Principle of Ag-Ab Reactions

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Principle of Ag-Ab Reactions
Mr. Said S. S. AlGhora CUCMS
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Immunological Assays

• 1. Precipitation
• 2. Agglutination
• 3. Radioimmunoassay
• 4. Enzyme-linked Immunosorbent Assay
• 5. Western Blotting
• 6. Immunostaining
• Immunofluorescence
• Immuno-gold EM
• 7. Flow Cytometry and Fluorescence
• 8. Immunoelectron Microscopy

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Nature of the Ag-Ab interaction Immunological
assays
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4 types of noncovalent forces
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Factors affecting measurement of antigen-antibody
reactions

• 1. Affinity The higher the affinity of the
  antibody for the antigen, the more stable will be
  the interaction. 2. Avidity Reactions between
  multivalent antigens and multivalent antibodies
  are more stable and thus easier to detect.3.
  Antigen to antibody ratio The ratio between the
  antigen and antibody influences the detection of
  antigen-antibody complexes because the size of
  the complexes formed is related to the
  concentration of the antigen and antibody.4.
  Physical form of the antigen The physical form of
  the antigen influences how one detects its
  reaction with an antibody.

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• In terms of infectious diseases, the following
  may act as antigens
• 1.Microbial structures (cell walls, capsules,
  flagella, pili, viral capsids, envelope-associated
  glycoproteins, etc.).
• 2. Microbial exotoxins
• Certain non-infectious materials may also act as
  antigens if they are recognized as "nonself" by
  the body. These include
• 1. Allergens (dust, pollen, hair, foods, dander,
  bee venom, drugs, and other agents causing
  allergic reactions).
• 2. Foreign tissues and cells (from transplants
  and transfusions).
• 3. The body's own cells that the body fails to
  recognize as "normal self" (cancer cells,
  infected cells, cells involved in autoimmune
  diseases).

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Preparation of known antisera in animals

• Preparation of known antiserum in animals
  involves inoculating animals with specific known
  antigens such as a specific strain of a
  bacterium. After the animal's immune responses
  have had time to produce antibodies against that
  antigen, the animal is bled and the blood is
  allowed to clot. The resulting liquid portion of
  the blood is the serum and it will contain
  antibodies specific for the injected antigen.
• However, one of the problems of using antibodies
  prepared in animals (by injecting the animal with
  a specific antigen and collecting the serum after
  antibodies are produced) is that up to 90 of the
  antibodies in the animal's serum may be
  antibodies the animal has made "on its own"
  against environmental antigens, rather than those
  made against the injected antigen. The
  development of monoclonal antibody technique has
  largely solved that problem.

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Preparation of known antibodies by monoclonal
antibody technique.

• Monoclonal antibodies are antibodies of a single
  specific type. In this technique, an animal is
  injected with the specific antigen for the
  antibody desired. After appropriate time for
  antibody production, the animal's spleen is
  removed. The spleen is rich in plasma cells and
  each plasma cell produces only one specific type
  of antibody. However, plasma cells will not grow
  artificially in cell culture. Therefore, a plasma
  cell producing the desired antibody is fused with
  a myeloma cell ,a cancer cell from bone marrow
  which will grow rapidly in cell culture, to
  produce a hybridoma cell. The hybridoma cell has
  the characteristics of both parent cells. It will
  produce the specific antibodies like the plasma
  cell and will also grow readily in cell culture
  like the myeloma cell. The hybridoma cells are
  grown artificially in huge vats where they
  produce large quantities of the specific
  antibody.
• Monoclonal antibodies are now used routinely in
  medical research and diagnostic serology and are
  being used experimentally in treating certain
  cancers and a few other diseases.

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Definition
Serology refers to using antigen-antibody
reactions in the laboratory for diagnostic
purposes. Its name comes from the fact that
serum, the liquid portion of the blood where
antibodies are found is used in testing.
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Serologic testing may be used in the clinical
laboratory in two distinct ways

• To identify unknown antigens (such as
  microorganisms). This is called direct serologic
  testing. Direct serologic testing uses a
  preparation known antibodies, called antiserum,
  to identify an unknown antigen.
• b. To detect antibodies being made against a
  specific antigen in the patient's serum. This is
  called indirect serologic testing. Indirect
  serologic testing is the procedure by which
  antibodies in a person's serum being made by that
  individual against an antigen associated with a
  particular disease are detected using a known
  antigen.

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Precipitation Reactions

• Precipitation in fluids Known antiserum is
  mixed with soluble test antigen and a cloudy
  precipitate forms at the zone of optimum
  antigen-antibody proportion.
• Precipitation in gels
• - radial immunodiffusion (Mancini method)
• - double immunodiffusion (Ouchterlony method)
• Immunoelectrophoresis

