Immunological Methods in Microbiological Testing

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Immunological Methods in Microbiological Testing
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Testing Methods
Total heterotrophic plate count
Growth on detection media
Most Probable Number
Culture-dependent techniques
Sample with Bugs
Culture-independent techniques
Nucleic Acid-Based
Direct Observation
Immunological Methods
Chemical
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• Antigen any thing, foreign to the immune
  system. e.g. bacteria, viruses, (or their parts),
  pollen, etc.

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• Antibody proteins produced by the immune system
  which help defend against antigens

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• Antiglobulins antibodies produced by one species
  against antibodies of another

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Immune system a quick overview
www.sfaf.org/aids101/ immunology.html
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Polyclonal vs monoclonal antibiodies

• Polyclonal antibodies
• derived from multiple B-cell lines
• A mixture of immunoglobulin molecules secreted
  against a specific antigen, each recognising a
  different epitope
• Monoclonal
• identical to each other
• Clones of a single parental cell

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Immunological TechniquesBackground
Mouse is injected with an antigen
Mouse antibodies injected into a goat.
Goat anti-mouse antibodies are harvested from
goat blood
Harvested goat anti-mouse antibodies are labeled
for easy detection
From Environmental Microbiology, Maier et al
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Immunological TechniquesBackground
Mouse is injected with an antigen
in response, mouse immune system
produces antibodies. Cell culture
Mouse antibodies injected into a goat.
Goat anti-mouse antibodies are harvested from
goat blood
Harvested goat anti-mouse antibodies are labeled
for easy detection
From Environmental Microbiology, Maier et al
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Immunological TechniquesBackground
Mouse is injected with an antigen
in response, mouse immune system
produces antibodies. Cell culture
Mouse antibodies injected into a goat.
Harvested goat anti-mouse antibodies are labeled
for easy detection
From Environmental Microbiology, Maier et al
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Immunological TechniquesBackground
Mouse is injected with an antigen
in response, mouse immune system
produces antibodies. Cell culture
Mouse antibodies injected into a goat.
Goat anti-mouse antibodies are harvested from
goat blood
Harvested goat anti-mouse antibodies are labeled
for easy detection
From Environmental Microbiology, Maier et al
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Immunological TechniquesBackground
Mouse is injected with an antigen
in response, mouse immune system
produces antibodies. Cell culture
Mouse antibodies injected into a goat.
Goat anti-mouse antibodies are harvested from
goat blood
Harvested goat anti-mouse antibodies are labeled
for easy detection
From Environmental Microbiology, Maier et al
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Immunological TechniquesLabels

• Fluorescent
• Radioisotopes
• Iodine
• Enzymes

fluorescently labeled Legionella cells under the
microscope
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Immunological TechniquesELISA
Direct Indirect
Antibody Antigen
From Environmental Microbiology, Maier et al
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Immunological TechniquesELISA
Direct Indirect
Antibody Antigen
antigen antibody sample
sample
From Environmental Microbiology, Maier et al
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Immunological TechniquesELISA
Direct Indirect
Antibody Antigen
enzyme-linked antibody is added
enzyme-linked antiglobulin is added
antigen antibody sample
sample
From Environmental Microbiology, Maier et al
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Immunological TechniquesELISA
Direct Indirect
Antibody Antigen
enzyme-linked antibody is added
enzyme-linked antiglobulin is added
antigen antibody sample
sample
substrate for the enzyme is added
From Environmental Microbiology, Maier et al
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Immunological TechniquesImmuno-Magnetic
Separation
Antibody magnetite
From Environmental Microbiology, Maier et al
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Immunological TechniquesAdvantages

• Very fast
• In line technology. Flow cytometry (fluorescently
  labeled bacteria)
• Kits are commercially available (your techs dont
  have to bleed mice and goats!)
• Very specific to a particular antigen.
• Very sensitive
• ELISA plates are suitable for field work

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Immunological TechniquesDisadvantages

• Some protocols require enrichment. Slows you
  down.
• In line technology. Flow cytometry. Requires
  expensive equipment
• Kits are commercially available, but not cheap
• Microscopes may be necessarily
• Very specific to a particular antigen. Wont
  recognize any other antigen
• False positives/negatives possible, especially
  with mutated/altered antigen

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Western Blot
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Western Blot
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Affinity Chromatography
Step 1. A column consists of a solid matrix to
which the antigen (shown in blue) has been
coupled (usually covalently). Step 2.The serum
is passed over the immunoadsorbent. Those
antibodies in the mixture specific for the
antigen (shown in red) will bind (noncovalently)
and be retained. Antibodies of other
specificities (green) and other proteins (yellow)
will pass through unimpeded. Step 3.Elution. A
reagent is passed into the column to release the
antibodies from the column. Buffers containing a
high concentration of salts and/or low pH are
often used to disrupt the noncovalent
interactions between antibodies and antigen. A
denaturing agent, such as 8 M urea, will also
break the interaction by altering the
configuration of the antigen-binding site of the
antibody molecule.
http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/A/AffinityChrom.html