Transcript Chapter 17 BauPP

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Chapter 17
Immunization and Immune Testing
Brief History of Immunization
12th century: smallpox; variolation (process of
infection with smallpox scab to induce
immunity)
1721: Lady Mary Montagu ---> England
1796: Edward Jenner
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Principles of Vaccine Preparation:
A vaccine must be effective, safe and easy to
administer. It must also provide a long-lasting
protection. Most vaccines are prepared as:
1. Killed whole cells or inactivated viruses.
2. Live, attenuated cells or viruses.
3. As parts of cells or viruses (these are called
subcellular, subunit or acellular vaccines).
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Live vaccines can be prepared by a process
called attenuation.
1. Attenuation can be achieved by long-term
cultivation.
2. Selection of mutant strains that grow at
colder temperatures (cold mutants).
3. Continued passage of microbes through
their non-natural hosts or tissue culture.
4. Removal of virulence genes.
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Types of Vaccines:
• Subunit vaccines:
• Subcellular vaccines:
• Acellular vaccines:
• Toxoid:
Fig.
15.19b
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Combination Vaccines:
Multiple vaccine:
DPT, MMR
Polyvalent vaccine:
Polio: type 1: Leon
type 2: Lansing
type 3: Brundhilde
Genetically Engineered Vaccines
Veggie vaccine:
1. The gene responsible for the surface protein of a
pathogen is isolated.
2. The gene is inserted into plant cells (banana,
potato, corn, tomato).
3. Plant cells are then able to produce the surface
protein of the pathogen.
4. When the plant along with the surface protein is
ingested, the protein will stimulate the immune
system to produce antibody against it. This
antibody should be able to react with the surface
protein of the pathogen to provide protection.
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• Subunit Vaccines:
A gene that codes for a surface protein of a
pathogen is inserted into a plasmid which is
introduced into a cloning host. The host is
able to produce the surface protein. This
protein is the subunit vaccine.
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DNA vaccine:
1. The gene coding for a surface protein (epitope)
of a pathogen is isolated and inserted into a
plasmid vector.
2. The plasmid is introduced into an appropriate
host cell, which can then be cloned.
3. The plasmids can be harvested from the cloning
host.
4. The plasmids suspended in saline solution are
used as a DNA vaccine.
5. When the plasmids are injected into a host, they
are incorporated in the host genome.
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6. The host is able to produce the surface protein
(epitope) of the pathogen, which will be
recognized as a foreign antigen by the host
immune system.
7. Some surface proteins are secreted and taken
up by phagocytes, which then present them to
the B lymphocytes, activating the B
lymphocytes to produce antibodies against the
surface protein.
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8. Some surface proteins are attached to
the surface of the host cells and activate
the T lymphocytes.
Thus, the DNA vaccine provides both
humoral immunity and cell-mediated
immunity to the host against the pathogen.
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Trojan horse vaccine:
1. The genes coding for the surface proteins of
HIV, herpes simplex type 2, Mycobacterium
tuberculosis, and M. leprae have been
incorporated into the genome of vaccinia
(cowpox virus) which is used as a carrier.
2. When the vaccinia with these surface proteins is
injected into a host, it will stimulate the immune
system to produce antibodies against the
pathogens with these surface proteins.
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• Adjuvant:
• Herd immunity:
• Immunotherapy:
Immunization
• Passive Immunotherapy
– Administration of antiserum that contains preformed
antibodies
– Provides immediate protection against a recent infection or
ongoing disease
– Antisera have several limitations
• Can trigger allergic reactions called serum sickness
• Antibodies of antisera are degraded relatively
quickly
• Individual not protected from subsequent infections
– Limitations are overcome through development of
hybridomas
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Monoclonal Antibodies:
1. Immunize an animal with a specific
antigen.
2. Harvest B cells from the spleen.
3. Mix B cells and myeloma cells (cancer
cells of the bone marrow), and add
polyethylene glycol to facilitate cell fusion.
4. Some cells fuse to form hybridoma cells.
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5. They are transferred to a selective
medium, which contains no histidine (an
amino acid).
6. Cancer cells need histidine to grow. So,
they are eliminated. B lymphocytes will
automatically die off as they cannot
propagate in vitro.
7. Hybridoma cells can survive in the
medium as they can synthesize the amino
acid.
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8. The hybridoma cells can be cloned in
vitro or in vivo.
Applications:
1. Diagnostic tests
2. Treatment of diseases and cancers
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Titer of Antibody:
1. The serum from a person is diluted (2-fold
dilution) in a series of test tubes or microtiter
plate.
2. An equal amount of an antigen is added to each
tube.
3. The tubes are mixed well and incubated.
4. The most diluted test tube that shows a positive
test (such as agglutination) is taken as the titer of
antibody. For example, if the last tube that
shows a positive result is 1:4, it is expressed as
the reciprocal of ¼ which is 4. That is, the titer
is 4.