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Precipitation Reactions
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Immunoelectrophoresis A complex mixture of
antigens is placed in a well punched out of an
agar gel and the antigens are electrophoresed so
that the antigen are separated according to their
charge. After electrophoresis, a trough is cut in
the gel and antibodies are added. As the
antibodies diffuse into the agar, precipitin
lines are produced in the equivalence zone when
an antigen/antibody reaction occurs.This tests
is used for the qualitative analysis of complex
mixtures of antigens, although a crude measure of
quantity (thickness of the line) can be obtained.
This test is commonly used for the analysis of
components in a patient' serum. Serum is placed
in the well and antibody to whole serum in the
trough. By comparisons to normal serum, one can
determine whether there are deficiencies on one
or more serum components or whether there is an
overabundance of some serum component (thickness
of the line). This test can also be used to
evaluate purity of isolated serum proteins.
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Immunoelectrophoresis
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Agglutination Reactions
Known antiserum causes bacteria or other
particulate antigens to clump together or
agglutinate. Molecular-sized antigens can be
detected by attaching the known antibodies to
larger, insoluble particles such as latex
particles or red blood cells in order to make the
agglutination visible to the naked eye.
Hemagglutination Bacterial Agglutination Passive
Agglutination Agglutination Inhibition
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HaemagglutinationHaemagglutination is visible
macroscopically and is the basis of
haemagglutination tests to detect the presence of
viral particles. The test does not discriminate
between viral particles that are infectious and
particles that are degraded and no longer able to
infect cells. Both can cause the agglutination
of red blood cells.-Influenza and other
viruses-Two spike proteins NEURAMINIDASE ,
HAEMAGGLUTININ ( Binds specifically to red blood
cells)Steps to haemagglutination1. Dispense
diluent.2. Add red blood cells and mix by gently
shaking.3. Allow the red blood cells to settle
and observe the pattern.4. Observe if the cells
have a normal settling pattern and there is no
auto-agglutination. This will be a distinct
button of cells in the micro test and an even
suspension with no signs of clumping in the rapid
test.
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Hemagglutination
Agglutination No Agglutination
No Ab
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Agglutination Inhibition
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Complement-fixation

• Known antiserum is mixed with the test antigen
  and complement is added. Sheep red blood cells
  and hemolysins (antibodies that lyse the sheep
  red blood cells in the presence of free
  complement) are then added. If the complement is
  tied up in the first antigen-antibody reaction,
  it will not be available for the sheep red blood
  cell-hemolysin reaction and there will be no
  hemolysis.
• A negative test would result in hemolysis.

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Radioactive binding techniques

• Test antigens from specimens are passed through a
  tube coated with the corresponding specific known
  antibodies and become trapped on the walls of the
  tube. Known antibodies to which a radioactive
  isotope has been chemically attached are then
  passed through the tube where they combine with
  the trapped antigens. The amount of
  antigen-antibody complex formed is proportional
  to the degree of radioactivity.

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Solid-phase Radioimmunoassay (RIA)
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Enzyme-linked Immunosorbent Assay (ELISA)
Test antigens from specimens are passed through a
tube (or a membrane) coated with the
corresponding specific known antibodies and
become trapped on the walls of the tube (or on
the membrane). Known antibodies to which an
enzyme has been chemically attached are then
passed through the tube (or membrane) where they
combine with the trapped antigens. Substrate for
the attached enzyme is then added and the amount
of antigen-antibody complex formed is
proportional to the amount of enzyme-substrate
reaction as indicated by a color change.
- Indirect ELISA - Sandwich ELISA
- Competitive ELISA - Chemiluminescence
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Elispot Assay
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Western Blot

• The western blot (alternatively, protein
  immunoblot) is an analytical technique used to
  detect specific proteins in a given sample of
  tissue homogenate or extract. It uses gel
  electrophoresis to separate native or denatured
  proteins by the length of the polypeptide
  (denaturing conditions) or by the 3-D structure
  of the protein (native/ non-denaturing
  conditions). The proteins are then transferred to
  a membrane (typically nitrocellulose or PVDF),
  where they are probed (detected) using antibodies
  specific to the target protein.

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Western Blotting
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• Immunostaining- Immunoflourescence
• -Immuno-gold EM
• Immunoflourscence A laboratory technique to
  identify specific antibodies or antigens.
  Antibody identification is usually performed on
  blood (serum).
• Antibody tagged with flourescent dye
• Antibody attached specifically to antigen
• View specimen under exciting light
• Flourscence microscope

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Immunofluorescence
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Immunogold Electron Microscopy Same principle
as immunoflourscenceGold particles attached to
antibodies (nanometer size particles) Viewed
under EM to localise specific proteins or antigens
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Flow Cytometry

• is commonly used in the clinical laboratory to
  identify and enumerate cells bearing a particular
  antigen. Cells in suspension are labeled with a
  fluorescent tag by either direct or indirect
  immunofluorescence. The cells are then analyzed
  on the flow cytometer.In a flow cytometer, the
  cells exit a flow cell and are illuminated with a
  laser beam. The amount of laser light that is
  scattered off the cells as they passes through
  the laser can be measured, which gives
  information concerning the size of the cells. In
  addition, the laser can excite the fluorochrome
  on the cells and the fluorescent light emitted by
  the cells can be measured by one or more
  detectors.

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Flow Cytometry
FACS Fluoresence-activated Cell sorter
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Immunoelectron microscopy
An electron microscope is a type of microscope
that uses electrons to illuminate a specimen and
create an enlarged image. Electron microscopes
have much greater resolving power than light
microscopes and can obtain much higher
magnifications.
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