T a t y ana Ivakhnyuk The D epartment of I nfectious D iseases and E pidemiology with C ourse of M icrobiology V irology and I mmunology ID: 1047016
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1. Serological reactions in MicrobiologyTatyana IvakhnyukThe Department of Infectious Diseases and Epidemiology with Course of Microbiology, Virology and Immunology Sumy State University Practical lesson 142010
2. 2SerologyA science that attempts to detect signs of infection in a patient’s serum such as Ab for a specific microbeSerological 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 serumAg-Ab reactions are visible by clumps, precipitates, color changes or release of radioactivity.The most effective tests have high specificity and sensitivity.
3. 3Types of serological testsAgglutination testsPrecipitation testsImmunoelectrophoresisWestern blot testsComplement fixation testsImmunofluorescence testingImmunoassays
4. Characteristics of serological reactionsReactants usedVisible and invisible reactionsDetermination of titerHapten reactionsSerology
5. 5Agglutination tests – Ab cross-links whole cell Ag, forming complexes that settle out and from visible clumps in the test chamberblood type, some bacterial & viral diseases
6. AgglutinationGeneral reactionExample in Widal testTube and slide agglutinationsPassive agglutinationHemagglutinationCoombs test
7. 7General reaction agglutination or Bacterial AgglutinationComponents:Specific agglutination serum (known Ab).Pure culture of bacteria (unknown Ag).NaCl solution.Used:In bacteriological method for identification pure culture of bscteria.
8. Table 1. Serological classification of some common Salmonella species. Salmonella species Serological group S. typhimurium B S. paratyphi C C1 S. enteriditis D S. anatum E agglutinationno agglutination?Antibody specificity known (S. enteriditis)Salmonella species identity not known (?)Agglutinationreaction between antibody and particulate antigen EXAPLE OF BACTERIAL AGGLUTINATIONis the reaction between a particulate antigen and its specific antibodiesPositive Negative
9. Bacterial agglutination?agglutinationno agglutination
10. 10Tube agglutinations test - Serological reaction in Serological methodComponents:Serum from patient (unknown Ab).Specific diagnosticum – killed known pure culture of bacteria (known Ag).NaCl solution.
11. 11Principleprecipitation reaction converted into agglutination - coating antigen onto the surface of carrier particles like red blood cells, latex, gelatin, bentonitebackground clearsExamples of typeslatex agglutinationco-agglutinationpassive hemagglutination (treated red blood cells made resistant)Passive (indirect) agglutination
12. 12Passive Hemagglutination Agglutination Test (PHAT)Components:Pair serum from patient (unknown Ab).Specific erythrocyte diagnosticum – (known Ag on surface erythrocytes from sheep ).NaCl solution.AntigenDilution of serum from patient 1:20 1:40 1:80 1:160 1:320 Control agglutination non agglutination
13. Precipitation TestsOne of the easiest of serological testsRelies on fact that antigens and antibody mixed in the proper proportion form large macromolecular complexes called precipitatesCorrect proportions are important to create precipitationTwo techniques determine optimal antibody and antigen concentrations ImmunodiffusionImmunelectrophoresis
14. Ring Precipitation TestComponents:Material from patient (unknown Ag).Specific precipitation serum – (known Ab).Used:For express-diagnosis of infection disease (ex. Anthrax)14
15. Immune Testing[INSERT FIGURE 17.7]Precipitation test in gel
16. Immune Testing[INSERT FIGURE 17.8]
17. Complement fixationComponents 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).Experiment systemIndicator system
18. Complement fixation: Principle 18 Complement fixation tests detect lysins- Ab that fix complement and can lyse target cells. Involves mixing test Ag and Ab with complement and then with sensitized sheep RBCs. If complement is fixed by the Ag-Ab, the RBCs remain intact and the test is positive. If RBCs are hemolyzed, specific Ab are lacking and the test is negative.
19. 19Immunofluorescence testing uses fluorescent Ab either directly or indirectly to visualize cells or cell aggregates that have reacted with the FAbs
20. Labeling techniqueCell infected with Dengue virusV. CholeraeImmuno-fluorescencePrincipleUse fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systemsRequires a fluorescent microscopeExamplesHerpes virus IgMDengue virusRabies virusScrub and murine typhus
21. Labeling techniqueTypes of immuno-fluorescenceDirect immuno-fluorescence Used to detect antigenIndirect and sandwich immuno-fluorescenceAntigen detectionAntibody detection
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23. ELISAEnzyme-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.ELISAsStands for enzyme-linked immunosorbent assayUses an enzyme as the labelReaction of the enzyme with its substrate produces a colored product indicative of a positive testMost common form of ELISA is used to detect the presence of antibodies in serum
24. ELISA
25. ELISA – general skim
26. 26Immunoelectrophoresis – migration of serum proteins in gel is combined with precipitation by Ab
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28. 28Western 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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