Practical Applications of Immunology

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Practical Applications of Immunology

• CHAPTER 18

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Vaccine

• Edward Jenner developed the modern practice of
  vaccination when he inoculated people with cowpox
  virus to protect them against smallpox (1798).
• A vaccine is
• A preparation that contains an antigen,
  consisting of whole disease-causing organisms
  (killed or weakened) or parts of such organisms,
  that is used to confer immunity against the
  disease that the organisms cause.

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Principles and EffectsVaccination

• Herd immunity results when most of a population
  is immune to a disease.

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Types of Vaccines

• Attenuated whole-agent vaccines
• Consist of attenuated (mutated, weakened)
  microorganisms. Attenuated virus vaccines provide
  lifelong immunity (Sabin polio, MMR, tuberculosis
  oral typhoid).
• Inactivated whole-agent vaccines
• Consist of killed (phenol, formalin) bacteria or
  viruses (Salk polio, rabies, influenza,
  pneumococcal pneumonia, cholera).

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Types of Vaccines

• Toxoids
• Inactivated toxins (tetanus diphtheria).
• Subunit vaccines
• Consist of antigenic fragments of a
  microorganism. Include recombinant vaccines
  (hepatitis B) and acellular vaccines (antigenic
  fraction pertussis or whooping cough).
• Conjugated vaccines
• Combine the desired antigen with a protein
  (diphtheria toxoid) that boosts the immune
  response (especially for up to 15 yr of age).
  Haemophilus influenza type b

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Types of Vaccines

• Nucleic acid vaccines
• Naked DNA or RNA (transcription and/or
  translation of antigen). Experimental.
• Edible vaccines
• Genetically engineered fruits (bananas) and
  vegetables (tomatoes , potatoes etc.) w.
  appropriate antigen. Experimental,

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The Development of New Vaccines

• Viruses for vaccines may be grown in animals,
  cell cultures, or chicken embryos.
• Recombinant vaccines and nucleic acid vaccines do
  not need to be grown in cells or animals.
• Edible vaccines
• Adjuvants (chemicals such as alum) improve the
  effectiveness of some antigens.
• Crystallized double sulfates of the typical
  formula M2SO4M32(SO4)324H2O, where M is the
  sign of an alkali metal (lithium, sodium,
  potassium, rubidium, ammonium or caesium), and
  M3 denotes one of the trivalent metal ions
  (typically aluminium, chromium, or iron (III)).

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Safety of Vaccines

• Vaccines are safe as they can be and most
  effective means of controlling infectious
  diseases
• Risk Benefit considerations
• Infant diarrhea rotavirus vaccine
• Withdrawn in 1999 life threatening intestinal
  obstruction.

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Diagnostic Immunology

• Many tests based on the interactions of
  antibodies and antigens have been developed to
  determine the presence of antibodies or antigens
  in a patient.
• Skin test for tuberculosis also known as purified
  protein derivative (PPD test)
• Delayed-type hypersensitivity skin reaction
  (swollen redness)

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Tuberculosis Positive Skin Test
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Precipitation Reactions

• Precipitation reactions
• The interaction of soluble antigens with the
  immunoglobulins (IgG or IgM) antibodies.
• Occur best when antigen and antibody are present
  in optimal proportions.
• The precipitin ring test is performed in a small
  tube.
• Immunodiffusion procedures are carried out in an
  agar gel medium.
• Immunoelectrophoresis (electrophoresis
  immunodiffusion) analyzes serum proteins.

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A Precipitation Curve
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The Precipitin Ring Test

• Soluble Antigens?

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Agglutination Reactions

• Agglutination reactions
• The interaction of particulate (cells that carry
  antigens) or insoluble antigens with antibodies.
• Diagnosis of diseases
• Combining the patient's serum with a known
  antigen.
• A rising titer (concentration) of antibodies or
• Seroconversion (from no antibodies to the
  presence of antibodies).

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Agglutination Reactions

• Direct agglutination reactions
• Used to determine antibody titer against large
  cellular antigens (i.e. RBC, bacteria fungi).
• Indirect or passive agglutination tests
• Used to determine antibody titer. Antibodies
  cause visible agglutination of soluble antigens
  affixed to latex spheres.
• Hemagglutination reactions
• Agglutination reactions using red blood cells.
• Used
• In blood typing.
• The diagnosis of certain diseases (i.e.
  infectious mononucleosis).
• The identification of viruses.

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Neutralization Reaction

• In neutralization reactions, the harmful effects
  of a bacterial exotoxin or a virus are eliminated
  by a specific antibody.
• An antitoxin is
• An antibody produced in response to a bacterial
  exotoxin
• An antibody produced in response to a toxoid
  (i.e. toxin of Corynebacterium diphtheriae) .
• In a virus neutralization test, the presence of
  antibodies against a virus can be detected by the
  antibodies' ability to prevent cytopathic effects
  (CPE) of viruses in cell cultures.
• Antibodies against certain viruses can be
  detected by their ability to interfere with viral
  hemagglutination in hemagglutination inhibition
  tests.
• CPE Morphological changes in the host cell
  cell rounding, disorientation,
• swelling or shrinking, death etc.
• Mumps, measles and influenza viruses can
  agglutinate RBC w/o antigen-
• antibody reaction.

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Complement-Fixation Reactions

• Complement-fixation reactions
• Complement (group of serum proteins) binds to
  antigen-antibody complex and is used up.
• Complement-fixation can be used to detect very
  small amounts of antibody.
• Wasserman test for syphilis (in the past)
• Certain viral, fungal rickettsial diseases.

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Fluorescent-Antibody (FA) Techniques

• FA techniques use antibodies labeled with
  fluorescent dyes (fluorescein isothiocyanate).
• Direct FA tests are used to identify specific
  microorganisms using a fluorescence microscope
  (yellow green fluorescence).
• Indirect FA tests are used to demonstrate the
  presence of antibody in serum.
• A fluorescence-activated cell sorter can be used
  to and count cells labeled with fluorescent
  antibodies.

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Citrus tristeza virus (CTV) detected with a
fluorescent (fluoresceinisothiocyanate) labeled
CTV specific antibody
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Enzyme-Linked Immunosorbent Assay (ELISA)

• ELISA techniques use antibodies linked to an
  enzyme as horseradish peroxidase or alkaline
  phosphatase.
• Antigen-antibody reactions are detected by the
  reaction enzyme-substrate. A color change
  indicates an antigen-antibody reaction has
  occurred.
• The direct ELISA
• Used to detect antigens against specific antibody
  bound in a test well.
• The indirect ELISA
• Used to detect specific antibodies (i.e. HIV in
  serum) against antigen bound in a test well.

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A 96-well ELISA plate (8 cm x 12 cm) The well
(1 cm high and 0.7 cm in diameter).
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Indirect ELISA To detect HIV antibodies in serum
Inactivated HIV antigens pre-coated onto an ELISA
plate
Patient serum
Anti-human immunoglobulin coupled to an enzyme.
This is the second antibody, and it binds to
human antibodies
Chromogen or substrate which changes color when
cleaved by the enzyme
Negative
Positive
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Optical density at 450 nm. The cutoff value
indicating a positive result is 0.500. Optical
densities of 0.300 to 0.499 are indeterminate and
need to be retested.