Diagnostic Immunology

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

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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.

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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).

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• Serological tests

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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.
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Precipitation reactions

• Visible soluble precipitate
• mix soluble antigen and antibody
• excess antigen or antibody--no precipitate
• zone of equivalence--precipitate forms

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Zone of equivalence

• change the amount of antigen
• constant amount of antibody

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Gel precipitation

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

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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

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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

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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

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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

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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)

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Qualitative agglutination

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

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Quantitative agglutination

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

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Complement fixation

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

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Complement fixation

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

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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

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ELISA

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

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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

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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

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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