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Diagnostic Immunology

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Immunology Tutorial Introduction & Course outline By: Moh d J. Al Khatatneh Immunological Reactions The ability to visualize Ab-Ag reactions is a powerful tool for ... – PowerPoint PPT presentation

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Title: Diagnostic Immunology


1
Immunology Tutorial Introduction Course
outline By Mohd J. Al Khatatneh
2
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.

3
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.

4
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).

5
  • Serological tests

6
Serology
Antibody 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.
7
Precipitation reactions
  • Visible soluble precipitate
  • mix soluble antigen and antibody
  • excess antigen or antibody--no precipitate
  • zone of equivalence--precipitate forms

8
Zone of equivalence
  • change the amount of antigen
  • constant amount of antibody

9
Gel precipitation
  • Agar dish
  • solid medium
  • One well contains antibody
  • Other well contains antigen
  • Allow diffusion
  • Form precipitate at zone of equivalence

10
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

11
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

12
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

13
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14
Agglutination reactions
  • Similar to precipitation reaction
  • Visible reaction because antigen or antibody is
    on larger molecule
  • cell
  • latex bead
  • Interaction of antigen and antibody
  • clumping of large particles

15
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)

16
Qualitative agglutination
  • Known antigen in fluid
  • Unknown specimen added
  • Agglutination
  • positive reaction
  • No agglutination
  • negative reaction

17
Quantitative agglutination
  • Similar to qualitative
  • Diluted samples of antibody
  • Measure amount of agglutination for each dilution

18
Complement fixation
  • Positive reaction
  • Antibody present in serum
  • Serum added to test antigen
  • Bound antibody fixes complement
  • No available complement to lyse indicator cells

19
Complement fixation
  • Negative reaction
  • No antibody in serum
  • Complement not fixed
  • Free complement lyses indicator cells

20
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

21
ELISA
  • Example of immunoassay
  • Indirect ELISA
  • antigen coated to plastic well
  • protein blocks remaining
  • plastic surface

22
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

23
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

24
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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