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LECTURE: 27 Title COMPLEX SEROLOGICAL LABORATORY TECHNIQUES LEARNING OBJECTIVES: The student should be able to: • Define the term "complex serological techniques". • Enumerate some serological applications in the immunology laboratory. • Explain the principle of the complement fixation test. • Explain the immunofluorescence (direct and indirect). • Explain the virus neutralization. • Enumerate procedures involving agglutination, and explaining the principle of each such as: - Hemagglutination inhibition. - Passive indirect agglutination. - Coombs (antiglobulin) "direct and indirect" tests. • Explain the principle of direct and indirect enzyme linked immunosorbent assay (ELISA). • Tests for rheumatoid factor such as: 1. Latex agglutination test (Latex coated with IgG). 2. Rose-Waaler test (tannic acid treated-SRBCs coated with rabbit IgG anti- human antibody). 3. Bis-diazotized Benzedine. 4. Test for soluble antigens (A two-step test can be employed to detect soluble antigens that react and neutralize a hemagglutinating antibody). • Explain the principle of direct and indirect radioimmunoassay (RIA). LECTURE REFRENCE: 1. TEXTBOOK: ROITT, BROSTOFF, MALE IMMUNOLOGY. 6th edition. Chapter 27. pg. 417-434. 2. TEXTBOOK: MARY LOUISE TURGEON. IMMUNOLOGY & SEROLOGY. IN LABORATORY MEDICINE. 2ND EDITION. Chapter 6. pg 111-131. 3. TEXTBOOK: Richard M. Hyde. NATIONAL MEDICAL SERIES FOR INDEPENDENT STUDY. pp 159-169. 1 COMPLEX SEROLOGIC PROCEDRURES I. Complex Serological Procedures Antigen – antibody reactions in which the visible manifestation requires the participation of accessory factors, indicator systems, and specialized equipment can be measured by several techniques. A) Complement, a protein constituent of normal blood serum, is consumed (i.e., fixed) during the interaction of antigens and antibodies. The phenomenon forms the basis for the complement fixation test, which is sensitive test that can be used to detect and quantitate antigens and antibodies. 1. The primary reacting ingredients are antigen, antibody, and complement. a. Normal guinea pig serum often is used as a primary source of complement because guinea pigs have high levels of complement with efficient lytic properties. b. Different sources of complement are used in different vitro test (e.g., rabbit complement is used in cytotoxicity tests performed for transplantation). 2. To use the complement fixation test to determine the presence of antibody to a known antigen in a patient's serum, a test system and an indicator system are used. a. Test system. The serum which is heated to 56oC to inactive native complement, has added to it measured amounts of antigen and complement (e.g., normal guinea pig serum). If antibody specific for the known antigen is present in the serum, antigen-antibody complexes will form that consume (or fix) all the complement. The initial reaction, however, cannot be seen. b. Indicator system. In a second step, an indicator system consisting of sheep red blood cell (SRBC) plus hemolysin, antibody specific for SRBC, is added to test for the presence of free complement. Interpretation of the test is based on the presence of hemolysis. 1) If all the complement has been fixed, none will be free to lyse the SRBCs, which constitutes a positive complement fixation test. 2) If no antibody is present in the patient's serum, then the complement is not fixed and is free to interact in the indicator system and lyse the SRBCs, which constitutes a negative complement fixation test. 3. Properly conducted complement fixation tests require the incorporation of appropriate controls of ensure that the result are not adversely affected by the presence of anticomplementary ingredients. The antigen or the serum itself may have anti- complementary properties (e.g., denatured or aggregated immunoglobulin, heparin, chelating agents, microbial contaminants), may fix all the complement in the system, or may remove calcium or magnesium ions (both of which are essential for complement- mediated lysis). B) Fluorescent dyes (e.g., fluorescein isothiocyanate) can be conjugated to antibody molecules to allow visualization of the molecules under ultraviolet light and a fluorescence microscope. Such labeled antibody may then be used to identify antigen. Both direct and indirect techniques are available. 