Diagnostic Immunology

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Diagnostic Immunology
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Objectives

• Know how the following work and some examples of
  how they might be used
• Enzyme immuno assays (EIAs)
• Particle agglutination tests work
• Fluorescence microscopy based detection
• Understand the Mantoux test for TB

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Types of antibodies used for diagnostic purposes

• Polyclonal antibodies Animals repeatedly
  immunized to develop very high antibody levels
  (the protein can be the organism of interest, a
  protein from its wall or human antibodies)
• Monoclonal antibodies developed when animal
  spleen cells are fused with malignant myeloma
  cells. These cells are then selected for those
  that produce only one kind of antibody in very
  high (and pure) amount.

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Measuring antibodies in a patients serum

• To detect infection
• IgM and bodies are usually a reflection of a
  recent infection eg. Measles, Rubella, Hepatitis
  A
• Rising levels of IgG antibodies often indicate
  recent infection (often this is a four fold
  increase but depends on the method) eg
  respiratory viruses
• Sometimes a very high titre of antibody will
  signal recent infection eg Legionnaires disease
• Any IgG antibody eg HIV or HCV
• To determine immunity

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An example of an EIA test to detect antibodies
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How are antibody levels detected?

• Usually by enzyme linked immunosorbent assays
  (ELISA).
• Coat a plastic well with proteins (antigens) from
  the organism you are interested in.
• Add the patients serum, if the patient has
  antibodies in the serum they will stick to the
  antigens.
• Wash the well to remove all the antibodies that
  are not specific (because they dont stick).
• Add an anti-antibody which is tagged with an
  enzyme this antibody sticks to the patients
  antibody.
• Add the substrate that the enzyme reacts on
• It turns the well yellow
• You measure the amount of yellow colour.
• More colourmore antibody. No colour no antibody.

Dont worry Ill explain in the class
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How are antigens detected in serum?

• By ELISA
• A well in a plate is coated with a specific
  antibody.
• The patients specimen is added to a tube
• The well is washed to remove non sticking Ag(
  error)
• A second antibody is added which is tagged with
  the enzyme (see Figure 18.12 in Tortora).
• The enzyme substrate is added and a color change
  is seen.

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Enzyme Immuno Assay to detect an antigen Eg.
Hepatitis B Surface Ag HBsAg
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How we detect the antigens that are in tissue as
a result of infection

• Fluorescence microscopy
• Using antibodies bound with fluorescein we can
  visualize infected cells or infecting bacteria.
• Put the specimen on a slide and dry it
• Add an antibody to the organism you are looking
  for
• Incubate it so that the antibody sticks
• Wash it off wellall of the unattached antibody
  is washed away.
• Look under a special fluorescent microscope to
  see if the cells are fluorescent.

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Detecting antigens using single use mini ELISA
kits

• Take a throat swab
• Treat it to extract the streptococcal antigens
• Put a drop in the device
• Let it diffuse along the filter until it comes up
  to the antibody in the filter paper. It will stop
  here and bind.
• Wash it to remove other garbage
• Add an enzyme tagged antibody to sandwich the
  antigen.
• Add a drop of the enzyme substrate and observe
  for a colour change.

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Detecting pregnancy using single use mini ELISA
kits

• Take a urine specimen
• Put a drop in the device
• Let it diffuse along the filter until it comes up
  to the antibody in the filter paper. It will stop
  here and bind.
• Wash it to remove other garbage
• Add an enzyme tagged antibody to sandwich the
  antigen.
• Add a drop of the enzyme substrate and observe
  for a colour change.

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How we detect the antigens that are in tissue as
a result of infection

• Fluorescence microscopy
• Put the specimen on a slide and dry it
• Overlay with specific antibodie bound with
  fluorescein
• Incubate it so that the antibody sticks
• Wash it off wellall of the unattached antibody
  is washed away.
• Look under a special fluorescent microscope to
  see if the cells are fluorescent.

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Examples of how fluorescent microscopy might be
used on patients specimens

• To detect influenza or RSV infected cells from
  naso-pharyngeal swabs.
• To detect Herpes virus infected cells scraped
  from the base of the ulcers
• To detect varicella zoster virus in cells scraped
  from the base of a chickenpox lesion.
• To detect Pneumocystis in a sputum sample.

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Examples of how fluorescence microscopy might be
used on cultures of patient specimens

• Occasionally, bacteriology and viruses need to be
  identified from cultures.
• Positive cultures (bacterial colonies or cells
  infected with virues) applied to slides can be
  examined by immunofluorescence.
• Herpes simplex virus (I or II) in tissue culture
• Respiratory viruses in tissue culture.
• Gonorrhoea and Legionella from agar culture
  plates

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Another method for detecting antigens in patient
specimens or on the surface of bacteria in the lab

• Particle agglutination tests
• The specimen (for instance spinal fluid or a
  bacteria from a culture in a lab) is mixed with
  tiny beads which have been coated with specific
  antibody
• If the specimen contains antigens, the particles
  come together and look quite granular, ie. They
  agglutinate
• Spinal antigen tests may be positive, even in
  patients who have received antibiotics after
  cultures have become negative

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An example of a particle agglutination test. The
particle can be a latex particle, a bead of
gelatin or a bacteria.
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How to use particle agglutination to detect
antibodies

• Instead of coating the beads with antibody you
  coat them with antigen
• You add the patients serum and if the patient has
  antibodies to that antigen the particles
  agglutinate.
• Examples
• Syphilis
• Mononucleosis (basis of the Monospot test)

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How to we use the cellular immune response to
diagnose infections?

• Skin testing is used most often (the TB skin test
  is the most common) (Mantoux test)
• TB antigens are injected under the skin (5 TU)
• Over 48 hours, cells migrate towards the injected
  antigen
• This produces local swelling (induration). The
  diameter of the induration is measured.
• Individuals without past TB have no induration