Chapter 18
Practical
Applications of
Immunology
© 2013 Pearson Education, Inc.
Lectures prepared by Christine L. Case
Vaccines
Learning Objectives
18-1
18-2
18-3
18-4
18-5
18-6
18-7
Define vaccine.
Explain why vaccination works.
Differentiate the following, and provide an example of
each: attenuated, inactivated, toxoid, subunit, and
conjugated vaccines.
Contrast subunit vaccines and nucleic acid vaccines.
Compare and contrast the production of
whole-agent vaccines, recombinant vaccines, and DNA
vaccines.
Define adjuvant.
Explain the value of vaccines, and discuss acceptable
risks for vaccines.
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History of Vaccines
ƒ Variolation: Inoculation of smallpox into skin
(18th century)
ƒ Vaccination:
ƒ Inoculation of cowpox virus into skin (Jenner)
ƒ Inoculation with rabies virus (Pasteur)
ƒ Vaccine: A suspension of organisms or fractions
of organisms that is used to induce immunity
ƒ If most of the population is immune called herd
immunity.
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Principles and effects of vaccination
ƒ Provoke a primary immune response in the
recipients leading to the formation of antibodies
and long-term memory cells.
ƒ When recipient encounter antigen, the memory
cells are stimulated to produce a rapid intense
secondary immune response.
ƒ Herd immunity results when most of a population is
immune to a disease.
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免疫反應的兩個階段
抗原
初級反應
抗原
次級反應
Primary
response
Secondary
response
初次入侵
再次入侵
免
疫
反
應
時間
© 2013 Pearson Education, Inc.
Adapted from Roitt et al (1985) Immunology. 1.9
免疫增幅
抗 原
初次入侵
初級反應
作用細胞
記憶細胞
抗 原
再次入侵
次級反應
作用細胞
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記憶細胞
Adapted from Roitt et al (1985) Immunology. 2.5
Chapter 18, unnumbered figure B, page 510, Reported numbers of measles cases in the United States,
1960–2010. (CDC, 2010)
140
Reported number of cases
120
Vaccine
licensed
500,000
450,000
Reported number of cases
400,000
100
80
60
40
20
350,000
0
2000
300,000
01
02
03
04
05
Year
06
07
08
09
10
250,000
200,000
150,000
100,000
50,000
0
1960
1965
1970
1975
1985
1980
1990
1995
2000
Year
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Chapter 18, unnumbered figure C, page 510, Countries with the highest measles mortality.
© 2013 Pearson Education, Inc.
2005
2010
Vaccines Used to Prevent Bacterial
Diseases
Disease
Diphtheria
Vaccine
Purified diphtheria toxoid
Meningococcal
meningitis
Pertussis (whooping
cough)
Pneumococcal
pneumonia
Purified polysaccharide from
Neisseria meningitidis
Inactivated toxin plus acellular
fragments of Bordetella pertussis
Purified polysaccharide from
seven strains of Streptococcus
pneumoniae
Purified tetanus toxoid
Tetanus
Haemophilus influenzae Polysaccharide from Haemophilus
type b meningitis
influenzae type b conjugated with
protein to enhance effectiveness
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Vaccines Used to Prevent Viral
Diseases
Disease
Influenza
Measles
Vaccine
Injected vaccine, inactivated virus
(nasally administered: attenuated
virus)
Attenuated virus
Mumps
Attenuated virus
Rubella
Attenuated virus
Chickenpox
Attenuated virus
Poliomyelitis
Killed virus
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Vaccines Used to Prevent Viral
Diseases
Disease
Vaccine
Rabies
Killed virus
Hepatitis B
Antigenic fragments of virus
Hepatitis A
Inactivated virus
Smallpox
Live vaccinia virus
Herpes zoster
Attenuated virus
Human papillomavirus
Antigenic fragments of virus
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Vaccines for Persons Aged 0–6 Years
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
Hepatitis B
Rotavirus
DTaP
Haemophilus influenzae type b
Pneumococcal
Inactivated poliovirus
Influenza
MMR
Varicella
Hepatitis A
Meningococcal
ANIMATION Vaccines: Function
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Types of Vaccines
ƒ Attenuated whole-agent vaccines
ƒ MMR vaccine
ƒ Inactivated whole-agent vaccines
ƒ Salk polio vaccine
ƒ Toxoids
ƒ Tetanus vaccine
ƒ Subunit vaccines
ƒ Acellular pertussis
ƒ Recombinant hepatitis B
ƒ Nucleic acid (DNA) vaccines
ƒ West Nile (for horses)
ANIMATION Vaccines: Types
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Attenuated whole-agent vaccines
ƒ Use living, but attenuated (weakened), microbes
ƒ The long-term effectiveness occurs because the
attenuated viruses replicate in the body
ƒ e.g. Sabin polio vaccine and MMR (measles,
mumps and rubella)
ƒ Usually derived from mutations accumulated
during long-term culture (live microbes can
backmutate to a virulent form).
