1. Serological reactions in Microbiology Tatyana Ivakhnyuk

2. 2 Serology A science that attempts to detect signs of infection in a patient’s serum such as Ab for a specific microbe Serological tests based on Abs specifically binding to Ag. Ag of known identity will react with Ab in an unknown serum sample. Known Ab can be used to detect Ag in serum Ag-Ab reactions are visible by clumps, precipitates, color changes or release of radioactivity. The most effective tests have high specificity and sensitivity.

3. 3 Types of serological tests Agglutination tests Precipitation tests Immunoelectrophoresis Western blot tests Complement fixation tests Immunofluorescence testing Immunoassays

4. Characteristics of serological reactions Reactants used Visible and invisible reactions Determination of titer Hapten reactions Serology

5. 5 Agglutination tests – Ab cross-links whole cell Ag, forming complexes that settle out and from visible clumps in the test chamber blood type, some bacterial & viral diseases

6. Agglutination General reaction Example in Widal test Tube and slide agglutinations Passive agglutination Hemagglutination Coombs test

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13. Precipitation Tests One of the easiest of serological tests Relies on fact that antigens and antibody mixed in the proper proportion form large macromolecular complexes called precipitates Correct proportions are important to create precipitation Two techniques determine optimal antibody and antigen concentrations Immunodiffusion Immunelectrophoresis

14. Ring Precipitation Test Components: Material from patient (unknown Ag). Specific precipitation serum – (known Ab). Used: For express-diagnosis of infection disease (ex. Anthrax) 14

15. Immune Testing

16. Immune Testing

17. Complement fixation Components of the CFT: 1. Serum from patient (unknown Ab). 2. Specific antigen (known Ag). 3. Complement. 4. Erythrocytes from sheep. 5. Hemolytic serum (in this serum present Ab against Erythrocytes from sheep).

18. Complement fixation: Principle  18

19. 19 Immunofluorescence testing uses fluorescent Ab either directly or indirectly to visualize cells or cell aggregates that have reacted with the FAbs

20. Immuno-fluorescence Principle Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems Requires a fluorescent microscope Examples Herpes virus IgM Dengue virus Rabies virus Scrub and murine typhus Photos public domainPhotos public domain

21. Types of immuno-fluorescence Direct immuno-fluorescence Used to detect antigen Indirect and sandwich immuno-fluorescence Antigen detection Antibody detection Graphic self preparedGraphic self prepared

23. ELISA Enzyme-linked immunosorbent assay (ELISA) can detect unknown Ag or Ab by direct or indirect means. A positive result is visualized when a colored product is released by an enzyme-substrate reaction. ELISAs Stands for enzyme-linked immunosorbent assay Uses an enzyme as the label Reaction of the enzyme with its substrate produces a colored product indicative of a positive test Most common form of ELISA is used to detect the presence of antibodies in serum

24. ELISA

25. ELISA – general skim

26. 26 Immunoelectrophoresis – migration of serum proteins in gel is combined with precipitation by Ab

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28. 28 Western blot test – separates Ag into bands. After the gel is affixed to a blotter, it is reacted with a test specimen and developed by radioactivity or with dyes

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