PowerPoint® Lecture

Presentations prepared by
Mindy Miller-Kittrell,
North Carolina State University

CHAPTER 17
Immunization
and Immune
Testing

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 Immunization

• Two Artificial Methods of Immunity

• Active immunization
• Administration of antigens so that patient actively mounts
an adaptive immune response

• Passive immunization
• Individual acquires immunity through the transfer of
antibodies formed by immune individual or animal

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 Immunization

• Brief History of Immunization

• Chinese noticed children who recovered from smallpox
did not contract the disease again

• They infected children with material from a smallpox

scab to induce immunity
• This process was known as variolation

• Variolation spread to England and America but was

stopped because of risk of death

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 Immunization

• Brief History of Immunization

• 1796 – Edward Jenner discovered process of
vaccination

• 1879 – Louis Pasteur developed a vaccine against

Pasteurella multocida

• Antibody transfer developed when it was discovered

that vaccines protect through the action of antibodies

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 Immunization

• Active Immunization
• Vaccine types
• Attenuated (modified live) vaccines
• Use pathogens with reduced virulence
• Process of reducing virulence is called attenuation
• Can result in mild infections
• Active microbes stimulate a strong immune response
• Can provide contact immunity
• Modified microbes may retain enough residual
virulence to cause disease in susceptible individuals
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 Immunization

• Active Immunization
• Vaccine types
• Inactivated (killed) vaccines
• Safer than live vaccines
• Whole agent vaccines
• Deactivated but whole microbes
• Subunit vaccines
• Antigenic fragments of microbes
• Often require multiple doses to achieve full immunity
• Often contain adjuvants
• Chemicals added to increase effective antigenicity
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 Immunization

• Active Immunization
• Vaccine types
• Toxoid vaccines

• Chemically or thermally modified toxins used to

stimulate active immunity

• Useful for some bacterial diseases

• Stimulate antibody-mediated immunity

• Require multiple doses because toxoids possess few

antigenic determinants

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 Immunization

• Active Immunization
• Vaccine types
• Combination vaccines
• Simultaneous administration of antigens from several
pathogens
• Vaccines using recombinant gene technology
• Research attempts to make vaccines more effective,
cheaper, and safer
• Recombinant DNA techniques used to improve
vaccines
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 Figure 17.2 Some uses of recombinant DNA technology for making improved vaccines.
Production of vaccine Components of vaccine

Virulence gene
is deleted

Virulent pathogen Harmless, attenuated pathogen

with antigens

Antigen Cell

Insertion of Cell Antigenic protein

Isolated DNA DNA into a cell releases molecules
coding for antigenic
antigenic protein protein

Antigen

Insertion of
Virus or cell
Isolated DNA DNA into virus
presenting pathogen's
coding for or cell
antigen
antigen

Antigen

Recombinant
Insertion of plasmids
DNA into produced
Isolated DNA plasmid
coding for vector
antigen
Plasmid in body's cell,
which synthesizes
pathogen's antigen

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 Immunization

• Active Immunization
• Vaccine manufacture
• Mass-produce many vaccines by growing microbes in
culture vessels

• Viruses are cultured inside chicken eggs

• Individuals with egg allergies must avoid some vaccines

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 Figure 17.3 The CDC's recommended immunization schedule for the general population.

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© 2015 Pearson Education, Inc.
© 2015 Pearson Education, Inc.
 Immunization

• Active Immunization
• Vaccine safety
• Problems associated with immunization

• Mild toxicity

• Risk of anaphylactic shock

• Residual virulence from attenuated viruses

• Allegations certain vaccines cause autism, diabetes,

and asthma

• Research has not substantiated these allegations

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 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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 Figure 17.4 The production of hybridomas.
1 Mouse is injected Long-lived myeloma cell
with antigen. lines are grown in culture.

2 Plasma cells,
which secrete
antibodies,
are removed.

Antibodies

3 Hybridomas are formed

by mixing and fusing
Hybridoma
plasma cells and myeloma
cells; hybridomas are
long lived and produce
antibodies.