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Inactivated whole-agent vaccines
ƒ Use microbes that have been killed by formalin or
phenol.
ƒ e.g. inactivated viruses: rabies, influenza and
polio (Salk polio vaccine); inactivated bacteria:
pneumococcal pneumonia and cholera.
ƒ New inactivated bacteria vaccines: pertussis
(whooping cough) and typhoid.
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Toxoids
ƒ Inactivated toxins
ƒ e.g. tetanus and diphtheria toxoids.
ƒ Require a series of injections for full immunity,
followed by boosters every 10 years.
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Subunit vaccines
ƒ Antigenic fragments of a microorganism that best
stimulate an immune response.
ƒ Produced by genetic engineering techniques
(other microbes are programmed to produce the
desired antigenic fraction (recombinant vaccines)
ƒ e.g. hepatitis B virus
ƒ Fewer adverse effects (little or no extraneous
material)
ƒ Acellular vaccines: fractions of a disrupted
bacterial cell retaining the desired antigenic
fractions, e.g. pertussis
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Conjugated vaccines
ƒ For poor immune response of children based on
capsular polysaccharides
ƒ Polysaccharides are T-independent antigens (no
response until 15-24 months)
ƒ Polysaccharide are combined with proteins such
as diphtheria toxoid.
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Nucleic acid vaccines
ƒ Plasmid of targeted DNA injected into muscle
results in the production of the protein
encoded in the DNA.
ƒ Disadvantage: DNA remains effective only
until it is degraded.
ƒ RNA might be a more effective agent.
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Figure 18.1 Influenza viruses are grown in embryonated eggs.
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Figure 13.7 Inoculation of an embryonated egg.
Shell
Amniotic
cavity
Chlorioallantoic
membrane
Chlorioallantoic
membrane
innoculation
Air
sac
Amniotic
innoculation
Yolk
sac
Shell
membrane
Albumin
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Allantoic
cavity
Allantoic
innoculation
Yolk sac
innoculation
The Development of New Vaccines
ƒ Culture pathogen
ƒ rDNA techniques
ƒ In plants (thick cell walls of plants help to protect the
cellular contents from degradation by stomach acids)
ƒ Adjuvants: chemicals added to improve the
effectiveness of antigens (e.g. alum)
ƒ Deliver in combination
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Safety of Vaccines
ƒ Cause and effect
Therapeutic index =
ƒ Risk vs. benefit
ƒ No vaccine will ever be perfectly safe or perfectly
effective
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Diagnostic Immunology
Learning Objectives
18-8
18-9
18-10
18-11
18-12
18-13
18-14
Differentiate sensitivity from specificity in a
diagnostic test.
Define monoclonal antibodies, and identify
their advantage over conventional antibody
production.
Explain how precipitation reactions and
immunodiffusion tests work.
Differentiate direct from indirect agglutination tests.
Differentiate agglutination from precipitation tests.
Define hemagglutination.
Explain how a neutralization test works.
© 2013 Pearson Education, Inc.
Diagnostic Immunology
Learning Objectives
18-15 Differentiate precipitation from neutralization
tests.
18-16 Explain the basis for the complement-fixation
test.
18-17 Compare and contrast direct and indirect
fluorescent-antibody tests.
