Applications of Immunology
Talaro
Chapter 16
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Acquired Immunity
• Natural Immunity
– Acquired as part of normal life experiences
• Artificial Immunity
– Acquired through vaccination
• Active Immunity
– Results when a person is challenged with Ag that stimulates production of Ab
– Memory
• Passive Immunity
– Preformed Ab are donated to an individual
– Acts immediately but is short term
– No memory
• Breast feeding
• Gamma globulin
• Monoclonal antibodies
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3
Vaccines
• Provide an antigenic stimulus that does not cause disease
– Attenuated strain
• Tissue culture or unnatural / unusual host
• Hypovirulent
– Dead whole cells or inactivated viruses
• Heat, formalin, UV irradiation
– Purified antigen subunits from cells or viruses
– Surface antigens produce via rDNA technology
– DNA vaccines
Why did the
vaccinia virus
work?
• Produces long lasting protective immunity
• Edward Jenner (page 476)
– www.sc.edu/library/spcoll/nathist/jenner2.html
– Cowpox
– Smallpox
• Variola
– Controlled experiments
• Vaccinia virus
– Cultured cow pox virus for many years
• Small pox eradicated in 1973
Immunization using a
closely related, less
pathogenic organism to
give protection against a
more pathogenic one.
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Smallpox
www.cdc.gov
Cowpox on human forearm
www.cdc.gov
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Vaccination Success
•Small pox
www.who.int/immunization_safety/en/
www.cdc.gov
•  2 million people a year died from small pox until 1967
• The World Health Organization initiated an immunization
campaign that eradicated small pox in 12 years
•Poliomyelitis (polio)
• This virus attacks the motor neurons of the brain and spinal cord
• Causes paralysis and death
• Immunization campaigns since the 1950s have virtually eradicated
polio in developed countries
news.bbc.co.uk
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Polio Eradication
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• Haemophilus influenzae type b
– Mistakenly believed to have
caused influenza
– Type b strains accounted for
majority of bacterial meningitis
www.cdc.gov
• Meningitis, pneumonia, and
epiglottitis
• Was the leading cause of
bacterial meningitis of children
5 years or younger
– 1/200 children contracted Hib
– Incidence has decreased 99%
since the vaccine was introduced
20,000 cases per year
in the early 1980’s
1,247 in 2000
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www.who.int/immunization_safety/aefi/immunization_misconceptions/en/index.html
Vaccines cause disease
Disease is no longer a threat in my country
Cost of vaccination
Difficulty reaching vaccination center
Not recommended by my physician
Protection from a disease
Safety concerns
among unvaccinated
Side effects
individuals occurs when 
SIDS
90% of a population is
Autism
immunized.
Depress the immune system
Mercury Poisoning
% depends on the disease &
Religious beliefs
vaccine
Herd Immunity
Content and Design Attributes of Antivaccination Web Sites
Robert M. Wolfe, MD; Lisa K. Sharp, PhD; Martin S. Lipsky, MD
JAMA 2002 287:3245-3248.
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Polio Outbreak Occurs Among Amish Families In Minnesota
by David Brown
Washington Post Staff Writer
October 14, 2005
The first outbreak of polio in the United States in 26 years occurred earlier
this fall in an Amish community in central Minnesota, state and federal health
officials reported yesterday. Four children have been infected with the virus,
although none has become paralyzed. The Amish typically decline to
vaccinate their children. The last large outbreak of polio occurred in
numerous Amish communities in several states in 1979.
Fears Rising Over Measles Outbreaks
by ROBERT A. HAMILTON
STATE health officials are preparing for what they fear could be ''a major outbreak''
of measles when students who attend Boston University return home next week for
spring break. Last week, a measles case was reported in Fairfield, an 18-year-old
Boston University freshman who returned home eight days ago with cold symptoms
and by Tuesday was in the hospital. ''We expect a lot more students will be returning
to Connecticut next week, for the spring break,'' the program director of the state
immunization program, Dr. Charles H. Alexander, .
March 3, 1985 The New York Times
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irradiation
hypovirulent
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Abs
Proteins
Clotting factors
Hormones
Nutrients
Ions
Serology
• A part of immunology
that attempts to detect
signs of infection in a
patient’s serum
Clear fluid
from clotted
blood
leukocytes
– Use Abs that
specifically bind to Ag
• Ag-Ab reactions are
visible by
–
–
–
–
Clumps
Precipitates
Color changes
Release of radioactivity
Isolate Ab from serum
• The most effective tests
have high specificity
and sensitivity.