2 1. Direct immunofluorescence uses antibody that is specific for a particular antigen or parasite labeled with a fluorescent dye, usually fluorescein. This allowed to react with an unknown tissue or organism. If the antibody reacts, it will be visualized as a green stain on the specimen when it is examined under ultraviolet light. To identify Treponema pallidum in exudates from a patient suspected of having syphilis, the following procedure is used. a. A slide of exudate is prepared and flooded with tagged, specific antibody. If organisms are present in the exudate, they will bind the tagged antibody. b. Excess antibody is then washed from the slide, and the slide is examined with a fluorescence microscope. T. pallidum will fluoresce against the black background. This is a remarkable rapid, useful test for the identification of unknown microorganisms. c. This same procedure is used in the identification of other human pathogens such as Legionella pneumophila and Mcoplasma pneumoniae. It also is useful in the detection of vial antigens in tissues. In the latter instance, horseradish peroxidase may be conjugated to the antibody. After the enzyme - antibody complex has reacted with the tissue, the excess reagent is washed away and an appropriate enzyme substrate is added to the tissue section. Bound antibody is detected by the presence of a dark precipitate at the site of antibody binding. d. Immunoperoxidase tests have advantages over immunofluorescent techniques. (1) The specimen can be stained with conventional histochemical dyes so structural detail can be used. (2) The tissue can be examined by standard light microscope procedures. 2. Indirect immunofluorescence procedures use antibody against antibody that has a fluorescent compound covalently coupled to it (e.g., rabbit antihuman gamma globulin antiserum). a. The "sandwich technique" allows for detection of antibody. (1) In the serodiagnosis of syphilis by the fluorescent treponemal antibody absorption (FTA- ABS) test, T. pallidum fixed to a slide is flooded with the patient's serum that is to be tested for antibody. If antibodies to the spirochete are present, they will bind to the organisms on the slide. (2) Excess antibody is removed by washing; to detect bound antibody, the preparation is overlain with fluorescein-tagged antibody to human gamma globulin. If the patient's serum contains antibody to T. pallidum, fluorescing organisms will be seen when the slide is examined with the fluorescence microscope. b. Indirect immunofluorescence also is used in detecting antinuclear antibodies (e.g., DNA, RNA, and histone). Antinuclear antibodies are present in systemic lupus erythematosus (SLE), and sometimes in rheumatoid arthritis and other autoimmune collagen – vascular diseases. For diagnosing SLE, the procedure is essentially identical to that described above for T. pallidum, except that the antigen is DNA in forms such as animal or human buffy coat cells, rat kidney sections, and beef thymus sections. C) Hemagglutination inhibition test. Hemagglutination involves the agglutination of red blood cells by antibodies (i.e., hemagglutinations), certain virus particles (e.g., influenza and mumps viruses), or other substances. Although viral hemagglutination inhibition test, which is extremely valuable as a viral diagnostic test (i.e., it demonstrates the presence of serum antibody to hemagglutinating viral substances). 3 1. Similar test can be used to detect soluble antigens that react with and neutralize a hemagglutinating antibody. 2. To examine the serum of a patient suspected of having influenza, the patient's serum is mixed with known influenza virus and red blood cells. a. If antibody is present, hemagglutination will be inhibited due to the ability of the antibody to bind to the virus and block its ability to Hemagglutinate: Serum antibody + Virus + Red blood cells = no Hemagglutination b. If no antibody is present, Hemagglutination will occur: Virus + Red blood cells = Hemagglutination D) Passive agglutination is the conversion of a reaction system from one that precipitates to one that agglutinates, thus yielding a more sensitive indication of antibody. 1. The use of latex particles in the diagnosis of rheumatoid arthritis is an example of passive agglutination. In this disease, the patient produces an antibody (mainly IgM) to his own IgG. The test consists of coating latex particles with IgG and reaching them with the patient's serum. Agglutination indicates the presence of antibodies (i.e., positive test). This antibody is called rheumatoid factor. 2. Bis-diazotized benzidine is a coupling reagent that can be used to conjugate proteins or haptens to red blood cells, thus allowing the detection of specific antibodies to these materials by passive hemagglutination procedures. 3. In the Rose-Waaler test, which detects rheumatoid factor in serum (an anti-IgG antibodies specific for the SRBCs. E) Coombs' (antiglobulin) test. In certain people, antibodies directed against antigenic determinants are unable to form visible aggregates when subjected to precipitation or agglutination procedures. To demonstrate the presence of antibody in such cases, the Coombs' (antiglobulin) test may be used. Coombs' test involves adding an
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