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To Confirm A Disease Using The Titers of
Paired Sera:
1. Acute phase serum is taken from a patient.
2. A 2-fold dilution is made in a series of test tubes
or a microtiter plate.
3. An equal amount of an antigen is added to each
tube.
4. The titer is read from the most diluted test tube.
For example, the titer is 4.
5. Convalescent serum is taken from the same
patient.
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6. An equal amount of the same antigen is added to
each tube.
7. A 2-fold dilution is made in a series of test tubes
or a microtiter plate.
8. The titer is read from the most diluted test tube.
For example, the titer is 16.
9. If there is a 4 folds or more increase in titer, it
confirms that the patient has been infected with
the disease. If not, it means that the patient was
not infected with the disease under testing. The
patient was infected with other disease.
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Serologic Tests:
Agglutination Test:
Particulate antigen + Serum (agglutinin?)
Agglutination: Positive
No agglutination: Negative
Agglutination Test
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Rapid Plasma Reagin Card (RPR) Test:
0.05 ml serum + 1 drop of charcoal-bound
cardiolipin
Rotate at 100 rpm for 8 minutes
Agglutination: Reactive
No agglutination: Non-reactive (Negative)
Agglutination Tests
• Widal Test: Typhoid fever (Salmonella typhi)
• Weil-Felix test: Rickettsial diseases
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Latex Agglutination Test
Pregnancy Test:
If a woman is pregnant she produces
human chorionic gonadotropin (HCG).
Purificed HCG is injected into an animal,
which then produces antibody against HCG.
This antibody is harvested and used as a
reagent in the test.
Latex particles are coated with HCG. It is
used as a second reagent.
Latex Agglutination Test
Urine + Antibody vs HCG
Mix well
Latex particles coated with HCH are added to
the mixture.
If agglutination occurs, the test is negative
If no agglutination occurs, the test is positive.
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If agglutination of latex particles occurs, it
means that the urine does not contain HCG.
Antibody vs HCG reacts with latex particles
and causes agglutination.
If no agglutination of latex particles occurs, it
means that the urine does contain HCG.
The HCG in the urine reacts with the
antibody vs HCG. No antibody is available
to react with latex particles. That is why
there is no agglutination. It means that the
woman is pregnant.
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Viral Hemagglutination
• Some viruses have the ability to cause
agglutination, called hemagglutination, of
red blood cells.
• In the presence of antibody, the virus cannot
agglutinate the RBC. The antibody
connects to the virus, preventing it from
clumping the RBC.
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Viral Hemagglutination
Viruses + RBC
Hemagglutination
(influenza, rubella, measles, mumps, vaccinia,
arboviruses, adenoviruses, reoviruses and
some enteroviruses)
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Hemagglutination-Inhibition (HI) Test:
Virus + Serum + RBC
Agglutination: No agglutination: +
Positive (+) means the serum must contain
antibody. That means the patient has been
infected with the virus.
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Precipitin Test
Precipitation: +
Soluble Antigen + Serum
No precipitation: Zone of antigen excess:
Zone of antibody excess:
Zone of optimum concentration:
Characteristics of precipitation
reactions
Figure 17.6
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Precipitin Test
Ring Test:
Antigen solution is slowly poured into a
tube containing serum (antibody?)
A ring of precipitation: +
No ring of precipitation: -
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Precipitin Test
VDRL (Venereal disease research laboratory) Test:
0.05 ml of heated serum + one drop of cardiolipin
rotate at 180 rpm for 4 minutes
Precipitation: Reactive
No precipitation: Non-reactive
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Precipitin Test
Immunodiffusion: If both antigen and antibody
are homologous, a line or band of precipitation
is formed.
Single Diffusion Technique (Oudin Technique):
A tube of antibody in agar is prepared.
Antigen solution is added.
If a band of precipitation occurs the test is
positive. If no precipitation occurs, the test is
negative.
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Double Diffusion Technique:
A. Oakley and Fulthorpe Technique:
A tube of antibody in agar with a layer of
plain agar on top is prepared. A solution
of antigen is added.
If a band of precipitation in the plain
agar occurs, the test is positive.
If no precipitation occurs, the test is
negative.
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B. Ouchterlony Technique (Plate Method):
Wells are cut from an agar plate. An
antibody is placed in the central well, and
different antigens are separately placed in
the wells around the central well.
If a line of precipitation occurs between
the antigen and the antibody, the test is
positive for the antigen.
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Immunodiffusion
Reaction of identity:
Reaction of partial identity:
Reaction of non-identity:
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Immunoelectrophoresis:
It is a combination of immunodiffusion and
electrophoresis.
