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1
Applications of Immunology
Talaro
Chapter 16
2
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
3
4
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
• 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
Why did the
vaccinia virus
work?
Immunization using a
closely related, less
pathogenic organism to
give protection against a
more pathogenic one.
5
Cowpox on human forearm
www.cdc.gov
Smallpox
www.cdc.gov
6
7
Vaccination Success
•Small pox
•  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
www.who.int/immunization_safety/en/
www.cdc.gov
news.bbc.co.uk
8
Polio Eradication
9
• Haemophilus influenzae type b
– Mistakenly believed to have
caused influenza
– Type b strains accounted for
majority of bacterial meningitis
• 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
www.cdc.gov
20,000 cases per year
in the early 1980’s
1,247 in 2000
10
Vaccines cause disease
Disease is no longer a threat in my country
Cost of vaccination
Difficulty reaching vaccination center
Not recommended by my physician
Safety concerns
Side effects
SIDS
Autism
Depress the immune system
Mercury Poisoning
Religious beliefs
www.who.int/immunization_safety/aefi/immunization_misconceptions/en/index.html
Herd Immunity
Protection from a disease
among unvaccinated
individuals occurs when 
90% of a population is
immunized.
% depends on the disease &
vaccine
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.
11
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
12
irradiation
hypovirulent
13
14
15
Serology
• A part of immunology
that attempts to detect
signs of infection in a
patient’s serum
– Use Abs that
specifically bind to Ag
• Ag-Ab reactions are
visible by
– Clumps
– Precipitates
– Color changes
– Release of radioactivity
• The most effective tests
have high specificity
and sensitivity.
leukocytes
Abs
Proteins
Clotting factors
Hormones
Nutrients
Ions
Clear fluid
from clotted
blood
Isolate Ab from serum
16
Agglutination Test
• Ab and Ag from
a whole cell
crosslink
– Forming
complexes that
settle out and
from visible
clumps in the
test chamber
Isolate Abs from
patient
17
Agglutination
Contains
patient’Abs
18
Agglutination
Contains Abs for the
specific pathogen
19
Precipitation Tests
• Soluble Ag is
precipitated
– Cloudy or opaque
zone
– Many variations are
used to maximize
this technique
– Measure optical
density
20
Calculate titer
The highest dilution of serum that shows a positive result
21
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
22
Perform a virtual ELISA courtesy of the Howard Hughes Medical Institute
www.hhmi.org/biointeractive/vlabs/
23
Radioimmunoassay
Ag or Abs are labeled with
radioactive isotopes and traced
24
ELISA
www.biology.arizona.edu
Positive Rxn Negative Rxn
Positive
Control
Negative
Control
Patient A Patient B Patient C
Assay
Control
1.689 0.153 0.055 0.412 1.999 0.123
+ is 0.5 or greater
Indeterminate
0.300 – 0.495
- is 0.300 or less
25
• More sensitive than ELSIA
– 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
Western Blot Western Blot for a HIV
on page 487
26
Western Blot for HIV
www.biology.arizona.edu
gp160 viral envelope precursor (env)
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 anti-
immunoglobulin conjugated to the primary Ab.
27
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
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
Patient A is –
Patient B is –
Patient C is +
28
29
• Fluorescent Ab
(FAbs) either
directly or indirectly
to visualize cells or
cell aggregates
Immunofluorescence
30
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
31
• Immunize animal
• Harvest spleen
• Fuse B & myeloma
• Hybridoma
• Immortal
• Screen hybridomas for Abs
directed against antigen of
interest
• Hybridomas produce antibody that
recognize single epitope
• Produce uniform, highly specific
Ab in large supply
• Multiple practical applications
• Diagnostic tests
• ELISA & Western Blots
• Immunosuppressive therapy for
transplants
• Prevent action of TC
•Anticancer drugs
• Antitoxins
• Identify a pathogen
• Purification of an important protein
• Precipitate
32
Zenapax
Prevent rejection in organ transplantation, especially in kidney transplants.
Mylotarg
Acute myeloid leukemia (AML)
Remicade
Auto-immune disorders like Crohn's disease and rheumatoid arthritis
Target is tumor necrosis factor
Rituxan
Treat a variety of non-Hodgkin's lymphomas.
Target and destroy non-Hodgkin's lymphoma cells.
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.