18-18 Explain how direct and indirect ELISA tests
work.
18-19 Explain how Western blotting works.
18-20 Explain the importance of monoclonal
antibodies.
© 2013 Pearson Education, Inc.
Diagnostic Immunology
ƒ Sensitivity: probability that the test is reactive if
the specimen is a true positive
ƒ Specificity: probability that a positive test will not
be reactive if a specimen is a true negative
ƒ Immunologic-based tests
ƒ Guinea pigs with TB injected with Mycobacterium
tuberculosis: site became red and slightly swollen
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Monoclonal Antibodies (Mabs)
ƒ Hybridoma: “immortal” cancerous B cell fused with
an antibody-producing normal B cell
ƒ Produces monoclonal antibodies
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Monoclonal Antibodies
31 2 4
抗 原
BA
1
3
2
4
LB /
c
免 疫
1
2
3
31 2 4
4
m
m
m
m
1
2 3
4
1
2
傳統抗血清
3
取出脾細胞
+
骨髓瘤細胞
細胞融合
4
單株抗體
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Figure 18.2.1-2 The Production of Monoclonal Antibodies.
Antigen
1 A mouse is injected with a specific antigen
that will induce production of antibodies
against that antigen.
2 The spleen of the mouse is removed and
homogenized into a cell suspension. The
suspension includes B cells that produce
antibodies against the injected antigen.
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Spleen
Figure 18.2.3-4 The Production of Monoclonal Antibodies.
Suspension of
spleen cells
3 The spleen cells are then mixed with myeloma
cells that are capable of continuous growth in
culture but have lost the ability to produce
antibodies. Some of the antibody-producing
spleen cells and myeloma cells fuse to form
hybrid cells. These hybrid cells are now
capable of growing continuously in culture
while producing antibodies.
Spleen cells
Myeloma cells
Suspension of
myeloma cells
Cultured myeloma cells
(cancerous B cells)
Hybrid cell
Hybrid cells
Myeloma cell
4 The mixture of cells is
placed in a selective
medium that allows only
hybrid cells to grow.
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Figure 18.2.5-6 The Production of Monoclonal Antibodies.
Hybridomas
5 Hybrid cells proliferate into
clones called hybridomas.
The hybridomas are
screened for production of
the desired antibody.
Desired
monoclonal
antibodies
6 The selected hybridomas are then
cultured to produce large quantities
of monoclonal antibodies. Isolated
antibodies are used for treating and
diagnosing disease.
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Spleen cell
Monoclonal Antibodies
ƒ Immunotoxins: Mabs conjugated with a toxin to
target cancer cells.
ƒ Chimeric mabs: Genetically modified mice that
produce Ab with a human constant region.
ƒ Humanized mabs: Mabs that are mostly human,
except for mouse antigen-binding.
ƒ Fully human antibodies: Mabs produced from a
human gene on a mouse.
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Monoclonal Antibodies (Mabs)
ƒ
ƒ
ƒ
ƒ
ƒ
Muromonab-CD3: for kidney transplant
Infliximab: for Crohn’s disease
Comalizumab: for allergic asthma
Rituximab: rheumatoid arthritis
Trastuzumab: Herceptin for breast cancer
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A Precipitation Curve
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Figure 18.3 A precipitation curve.
Precipitation Reactions
ƒ Reaction of soluble antigens with antibodies
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Figure 18.4 The precipitin ring test.
Immunoelectrophoresis
If Lyme disease is suspected in a patient: Electrophoresis
is used to separate Borrelia burgdorferi proteins in the
serum. Proteins move at different rates based on their
charge and size when the gel is exposed to an electric
current.
Lysed
bacteria
Polyacrylamide
gel
Proteins
Larger
Paper towels
The bands are transferred to a nitrocellulose filter by
blotting. Each band consists of many molecules of a
particular protein (antigen). The bands are not visible at
this point.
Smaller
Sponge
Salt solution
Gel
Nitrocellulose
filter
The proteins (antigens) are positioned on the filter
exactly as they were on the gel. The filter is then
washed with patient’s serum followed by anti-human
antibodies tagged with an enzyme. The patient
antibodies that combine with their specific antigen are
visible (shown here in red) when the enzyme’s
substrate is added.
