; the 1:4 dilution is again diluted by half to give a 1:8 dilution; and so on, to produce dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1,024, and so forth. To each of these dilutions of antibody-containing serum is added an equal volume of antigen, further diluting the amount of serum present. For example, if the volume of serum dilution is 0.1 ml, then 0.1 ml of antigen is added. The final serum dilutions would then be 1:4, 1:8, and so on through 1:2,048. After waiting enough time for the antigen-antibody interaction to occur, a lab worker examines each dilution for evidence of a reaction. The reciprocal of the last dilution showing a positive reaction is taken as the titer. Thus, if a positive

17.3 Principles of Immunological Testing

PERSPECTIVE 17.1 Monoclonal Antibodies

In 1975, an exciting breakthrough occurred in immunology. Georges Köhler and Cesar Milstein developed techniques that fused normal antibody-producing B lymphocytes with malignant plasma cells (myeloma tumor cells), resulting in clones of cells they termed hybridomas. Since these hybridomas are clones, they produce antibodies with a single specificity that, therefore, are known as monoclonal antibodies (figure 1).

Plasma cells produce large amounts of antibody, and when they become malignant they grow profusely and indefinitely. Special myeloma cells are used to make hybridomas; they have lost the ability to make their own specificity of antibody but have retained the ability to produce large amounts of immunoglobulin.The normal B cell in the hybridoma supplies the genes for the specific antibody to be produced; the myeloma cell supplies the cellular machinery, the rough endoplasmic reticulum, for producing the antibodies.

Usually, when an animal is injected with an immunizing agent, it responds by making a variety of antibodies directed against different epitopes on the antigen. Therefore, even though there is a single antigen, the result is a mixture of different antibodies.When these antisera are used in immunological tests, standardizing the results is difficult, since there are differences each time the antiserum is made. Monoclonal antibodies, however, will be of the same immunoglobulin class and have the same variable regions and, thus, the same specificity and other characteristics. With such specificity, tests can be standardized much more easily and with greater reliability.

It was hoped that monoclonal antibodies would also become a useful therapeutic tool. In theory, it should be possible to make monoclonal antibodies that are specific for, say, a cancer cell.

Hyperimmunize mouse with antigen a to produce many lymphocytes making anti-a antibodies.

Remove spleen and make a suspension of lymphocytes.

Antibody-producing lymphocytes (B cells) from spleen.

Add a chemical that induces fusion of the two cell types to form hybridomas.

Select single hybridoma cells producing anti-a antibody and clone the hybridomas.

Figure 1 Production of Monoclonal Antibodies

Antibody-producing cells

Radioactive materials that destroy cancer cells could be attached to the monoclonals, making a sort of "magic bullet." These antibodies would search and find the cancer cells and attach specifically to them, allowing the radioactive material to destroy the cells. In reality, many problems have arisen when the monoclonal antibodies have been used to treat humans. One problem was that mouse cells were used to produce the hybridomas, and humans reacted to the mouse antigens on the hybridoma antibodies, causing them to be rapidly removed from the body.This limited the effectiveness of the monoclonal antibodies to one or a few doses.This and many other unforeseen difficulties are being addressed now, with some encouraging results. For example, from the spleen of a mouse immunized with the desired antigen are fused with myeloma tumor cells.The hybrid clone is grown as a line of cells in vitro, all producing large amounts of homogeneous antibody.

Hyperimmunize mouse with antigen a to produce many lymphocytes making anti-a antibodies.

Remove spleen and make a suspension of lymphocytes.

Antibody-producing lymphocytes (B cells) from spleen.

Add a chemical that induces fusion of the two cell types to form hybridomas.

Select single hybridoma cells producing anti-a antibody and clone the hybridomas.

Figure 1 Production of Monoclonal Antibodies

Antibody-producing cells

Principles Cell Fusion

Myeloma cell culture

Mix lymphocytes with special cultured myeloma cells that have lost the ability to produce their specific antibody and that lack a particular enzyme.

Hybridomas grow indefinitely because missing enzyme was supplied by the fused lymphocyte.

Myeloma cells

Grow hybridomas in cultures and purify large amounts of antibody from the culture medium.

Myeloma cell culture

Myeloma cells

Mix lymphocytes with special cultured myeloma cells that have lost the ability to produce their specific antibody and that lack a particular enzyme.

Grow hybridomas in a special medium. Spleen lymphocytes only grow a few days in culture. Myeloma cells will not survive in the special medium because of the lacking enzyme.

Hybridomas grow indefinitely because missing enzyme was supplied by the fused lymphocyte.

Grow hybridomas in cultures and purify large amounts of antibody from the culture medium.

genetic engineering is being used to construct antibody genes from human DNA that can be put into hybridomas to produce monoclonal antibodies useful in treating humans.

In the laboratory, monoclonal antibodies are the basis of a number of diagnostic tests. For example, monoclonal antibodies against a hormone can detect pregnancy only 10 days after conception. Specific monoclonal antibodies are used for rapid diagnosis of hepatitis, influenza, herpes simplex, and chlamydia infections. Köhler and Milstein won the Nobel Prize in 1984 for their work.

Chapter 17 Applications of Immune Responses

Well number

Figure 17.2 Quantitation of Immunologic Tests (a) Microtiter plates used to quantitate immunoassays. (b) Hemagglutination inhibition tests done in a microtiter plate. Agglutinated cells form a rough pattern over the bottom of the well, while unagglutinated cells fall into a small button at the bottom. Eight tests (A-H) can be done in a single plate.

Dilutions:

Well number

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