Additional information at www.fda.gov

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Immunoblott technique for protein detection.ppt

  • 2. 2 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
  • 3. 3
  • 4. 4 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 • 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 Why did the vaccinia virus work? Immunization using a closely related, less pathogenic organism to give protection against a more pathogenic one.
  • 5. 5 Cowpox on human forearm www.cdc.gov Smallpox www.cdc.gov
  • 6. 6
  • 7. 7 Vaccination Success •Small pox •  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 www.who.int/immunization_safety/en/ www.cdc.gov news.bbc.co.uk
  • 9. 9 • Haemophilus influenzae type b – Mistakenly believed to have caused influenza – Type b strains accounted for majority of bacterial meningitis • 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 www.cdc.gov 20,000 cases per year in the early 1980’s 1,247 in 2000
  • 10. 10 Vaccines cause disease Disease is no longer a threat in my country Cost of vaccination Difficulty reaching vaccination center Not recommended by my physician Safety concerns Side effects SIDS Autism Depress the immune system Mercury Poisoning Religious beliefs www.who.int/immunization_safety/aefi/immunization_misconceptions/en/index.html Herd Immunity Protection from a disease among unvaccinated individuals occurs when  90% of a population is immunized. % depends on the disease & vaccine 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.
  • 11. 11 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
  • 13. 13
  • 14. 14
  • 15. 15 Serology • A part of immunology that attempts to detect signs of infection in a patient’s serum – Use Abs that specifically bind to Ag • Ag-Ab reactions are visible by – Clumps – Precipitates – Color changes – Release of radioactivity • The most effective tests have high specificity and sensitivity. leukocytes Abs Proteins Clotting factors Hormones Nutrients Ions Clear fluid from clotted blood Isolate Ab from serum
  • 16. 16 Agglutination Test • Ab and Ag from a whole cell crosslink – Forming complexes that settle out and from visible clumps in the test chamber Isolate Abs from patient
  • 18. 18 Agglutination Contains Abs for the specific pathogen
  • 19. 19 Precipitation Tests • Soluble Ag is precipitated – Cloudy or opaque zone – Many variations are used to maximize this technique – Measure optical density
  • 20. 20 Calculate titer The highest dilution of serum that shows a positive result
  • 21. 21 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
  • 22. 22 Perform a virtual ELISA courtesy of the Howard Hughes Medical Institute www.hhmi.org/biointeractive/vlabs/
  • 23. 23 Radioimmunoassay Ag or Abs are labeled with radioactive isotopes and traced
  • 24. 24 ELISA www.biology.arizona.edu Positive Rxn Negative Rxn Positive Control Negative Control Patient A Patient B Patient C Assay Control 1.689 0.153 0.055 0.412 1.999 0.123 + is 0.5 or greater Indeterminate 0.300 – 0.495 - is 0.300 or less
  • 25. 25 • More sensitive than ELSIA – 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 Western Blot Western Blot for a HIV on page 487
  • 26. 26 Western Blot for HIV www.biology.arizona.edu gp160 viral envelope precursor (env) 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 anti- immunoglobulin conjugated to the primary Ab.
  • 27. 27 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 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 Patient A is – Patient B is – Patient C is +
  • 28. 28
  • 29. 29 • Fluorescent Ab (FAbs) either directly or indirectly to visualize cells or cell aggregates Immunofluorescence
  • 30. 30 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
  • 31. 31 • Immunize animal • Harvest spleen • Fuse B & myeloma • Hybridoma • Immortal • Screen hybridomas for Abs directed against antigen of interest • Hybridomas produce antibody that recognize single epitope • Produce uniform, highly specific Ab in large supply • Multiple practical applications • Diagnostic tests • ELISA & Western Blots • Immunosuppressive therapy for transplants • Prevent action of TC •Anticancer drugs • Antitoxins • Identify a pathogen • Purification of an important protein • Precipitate
  • 32. 32 Zenapax Prevent rejection in organ transplantation, especially in kidney transplants. Mylotarg Acute myeloid leukemia (AML) Remicade Auto-immune disorders like Crohn's disease and rheumatoid arthritis Target is tumor necrosis factor Rituxan Treat a variety of non-Hodgkin's lymphomas. Target and destroy non-Hodgkin's lymphoma cells. 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. Additional information at www.fda.gov