© 2004 wadsworth – thomson learning immunology tutorial introduction & course outline by:...
TRANSCRIPT
© 2004 Wadsworth – Thomson Learning
Immunology TutorialImmunology Tutorial
Introduction & Course outlineIntroduction & Course outline
By: Moh’d J. Al KhatatnehBy: 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
SerologyAntibody Type Antigen Called Reaction Called Definition
Precipitin Precipitinogen Precipitation The formation of an insoluble complex composed of a soluble Ag and a soluble Ab.
Agglutinin Agglutinogen Agglutination The cross-linking of a particulate or insoluble Ag by the corresponding Ab.
Hemolysin Hemolysis A 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.
Cytolysin Cytolysis A 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.
Antitoxin Toxin In 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