4 Hybridomas are
placed individually
in small wells, and
their antibodies are
tested for reactivity
against the antigen.

5 A hybridoma that makes

antibodies that react
with the antigen is cloned.
Monoclonal
antibodies

Hybridoma clone

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 Figure 17.5 The characteristics of immunity produced by active immunization (red) and passive immunotherapy
(green).

Passive
immunotherapy

Injection

Active
Boosters immunization

Initial
inoculation

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 Immunization

• Tell Me Why
• Vaccines have drastically reduced the number of cases
of many diseases, such as measles and whooping
cough. Why should parents have their children
vaccinated given that there are so few cases?

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Serology is the determination of the presence of
specific antigens or antibodies in blood serum

• Serological tests are available to identify a variety

of antigens and antibodies in serum

• Serological tests have several uses

• Monitor the spread of infection within a population

• Establish diagnosis of disease

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Precipitation Tests
• Among the simplest of serological tests

• Antigens and antibody are mixed in the proper

proportion form large complexes called precipitates

• Antigen-antibody complexes are also called immune

complexes

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 Figure 17.6 Characteristics of precipitation reactions.

No precipitate Precipitate No precipitate

Amount of antibody
precipitated

Increasing amount of antigen

(a)

Antibody

Antigen

Antibody excess Optimal proportions Antigen excess

(b)
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 Figure 17.7 Immunodiffusion, a type of precipitation reaction.
Well containing Line of immune precipitation Well containing
antigen molecules antibodies against
the antigen

Agar

Zone of Zone of Zone of

antigen optimal antibody
(a) excess precipitation excess

Well containing Lines of immune precipitation Well containing

four different a mixture of
antigens antibodies, each
reacting to a
different antigen

(b)
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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Turbidimetric and Nephelometric Tests
• Turbidimetry and nephelometry measure the cloudiness
of a solution

• Turbidimetry measures the light passing through a

solution

• Nephelometry measures the light reflected from a

solution

• Can be used to quantify the amounts of proteins in serum

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Agglutination Tests
• Agglutination results from the cross-linking of antibodies with
particulate antigens

• Agglutination is the clumping of insoluble particles

• Precipitation involves the aggregation of soluble molecules

• Reactions can be easy to see and interpret with the unaided eye

• Hemagglutination

• Agglutination of red blood cells

• Can be used to determine blood type

• Titration measures antibody levels in serum by agglutination

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 Figure 17.8 The use of hemagglutination to determine blood types in humans.
Anti-A antibody added Anti-B antibody added

Blood sample

A B

A B

Negative result: no agglutination Positive result: agglutination

of blood cells of blood cells

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 Figure 17.9 Titration, the use of agglutination to quantify the amount of antibody in a serum sample.
Serum added in increasing dilutions
Control (no
1:1 1:10 1:100 1:1000 1:10,000 specimen added)

Antigen (identical
in each well)

++++ +++ ++ + – Control

Very strong No
agglutination agglutination
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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Neutralization Tests
• Viral neutralization

• Cytopathic effect

• Viruses introduced into appropriate cell cultures will kill the cells

• Ability of virus to kill culture cells is neutralized when virus is first mixed
with antibodies against it

• Viral neutralization test

• Mixture of virus and serum added to cell culture

• Absence of cytopathic effect indicates presence of antibodies

against the virus in the serum

• Identifies whether individual has been exposed to a particular virus

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Neutralization Tests
• Viral hemagglutination inhibition test
• Useful for viruses that are not cytopathic

• Based on viral hemagglutination

• Some viral surface proteins can clump red blood cells

• A serum sample that contains antibodies against a

specific virus will inhibit viral hemagglutination

• Commonly used to detect antibodies against influenza,

measles, and mumps

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• The Complement Fixation Test
• Based on generation of membrane attack complexes
during complement activation

• Used to detect the presence of specific antibodies in an

individual's serum

• Can detect antibody amounts too small to detect by

agglutination

• Replaced by other serological methods

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Labeled Antibody Test
• Uses antibody molecules linked to some "label" that
enables them to be easily detected

• Used to detect either antigens or antibodies

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Labeled Antibody Test
• Fluorescent antibody tests
• Use fluorescent dyes as labels

• Fluorescently labeled antibodies used in two types of

tests

• Direct fluorescent antibody tests

• Indirect fluorescent antibody tests

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 Figure 17.10 A direct fluorescent antibody test.