Positively charged proteins migrate to the
cathode, and the negatively charged
proteins migrate to the anode.
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Complement-Fixation (CF) Test:
(Antigen + Complement + Serum)
+
(RBC + Hemolysin)
Mix, Incubate
No hemolysis: Positive test
Hemolysis:
Negative test
Wassermann test: for syphilis
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Treponema pallidum immobilization (TPI)
Test:
Live Treponema + Serum + Complement
Immobilized: Positive test
Motile: Negative test
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Neutralization Test (NT):
Antigen (Virus or Toxin) + Serum
Inoculate into animals or tissue culture
Animals die:
Negative test
Animals survive: Positive test
CPE (cytopathic effect) in tissue
culture: Negative test
No CPE:
Positive test
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Quellung Test:
It is used to identify different serotypes of
pneumococci.
Unknown pneumococcus + Specific antiserum
Capsule swelling (due to light effect): +
No capsule swelling: -
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Fluorescent-Antibody Technique:
1. Direct method: to detect an unknown antigen
by using a known antibody.
Unknown antigen + Labeled antibody
Presence of brilliant greenish color: +
Absence of brilliant greenish color: -
Fig.
17.15
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2. Indirect method: to detect an unknown
antibody by using a known antigen.
Antigen + Serum + Labeled anti-antibody
Presence of brilliant greenish color: +
Absence of brilliant greenish color: -
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Enzyme-Linked Immunoabsorbent Assay
(ELISA):
1. Direct method (sandwich technique): to
detect an unknown antigen by using a
known antibody.
Known antibody + Unknown antigen
+ Enzyme-linked antibody + Substrate
Color change: +
No color change: -
Fig.
17.16.c
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2. Indirect method: to detect an unknown
antibody by using a known antigen.
Known antigen + Unknown antibody
+ Enzyme-linked anti-antibody + Substrate
Color change: +
No color change: -
Immune Testing
• Recent Developments in Immune Testing
– Immunochromatography
• Very rapid and easy to read ELISAs
• Antigen solution flows through a porous strip,
where it encounters labeled antibody
• Visible line produced when antigen-antibody
immune complexes encounter antibody against
them
• Used in pregnancy testing and for rapid
identification of some infections
Figure 17.15 Immunochromatographic dipstick.
Zone of antibodies
linked to colloidal metal,
color too diffuse to see
Line of fixed
anti-antibody
Anti-antibodies stop
movement of antibodyantigen complexes. Color
becomes visible because
of density of complexes.
Movement of
fluid containing
complexes of
antibodies
bound to
antigen
Prepared antigen
extract from patient’s
nasal sample
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Western Blot (to detect a specific protein):
1. A sample is separated by electrophoresis.
2. It is transferred (transblotted) to a
nitrocellulose paper.
3. The transblot is cut into strips, which are
incubated with a solution of antibody vs
the test agent to allow antigen-antibody
reaction.
4. The strips are washed to remove the
unbound antibody molecules.
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5. The strips are treated with an enzyme
linked antihuman immunoglobulin
(enzyme-labeled anti-antibody). The
strips are washed.
6. Substrate of the enzyme is added.
7. If a visible color change occurs, the test is
positive. If not, the test is negative.
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Southern Blot (to detect a specific DNA sequence):
It was devised by Ed. Southern in 1975.
1. DNA is cleaved by restriction endonuclease.
2. The DNA fragments are separated by
electrophoresis.
3. The DNA fragments are denatured.
4. The single-stranded DNA fragments are
transferred (transblotted) to a nitrocellulose or
nylon membrane.
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5. A labeled DNA probe or RNA probe is added.
The probe can be labeled with radioactive
isotope, fluorescent dye or enzyme.
6. Rinse thoroughly to remove the unbound
labeled probe.
7. The DNA sequence of interest can be detected.
Autoradiography is used for radioactive
isotope-labeled probe, ultraviolet light is used
for fluorescent dye-labeled probe, and
substrate is used for enzyme-labeled probe.
Northern blot is used to detect a specific
RNA sequence using a labeled DNA probe or
RNA probe.
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In Vivo Testing:
Skin Test
Tuberculin
Lepromin
Schick
Dick
Disease
Tuberculosis
Leprosy
Diphtheria
Agent
M. tuberculosis
M. leprae
Corynebacterium
diphtheriae
Scarlet fever Streptococcus
pyrogens
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Skin Test
Disease
Brucellergen Brucellosis
Frei
Lymphogranuloma
venereum
Blastomycin Blastomycosis
Agent
Brucella abortus
Chlamydia
trachomatis
Blastomyces
dermatitidis
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Skin Test
Histoplasmin
Leishmanin
Disease
Histoplasmosis
Leishmaniasis
Agents
Histoplasma
capsulatum
Leishmania
donovani