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Nitrocellulose sheet is cut into strips.

Proteins are transferred onto a sheet of nitrocellulose.

Detection of specific antibody is based on enzyme-substrate colored reaction product, which occurs in a band pattern based on the position of the proteins on the strip.

Radioimmunoassays

Radioimmunoassay (RIA) is an automated method of detecting antibodies that is usually performed in a chemistry laboratory, rather than in a serology laboratory. RIA tests were originally used to detect antibody to hepatitis B viral andgens. Radioactively labeled antibody competes with the patient's unlabeled antibody for binding sites on a known amount of antigen, A reduction in radioactivity of the antigen-patient antibody complex compared with the radioactive counts measured in a control test with no antibody is used to quantitate the amount of patient antibody bound to the antigen. The development of new marker substances, such as ELISA systems, chemiluminescence, and fluorescence, resulted in the production of diagnostic tests as sensitive as RIA without the hazards and associated disposal problems of radioactive reagents.

Fluorescent Immunoassays

Because of the inconveniences associated with RIA (radioactive substances and expensive scintillation counters), fluorescent immunoassays (FIA) were developed. These tests, which use fluorescent dyes or molecules as markers instead of radioactive labels, are based on the same principle as RIA. The primary difference is that in RIA systems the competitive antibody is labeled with a radioisotope and in FIA the antigen is labeled with a compound that fluoresces under the appropriate light rays. Binding of patient antibody to a fluorescent-labeled antigen can reduce or quench the fluorescence, or binding can cause fluorescence by allowing conformational change in a fluorescent molecule. Measurement of fluorescence is thus a direct measurement of antigen-antibody binding, not dependent on a second marker system such as that in ELISA tests. Systems are commercially available to measure antibody developed against numerous infectious agents, as well as against self-antigens (autoimmune antibodies).

Western Blot Immunoassays

Requirements for the detection of very specific antibodies have driven the development of the Western blot immunoassay system (Figure 10-7). The method is based on the electrophoretic separation of major proteins of an infectious agent in a two-dimensional agarose gel matrix. A suspension of the organism against which antibodies are being sought is mechanically or chemically disrupted, and the solubilized antigen suspension is placed at one end of a polyacrylamide (polymer) gel. Under influence of an electrical current, the proteins migrate through the gel. Most bacteria or viruses contain several major proteins that can be recognized based on their position in the gel after electrophoresis. Smaller proteins travel faster and migrate

Figure 10-8 Human immunodeficiency virus type 1 (HIV-1) Western blot. Samples are characterized as positive, indeterminate, or negative based on the bands found to be present in significant intensity. A positive blot has any two or more of the following bands: p24, gp41, and/or gp120/160. An indeterminate blot contains some bands but not the definitive ones. A negative blot has no bands present. Lane 16 shows antibodies from a control serum binding to the virus-spedfic proteins (p) and glycoproteins (gp) transferred onto the nitrocellulose paper. (Courtesy Calypte Biomedical Corporation, Pleasanton, Calif.)

Figure 10-8 Human immunodeficiency virus type 1 (HIV-1) Western blot. Samples are characterized as positive, indeterminate, or negative based on the bands found to be present in significant intensity. A positive blot has any two or more of the following bands: p24, gp41, and/or gp120/160. An indeterminate blot contains some bands but not the definitive ones. A negative blot has no bands present. Lane 16 shows antibodies from a control serum binding to the virus-spedfic proteins (p) and glycoproteins (gp) transferred onto the nitrocellulose paper. (Courtesy Calypte Biomedical Corporation, Pleasanton, Calif.)

farther in the lanes of the gel than do the larger proteins. The protein bands are transferred from the gel to a jditrocqllulose or other type of thin membrane, and the membrane is treated to immobilize the proteins. The jxiembrane is then cut into many thin strips, each carrying the pattern of protein bands. When patient serum is layered over the strip, antibodies will bind to each of the protein components represented by a band on the strip. The pattern of antibodies present can be used to determine whether the patient is infected with the agent (Figure 10-8). Antibodies against microbes with numerous cross-reacting antibodies, such as T. pallidum, B. burgdorferi, herpes simplex virus types 1 and 2, and HIV, are identified more specifically using this technology than a method that tests for only one general antibody type. For example, CDC defines an EIISA or immunofluorescence assay as a first-line

Lyme disease antibody test but positive or equivocal results must be confirmed by a Western blot test.

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