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 Figure 17.11 The indirect fluorescent antibody test.
1 Cells with antigen are attached to slide
and flooded with patient's serum.

IgG from
patient's serum
Antigen
on cell
bound
to slide

2 Fluorescent-labeled anti-Ig antiglobulin is added.

Fluorescent label

Anti-IgG (antiglobulin,
anti-antibody)

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Labeled Antibody Test
• ELISAs (EIAs)
• Stands for enzyme-linked immunosorbent assay

• Uses an enzyme as the label

• Reaction of enzyme with its substrate produces a

colored product, indicating a positive test

• Commonly used to detect the presence of serum

antibodies

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 Figure 17.12 Enzyme-linked immunosorbent assay (ELISA), also known as enzyme immunoassay (EIA).

1 Antigen is attached to well in plate.

2 A protein such as gelatin is added to block the uncoated surface.

3 Patient serum is added; complementary antibody binds to antigen.

Enzyme

Anti-antibody

4 Enzyme-linked anti-antibody is added and binds to bound antibody.

Substrate Colored product

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5 Enzyme's substrate is added, and reaction produces a visible color change.
 Serological Tests That Use Antigens and
Corresponding Antibodies
• Labeled Antibody Test
• ELISAs
• Advantages of the ELISA
• Can detect either antibody or antigen
• Sensitive
• Can quantify amounts of antigen or antibody
• Easy to perform and can test many samples quickly
• Relatively inexpensive and easy to automate
• Plates coated with antigen can be stored for later
testing
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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Labeled Antibody Test
• ELISAs
• Antibody sandwich ELISA

• Modification of the ELISA technique

• Commonly used to detect antigen

• Antigen being tested for is "sandwiched" between two

antibody molecules

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 Figure 17.13 An antibody sandwich ELISA.

Colored
product
Gelatin
Enyme-linked
Substrate antibody
Antigen in
patient's serum
Antibody bound
to microwell

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Labeled Antibody Test
• Immunoblots
• Also called a western blot

• Technique to detect antibodies against multiple antigens

• Used to confirm the presence of proteins

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 Figure 17.14 Immunoblotting.
Wells containing antigens

Polyacrylamide gel

1 Electrophoresis (not shown)

Polyacrylamide gel

2 Blotting
Filter paper
Nitrocellulose
membrane

Patient
Polyacrylamide gel
1
Absorbant paper
2

Nitrocellulose (blot) 4
is cut into strips
5
3 ELISA is performed on strips
6

Positive
control
Negative
control
Polypeptides

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Point-of-Care Testing
• Simple immunoassays that give results in minutes

• Useful in determining a quick diagnosis

• Common tests
• Immunofiltration assay

• Immunochromatography assay

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Point-of-Care Testing
• Immunofiltration
• Rapid ELISA that uses antibodies bound to membrane
filters rather than plates

• Large surface area of the membrane filter reduces time to

complete the assay

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 Serological Tests That Use Antigens and
Corresponding Antibodies
• Recent Developments in Immune Testing
• Immunochromatography
• Very rapid and easy-to-read ELISAs

• Antigen solution flows through a porous strip

• 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

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 Figure 17.15 Immunochromatographic dipstick.
Zone of antibodies Line of fixed
linked to colloidal metal, anti-antibody
color too diffuse to see

Anti-antibodies stop
movement of antibody-
antigen complexes. Color
becomes visible because
of density of complexes.

Movement of
fluid containing
complexes of
antibodies Prepared antigen
bound to extract from patient's
antigen nasal sample

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© 2015 Pearson Education, Inc.
 Serological Tests That Use Antigens and
Corresponding Antibodies
• Tell Me Why
• A diagnostician used an ELISA to show that a newborn
had antibodies against HIV in her blood. However,
six months later the same test was negative. How can
this be?

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