Serological reactions in Microbiology Tatyana Ivakhnyuk The Department of Infectious Diseases and Epidemiology with Course of Microbiology, Virology and Immunology Sumy State University Practical lesson

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 Types of serological tests 1.Agglutination tests 2.Precipitation tests 3.Immunoelectrophoresis 4.Western blot tests 5.Complement fixation tests 6.Immunofluorescence testing 7.Immunoassays

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

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

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

7 General reaction agglutination or Bacterial Agglutination Components: 1.Specific agglutination serum (known Ab). 2.Pure culture of bacteria (unknown Ag). 3.NaCl solution. Used: In bacteriological method for identification pure culture of bscteria.

Table 1. Serological classification of some common Salmonella species. Salmonella speciesSerological group S. typhimuriumB S. paratyphi CC1 S. enteriditisD S. anatumE agglutination no agglutination ? Antibody specificity known (S. enteriditis) Salmonella species identity not known (?) Agglutination  reaction between antibody and particulate antigen EXAPLE OF BACTERIAL AGGLUTINATION is the reaction between a particulate antigen and its specific antibodies Positive Negative

Bacterial agglutination ? agglutinationno agglutination

10 Tube agglutinations test - Serological reaction in Serological method Components: 1.Serum from patient (unknown Ab). 2.Specific diagnosticum – killed known pure culture of bacteria (known Ag). 3.NaCl solution.

11 Principle –precipitation reaction converted into agglutination - coating antigen onto the surface of carrier particles like red blood cells, latex, gelatin, bentonite background clears Examples of types –latex agglutination –co-agglutination –passive hemagglutination (treated red blood cells made resistant) Passive (indirect) agglutination

12 Passive Hemagglutination Agglutination Test (PHAT) Components: 1.Pair serum from patient (unknown Ab). 2.Specific erythrocyte diagnosticum – (known Ag on surface erythrocytes from sheep ). 3.NaCl solution. Antigen Dilution of serum from patient 1:20 1:40 1:80 1:160 1:320 Control agglutination non agglutination

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

Ring Precipitation Test Components: 1.Material from patient (unknown Ag). 2.Specific precipitation serum – (known Ab). Used: For express-diagnosis of infection disease (ex. Anthrax) 14

Immune Testing [INSERT FIGURE 17.7] Precipitation test in gel

Immune Testing [INSERT FIGURE 17.8]

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). Experiment system Indicator system

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

Labeling technique Cell infected with Dengue virus V. Cholerae 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

Labeling technique Types of immuno-fluorescence Direct immuno- fluorescence –Used to detect antigen Indirect and sandwich immuno-fluorescence –Antigen detection –Antibody detection

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

ELISA

ELISA – general skim

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

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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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