The test is read. If the tagged antibodies stick to the
filter, evidence of the presence of the microorganism in
question—in this case, B. burgdorferi—has been found
in the patient’s serum.
Figure 10.12 The Western blot.
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Agglutination Reactions
ƒ Precipitation reactions involve soluble antigens
ƒ Agglutination reaction involve either particulate
antigens or soluble antigens adhering to
particles.
ƒ Agglutination: antigens are linked together by
antibodies
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Agglutination Reactions
ƒ Particulate antigens
and antibodies
ƒ Antigens on a
cell (direct
agglutination)
Figure 18.5 An agglutination reaction.
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Indirect Agglutination
Antigen attached
to bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Latex
bead
Insert Fig 18.7
Latex
bead
Latex
bead
IgM antibody
Reaction in a positive indirect test for
antibodies. When particles (latex beads here)
are coated with antigens, agglutination
indicates the presence of antibodies, such as
the IgM shown here.
Figure 18.7 Reactions in indirect agglutination tests.
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Antibody
attached to bead
Latex
bead
Bacterial
antigen
Reaction in a positive indirect test for
antigens. When particles are coated with
monoclonal antibodies, agglutination
indicates the presence of antigens.
Antibody Titer
ƒ Concentration of
antibodies against
a particular antigen
Figure 18.6 Measuring antibody titer with the direct agglutination test.
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Hemagglutination
ƒ Hemagglutination involves agglutination of RBCs
ƒ Some viruses agglutinate RBCs in vitro
Figure 18.8 Viral hemagglutination.
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Viral Hemagglutination-Inhibition
ƒ Hemagglutination inhibition: antibodies prevent
hemagglutination
Figure 18.9b Reactions in neutralization tests.
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Neutralization Reactions
ƒ Neutralization is an antigen-antibody reaction in which the
harmful effects of a bacterial exotoxin or a virus are
blocked by specific antibodies.
ƒ A neutralizing substance, which is called an antitoxin, is a
specific antibody produced by a host as it responds to a
bacterial exotoxin or its corresponding toxoid.
ƒ Antitoxins can provide passive immunity against a toxin.
ƒ Viruses that exhibit their cytopathic effect (cell-damaging)
in cell culture or embryonated eggs can be used to detect
the presence of neutralizing viral antibodies.
ƒ In vitro neutralization test: Serum to be tested contains
antibodies against the particular virus, the antibodies will
prevent that virus from infecting cells in the cell culture or
eggs (no cytopathic effects will be seen).
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Neutralization Reactions
ƒ Eliminate the harmful effect of a virus or exotoxin
Figure 18.9a Reactions in neutralization tests.
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Complement Fixation
ƒ Complement fixation: complement binds to the
antigen-antibody complex and is fixed.
ƒ Complement fixation can be used to detect very
small amounts of antibody.
ƒ Antibodies that do not produce a visible reaction can
be demonstrated by the fixing of complement during
the antigen-antibody reaction.
ƒ Execution of the complement-fixation test requires
great care and good controls.
ƒ The test is performed in two stages: complement
fixation and indicator.
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Figure 18.10 The complement-fixation test.
Antigen
Antigen
Complement
Complement
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Complement
fixation
No
complement
fixation
Sheep RBC
Sheep RBC
Antibody to
sheep RBC
Antibody to
sheep RBC
No hemolysis
(complement
tied up in
antigen–
antibody
reaction)
Hemolysis
(uncombined
complement
available)
(a) Positive test. All available
complement is fixed by the antigen–
antibody reaction; no hemolysis
occurs, so the test is positive for the
presence of antibodies.
Complement-fixation stage
Serum
without
antibody
Indicator stage
Serum with
antibody
against antigen
(b) Negative test. No antigen–
antibody reaction occurs. The
complement remains, and the red
blood cells are lysed in the indicator
stage, so the test is negative.
