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© 2004 Wadsworth – Thomson Learning Immunology Tutorial Introduction & Course outline By: Moh’d J. Al Khatatneh
© 2004 Wadsworth – Thomson Learning Immunological Reactions The ability to visualize Ab-Ag reactions is a powerful tool for detecting, identifying, and quantifying antibodies or antigens. One may detect or identify an unknown antibody using a known antigen, or vice versa, one may use an antibody of known specificity to detect or identify an unknown antigen. This is the underlying basis of immunological testing.
© 2004 Wadsworth – Thomson Learning Characteristics aimed for in immunological testing: Specificity is the ability of a test to measure what is intended. A test with good specificity gives few false positive reactions. For example, an antibody which is specific will not cross-react. Sensitivity is the ability of a test to detect very, very small amounts of what is intended. A test with good sensitivity gives few false negative reactions. For example, an antibody that is very sensitive will detect minute quantities of its corresponding antigen.
© 2004 Wadsworth – Thomson Learning Serology Serology is a branch of immunology that deals with the in vitro diagnostic testing of antibodies and/or antigens (most often serum is used).
© 2004 Wadsworth – Thomson Learning Serological tests
© 2004 Wadsworth – Thomson Learning Serology Antibody TypeAntigen CalledReaction CalledDefinition PrecipitinPrecipitinogenPrecipitationThe formation of an insoluble complex composed of a soluble Ag and a soluble Ab. AgglutininAgglutinogenAgglutinationThe cross-linking of a particulate or insoluble Ag by the corresponding Ab. HemolysinHemolysisA reaction where the antibody is directed towards a red cell antigen and complement is activated to the C9 stage and the red cells are lyzed. CytolysinCytolysisA reaction where the antibody is directed towards a cell other than a red cell and complement is activated to the C9 stage and the cell is destroyed. AntitoxinToxinIn this reaction the antibody is directed towards a toxin, usually bacterial, and it neutralizes the toxin making it harmless.
© 2004 Wadsworth – Thomson Learning Precipitation reactions Visible soluble precipitate – mix soluble antigen and antibody – excess antigen or antibody--no precipitate – zone of equivalence--precipitate forms
© 2004 Wadsworth – Thomson Learning Zone of equivalence change the amount of antigen constant amount of antibody
© 2004 Wadsworth – Thomson Learning Gel precipitation Agar dish – solid medium One well contains antibody Other well contains antigen Allow diffusion Form precipitate at zone of equivalence
© 2004 Wadsworth – Thomson Learning Double immunodiffusion Two antigens and one antibody Place in separate wells Allow diffusion Lines of precipitation – continuous identical antigens – crossing lines completely different antigens – continuous with spur partial identity
© 2004 Wadsworth – Thomson Learning Single immunodiffusion Antibody mixed into gel specimens in well – screening for presence of antigen precipitate forms band around well – indicate presence of antigen – size of band relative to concentration of antigen
© 2004 Wadsworth – Thomson Learning Immunoelectrophoresis Separate antigens before testing put antigen in well expose to electrical field antigens are separated by size and charge add antibody and allow diffusion and precipitation precipitation with specific antibody gives identity of antigen
© 2004 Wadsworth – Thomson Learning
Agglutination reactions Visible reaction because antigen or antibody is on larger molecule – cell – latex bead Interaction of antigen and antibody – clumping of large particles Similar to precipitation reaction
© 2004 Wadsworth – Thomson Learning Agglutination reactions Direct--detect antibodies – using cells with antigen on them Indirect--detect antigen or antibody – coated spheres or cells – observe agglutination Hemagglutination – Red blood cells agglutinate – certain viruses (influenza)
© 2004 Wadsworth – Thomson Learning Qualitative agglutination Known antigen in fluid Unknown specimen added Agglutination – positive reaction No agglutination – negative reaction
© 2004 Wadsworth – Thomson Learning Quantitative agglutination Similar to qualitative Diluted samples of antibody Measure amount of agglutination for each dilution
© 2004 Wadsworth – Thomson Learning Complement fixation Positive reaction: – Antibody present in serum – Serum added to test antigen – Bound antibody “fixes” complement – No available complement to lyse indicator cells
© 2004 Wadsworth – Thomson Learning Complement fixation Negative reaction – No antibody in serum – Complement not “fixed” – Free complement lyses indicator cells
© 2004 Wadsworth – Thomson Learning Immunoassays Detect antigen or antibody – use a secondary antibody – tagged with marker radioactive fluorescent enzyme Multiple samples tested at once Great sensitivity – dependent on type of tag – much greater than other tests
© 2004 Wadsworth – Thomson Learning ELISA Example of immunoassay Indirect ELISA – antigen coated to plastic well – protein blocks remaining plastic surface
© 2004 Wadsworth – Thomson Learning ELISA – Serum added primary antibody if antibodies – bind antigen if no antibodies – antigen not bound – Indicator antibody enzyme-linked anti-Ig antibody binds primary antibody
© 2004 Wadsworth – Thomson Learning ELISA – Substrate specific for enzyme linked to secondary antibody enzyme causes substrate to change color – Reactions color change – antibody in serum no color change – no antibody in serum
© 2004 Wadsworth – Thomson Learning Immunofluorescence Antibody with fluorescent label – Bind to cell – Visualize under UV light Purpose – detect specific proteins in cells – detect viruses in cells – identify microbial cells – identify and sort cells fluorescent activated cell sorter
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Measurement of Immune function:. Immunological tests rely upon: Ability of antibodies to aggregate particulate antigens (agglutination) Or to precipitate.
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