Sumy State University The Department of Infectious Diseases

Sumy State University The Department of Infectious Diseases and Epidemiology with Course of Microbiology, Virology and Immunology Serological reactions in Microbiology Practical lesson 14 Tatyana Ivakhnyuk 2010

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. 2

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

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

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 5

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

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

EXAPLE OF BACTERIAL AGGLUTINATION is the reaction between a particulate antigen and its specific antibodies Antibody specificity known (S. enteriditis) Salmonella species identity not known (? ) Agglutination reaction between antibody and particulate antigen agglutination ? Positive Negative no agglutination Table 1. Serological classification of some common Salmonella species Serological group S. typhimurium B S. paratyphi C C 1 S. enteriditis D S. anatum E

Bacterial agglutination no agglutination ?

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. Na. Cl solution. 10

Passive (indirect) agglutination 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) 11

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. Na. Cl solution. Antigen Dilution of serum from patient 1: 20 1: 40 1: 80 1: 160 1: 320 agglutination Control non agglutination 12

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

Precipitation test in gel Immune Testing [INSERT FIGURE 17. 7]

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 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. 18

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

Labeling technique Immuno-fluorescence • Principle – Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems – Requires a fluorescent microscope • Examples – Herpes virus Ig. M – Dengue virus – Rabies virus – Scrub and murine typhus Cell infected with Dengue virus V. Cholerae

Labeling technique Types of immuno-fluorescence • Direct immunofluorescence – 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 enzymesubstrate 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

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

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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 28

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