Application of immunological tests

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• Application of immunological tests
• in diagnosis.

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Antigen -Antibody Reactions .

• Antigen antibody reactions are performed
  to determine the presence of either the
  antigen or antibody. ( serological tests ).
• One of the two components has to be
  known.
• e.g. with a known antigen, such as
  influenza virus , a test can determine
  whether antibody to the virus is present
  or not .

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Types of serological Tests

• Agglutination
• In this test the antigen is particulate (e.g.
  bacteria and red blood cells) or an inert
  particle (latex beads) coated with antigen.
• Antibody is divalent and cross links the
  multivalent antigen to form a lattice
  network or clumps
  (agglutination).
• This reaction can be performed in a tube
  or on a glass slide e.g. ABO blood
  grouping.

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Agglutination Test positive.
negative.
Antibody.
antigen
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Antigen Antibody Reactions

• Haemaggultination Tests
• Viruses can clump red blood cells from one
  species or another (active hemagglutination)
  .
• This can be inhibited by specific anti-viral
  antibodies.
• Red cells can also absorb many antigens and when
  mixed with specific antibodies will form
  clumps (passive hemagglutination) i.e. red
  cells are passive carriers .

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Haemaggultination Tests
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Precipitation test

• In this test antigen is in soluble form
  (solution).
• Antibody cross -links antigen molecules to
  form aggregates (precipitates) in the
  zone of equivalence optimal proportion of
  antigen and antibody.
• Precipitation test can be performed in
  solution or in semi- solid medium (agar).

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Zone of Equivalence
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Precipitation in Agar.

• Single radial immunodiffusion
• Antibody is incorporated into agar and
  antigen introduced into the well.
• As antigen diffuses into agar precipitation
  rings form depending on the concentration
  of the antigen.
• Radial Immunodiffusion is used to measure
  IgG, IgM and complement components.

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Single Radial Immunodiffusion.
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Double immunodiffusion.

• Antigen and antibody are placed in different
  wells in agar and allowed to
  diffuse and form precipitation lines at
  the points of optimal concentrations.
• This method is used to determine whether
  antigens are related, identical or
  non identical.

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Precipitation Test
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Double immunodiffusion
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RAST
Radioimmunoassay ( RAST) measure specific IgE.

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Enzyme Linked Immunosorbent Assay (ELISA)

• This method is used for measuring either
  antigen or antibody in patient serum ..
• For measurement of antibody, known antigen is
  fixed to a surface i.e. bottom of small wells on
  a plastic plate.
• Incubated with dilutions of the patients
  serum.
• Washed and then re-incubated with anti-human
  antibody labeled with an enzyme i.e.
  horseradish peroxidase.

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ELISA .
antigen
Antibody.
Enzyme Labelled antibody
Enzyme substrate.
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ELISA .

• Enzyme activity is measured by adding the
  substrate for the enzyme that leads
  to development of a color.
• Color reaction is estimated in a
  spectrophotometer.
• The amount of antibody bound is
  proportional to the enzyme activity.
• The titer of antibody in patients serum is
  the highest dilution of the serum
  that gives a positive color reaction .

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ELISA
Intensity of color correspond to concentration of
antibody.
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Immunofluoresence

• Fluorescent dyes e.g. fluorescein and
  rhodamine can be covalently attached to
  antibody molecules and made visible by
  ultraviolet (UV) light in a
  fluorescent microscope.
• Such labeled antibody can be used to
  identify antigens on surface of
  microorganisms ( e.g. treponemes), in
  histological section or in other
  specimens.

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Immunofluoresence

• Immunofluoresence reaction is called direct when
  a known labeled antibody interacts
  directly with unknown antigen .
• Indirect Immunofluoresence involves a two
  stage process
• Patients serum is added, incubated and the
  preparation is washed.
• Antigen is attached to a slide.
• Antibody of interest if present will
  remain attached and can be detected by
  addition of fluorescent dye labeled
  antibody under UV light.

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Immunofluoresence .
Antigen fixed on slide e.g. nuclear antigen .
Biopsy specimen from patient.
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Antigen Antibody Reactions
Immunofluoresence .
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Antigen Antibody Reactions

• Complement Fixation
• Based on the principle that antigen and
  antibody reaction activates complement .
• Antigen and antibody, one known and the other
  unknown are mixed.
• A measured amount of complement is added .
• If antigen-antibody reaction has occurred it
  will combine fix complement.

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Complement Fixation

• An indicator system consisting of sensitized
  red blood cells (red blood cells plus anti-red
  blood cell antibody) is added.
• If the complement was fixed because of antigen
  antibody reaction red cells will not be
  hemolyzed i.e. the test is positive.
• If the antigen antibody reaction did not occur in
  the first step complement will not be fixed and
  will be available to lyse RBCs a negative
  test.

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Complement Fixation Test
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Diagnosis of cell-mediated responses

• 1. Delayed hypersensitivity reactions .
• - delayed skin test.
• - patch test.
• 2. Lymphocyte transformation test .
• lymphocyte activation test.
• ( detect markers by flow cytometry .)

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contact dermatitis diagnosed by patch test .
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Patch test for contact dermatitis .
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Type 1 allergy diagnosed by skin prick test .