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Diagnostic Immunology

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Diagnostic Immunology. Objectives. Know how the following work and ... This produces local swelling (induration). The diameter of the induration is measured. ... – PowerPoint PPT presentation

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Title: Diagnostic Immunology


1
Diagnostic Immunology
2
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

3
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
12
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.

13
Enzyme Immuno Assay to detect an antigen Eg.
Hepatitis B Surface Ag HBsAg
14
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.

15
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.

16
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.

17
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.

20
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

21
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

22
An example of a particle agglutination test. The
particle can be a latex particle, a bead of
gelatin or a bacteria.
23
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)

24
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
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