it describes the basics of immunoblotting

1. 1
Applications of Immunology
Talaro
Chapter 16

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

8. 8
Polio Eradication

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

12. 12
irradiation
hypovirulent

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

17. 17
Agglutination
Contains
patient’Abs

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