6.immunologicaltechniques

8/7/2019 6.ImmunologicalTechniques

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Immunological Methods in

Microbiological Testing


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Testing Methods

Culture-dependent techniques

Culture-independent techniques

Total heterotrophicplate count

Growth on detection

media

Samplewith Bugs

Direct Observation

ImmunologicalImmunological

MethodsMethods

Nucleic AcidNucleic Acid--BasedBased

Chemical

Most Probable

Number


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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 ofimmunoglobulin molecules secreted against aspecific antigen, each recognising a different epitope

Monoclonal

identical to each other

Clones of a single parental cell


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Immunological Techniques:

Background

From Environmental Microbiology, Maier et al

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


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Background


Mouse is injected

with an antigen

in response, mouse

immune system produces

antibodies. Cell culture

Mouse antibodies



from goat blood



easy detection


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Background


Mouse is injected

with an antigen

in response, mouse



Mouse antibodies

injected into a goat.



easy detection


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Background


Mouse is injected

with an antigen

in response, mouse



Mouse antibodies



from goat blood



easy detection


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Background


Mouse is injected

with an antigen

in response, mouse



Mouse antibodies



from goat blood



easy detection


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Fluorescent

Radioisotopes

Iodine

Enzymes

fluorescently labeled Legionella

cells under the microscope


Labels


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Direct Indirect

Antibody Antigen


ELISA



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Direct Indirect

Antibody Antigen

+ antigen + antibody

sample sample


ELISA



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Direct Indirect

Antibody Antigen


sample sample

enzyme-linked

antibody is added

enzyme-linked

antiglobulin is added


ELISA



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Direct Indirect

Antibody Antigen


sample sample

enzyme-linked

antibody is added

enzyme-linked

antiglobulin is added

substrate for the enzyme is added


ELISA



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Antibody

+ magnetite


Immuno-Magnetic Separation



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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. Wontrecognize any other antigen

False positives/negatives possible, especially withmutated/altered antigen


Disadvantages


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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 antibodiesin 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 pHare often used to disrupt the noncovalent interactions between antibodiesand antigen. A denaturing agent, such as 8 M urea, will also break theinteraction by altering the configuration of the antigen-binding site of theantibody molecule.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/A/AffinityChrom.html

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