Fluorescent-antibody (FA) techniques
ƒ FA can identify microorganisms in clinical
specimens and can detect the presence of a
specific antibody in serum
ƒ The technique combine fluorescent dyes such as
fluorescein isothiocyanate (FITC) with antibodies
to make them fluoresce when exposed to UV
light
ƒ Advantages: quick, sensitive, and specific
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Fluorescent-antibody (FA) techniques
ƒ Two types: direct and indirect.
ƒ Direct: to identify a microorganism in a clinical
specimen.
ƒ Indirect: to detect the presence of a specific
antibody in serum following exposure to a
microorganism. (often more sensitive than direct)
ƒ Fluorescein-labeled antihuman immune serum
globulin (anti-HISG), an antibody that reacts
specifically with any human antibody.
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Fluorescent-Antibody (FA) Techniques
ƒ Direct FA
Figure 18.11a Fluorescent-antibody (FA) techniques.
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Indirect Fluorescent-Antibody Test
Figure 18.11b Fluorescent-antibody (FA) techniques.
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Fluorescence-activated cell sorter
(FACS)
ƒ T cells carry antigenically specific receptors,
e.g. CD4 and CD8, on their surface.
ƒ The depletion of CD4 T cells is used to follow
the progression of diseases such as AIDS; their
populations can be determined with a FACS.
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Figure 18.12 The fluorescence-activated cell sorter (FACS).
1 A mixture of cells is
treated to label cells
that have certain
antigens with
fluorescent-antibody
markers.
2 Cell mixture leaves
Fluorescently
labeled cells
nozzle in droplets.
3 Laser beam strikes
each droplet.
Laser beam
Detector of
scattered light
Laser
Electrode
4 Fluorescence detector
identifies fluorescent
cells by fluorescent
light emitted by cell.
Fluorescence
detector
5 Electrode gives
positive charge to
identified cells.
Electrically
charged
metal plates
6 As cells drop between
electrically charged
plates, the cells with
a positive charge
move closer to the
negative plate.
7 The separated cells
fall into different
collection tubes.
Collection
tubes
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6
Enzyme-linked immunosorbent assay
(ELISA)
ƒ A group of enzyme immunoassay (EIA)
ƒ Two basic methods: direct and indirect ELISA
ƒ Advantages: little interpretive skill required, clear
positive or negative result presented
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Serological Tests
ƒ Direct tests detect antigens (from patient sample)
ƒ Indirect tests detect antibodies (in patient's
serum)
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Direct ELISA
1 Antibody is adsorbed to well.
2 Patient sample is added;
complementary antigen
binds to antibody.
3 Enzyme-linked antibody specific
for test antigen is added and binds
to antigen, forming sandwich.
4 Enzyme's substrate ( ) is added,
Figure 18.14a The ELISA method.
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and reaction produces a product that
causes a visible color change ( ).
A positive direct ELISA to detect antigens
Indirect ELISA
1 Antigen is adsorbed to well.
2 Patient serum is added;
complementary antibody
binds to antigen.
3 Enzyme-linked anti-HISG
(see page 518) is added and
binds to bound antibody.
Figure 18.14b The ELISA method.
4 Enzyme's substrate ( ) is added,
and reaction produces a product that
causes a visible color change ( ).
A positive indirect ELISA to detect antibodies
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Serological Tests
Control
windows
1 Free monoclonal
antibody specific
for hCG, a hormone
produced during
pregnancy.
Test
windows
2 Capture monoclonal
antibody bound to
substrate.
3 Sandwich formed by combination of
capture antibody and free antibody
when hCG is present, creating a
color change.
Not pregnant
Pregnant
Figure 18.13 The use of monoclonal antibodies in a home pregnancy test.
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Serological Tests
ƒ Precipitation: soluble antigens
ƒ Agglutination: particulate antigens
ƒ Hemagglutination: agglutination of RBCs
ƒ Neutralization: inactivates toxin or virus
ƒ Fluorescent-antibody technique: antibodies linked to
fluorescent dye
ƒ Complement fixation: RBCs are indicator
ƒ ELISA: peroxidase enzyme is the indicator
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The future of diagnostic immunology
ƒ Monoclonal antibodies: more sensitive, specific,
rapid, and simpler
ƒ Nonimmunological tests: PCR, DNA chip
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