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Agglutination Test
•
Isolate Abs from
patient
Ab and Ag from
a whole cell
crosslink
–
Forming
complexes that
settle out and
from visible
clumps in the
test chamber
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Contains
patient’Abs
Agglutination
17
Contains Abs for the
specific pathogen
Agglutination
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Precipitation Tests
• Soluble Ag is
precipitated
– Cloudy or opaque
zone
– Many variations are
used to maximize
this technique
– Measure optical
density
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Calculate titer
The highest dilution of serum that shows a positive result
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Enzyme-linked immunosorbent assay (ELISA)
• Detect unknown Ag or Ab
• A positive result is visualized when a colored
product is released by an enzyme-substrate
reaction
Anti-immunoglobulin Ab complexed to an enzyme
conjugate
21
Perform a virtual ELISA courtesy of the Howard Hughes Medical Institute
www.hhmi.org/biointeractive/vlabs/
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Radioimmunoassay
Ag or Abs are labeled with
radioactive isotopes and traced
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ELISA
www.biology.arizona.edu
Positive Rxn
Positive
Control
1.689
Negative
Control
0.153
Negative Rxn
Patient A
Patient B
Patient C
0.055
0.412
1.999
Assay
Control
0.123
+ is 0.5 or greater
Indeterminate
0.300 – 0.495
- is 0.300 or less
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Western Blot
• More sensitive than ELSIA
Western Blot for a HIV
on page 487
– Less chance of a false +
•
•
•
•
Detects specific Ag or Ab proteins in serum
Separates proteins into bands via gel electrophoresis
Protein bands are transferred to a membrane
Specific Abs are used to “probe” the membrane
– Primary Ab are from patient
– Secondary Ab
• Anti-immunoglobulin Ab complex to an enzyme
– Colorless substrate
• Colormetric
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Western Blot for HIV gp160
viral envelope precursor (env)
www.biology.arizona.edu
gp120
viral envelope protein (env) binds to CD4
p24
viral core protein (gag)
p31
reverse transcriptase (pol)
Serum proteins (virus) separated via electrophoresis.
Transferred to a membrane.
The primary Abs from the serum of a patient is added
and will react to the HIV proteins (above).
The secondary Ab is an anti-immunoglobulin
conjugated to an enzyme. This Ab is specific for the
primary Abs.
A colorless substrate is cleaved if the antiimmunoglobulin conjugated to the primary Ab.
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Band Pattern Interpretation
Lane 1, HIV+ serum (positive control)
Lane 2, HIV- serum (negative control)
Lane A, Patient A
Lane B, Patient B
Lane C, Patient C
Patient A is –
Patient B is –
Patient C is +
No bands present
-
Bands at either p31
OR p24 AND
bands present at
either gp160 OR
gp120
+
Bands present, but
pattern does not
meet criteria for
positivity
Indeterminate
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Immunofluorescence
• Fluorescent Ab
(FAbs) either
directly or indirectly
to visualize cells or
cell aggregates
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Monoclonal Antibodies (Mab)
• Single specificity antibodies
formed by fusing a mouse B
cell with a myeloma cell
– A malignant tumor formed by the
cells of the bone marrow
• Used in diagnosis of disease,
identification of microbes
and therapy
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• Hybridomas produce antibody that
recognize single epitope
• Produce uniform, highly specific
Ab in large supply
• Immunize animal
• Harvest spleen
• Fuse B & myeloma
• Hybridoma
• Multiple practical applications
• Immortal
• Screen hybridomas for Abs • Diagnostic tests
• ELISA & Western Blots
directed against antigen of
• Immunosuppressive therapy for
interest
transplants
• Prevent action of TC
• Anticancer drugs
• Antitoxins
• Identify a pathogen
• Purification of an important protein
• Precipitate
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Monoclonals on the Market
Herceptin
Blocks the effects of the growth factor protein HER2,
which transmits growth signals to breast cancer cells.
Causes tumor shrinkage.
Rituxan
Mylotarg
Acute myeloid leukemia (AML)
Treat a variety of non-Hodgkin's lymphomas.
Target and destroy non-Hodgkin's lymphoma cells.
Remicade
Auto-immune disorders like Crohn's disease and rheumatoid arthritis
Target is tumor necrosis factor
Zenapax
Prevent rejection in organ transplantation, especially in kidney transplants.
Additional information at www.fda.gov
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