Transcript Applications of Immune Responses

Applications of
Immune Responses
Chapter 17
Principles of Immunization
 Naturally acquired immunity is
acquisition of adaptive immunity
through natural events
 Immunization mimics these events
by inducing artificially acquired
immunity
 Natural or artificial immunity can
be divided into
 Active immunity
 Passive immunity
Principles of Immunization
 Active immunity
 Result from immune response
upon exposure to an antigen
 Active immunity can develop
naturally
 Following illness
 Or artificially
 After immunization
Principles of Immunization
 Passive Immunity
 Occurs naturally during pregnancy
 IgG from mother crosses placenta
 Inferres protection to the baby
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Occurs naturally as result of breast
feeding
 IgA antibodies in breast milk given
to child
Artificial passive immunity involves
transfer of antibodies produced by
another person or animal
 Can be used to prevent disease
before or after likely exposure
Vaccines and Immunization
 Attenuated vaccines
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Weakened form of pathogen
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Generally unable to cause disease
Strain replicates in vaccine recipient
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Causes infection with undetectable or mild
symptoms
Results in long lasting immunity
Vaccines and Immunization
 Attenuated vaccines
 Advantages
 Single dose usually
sufficient to induce longlasting immunity

 Due to multiplication of
microbe in body
 Continued stimulation
of immune system
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Vaccine as added potential
for being spread
 “Disease” after
immunization could be
spread to un-immunized
individuals inadvertently

Disadvantages
 Have potential to
cause disease in
immunocompromised
individuals
 Pregnant women
should also avoid
immunization with
attenuated vaccine
Attenuated vaccines in
use include
 Sabin polio vaccine
 MMR
 Yellow fever
Vaccines and Immunization
 Inactivated vaccines
 Unable to replicate in vaccinated individual
 Retains immunogenicity of infectious agent
 Immunogenic not pathogenic
 Inactivated vaccines fall into two categories
 Whole agents
 Contain killed organisms of inactivated virus
 Does not change epitopes
 Cholera, plague, influenza and Salk polio are whole agents
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Fragments
 Portions of organisms or agents including toxins proteins and
cell wall components
 Includes toxoids, protein subunit vaccines and polysaccharide
vaccines
Principles of Immunological Testing
 Terms
 Seronegative
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Seropositive
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Person not yet exposed to antigen and has no
specific antibodies
Person with exposure and actively producing
antibody
Titer
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Concentration of antibody in serum
Indicates previous exposure
Principles of Immunological Testing
 Obtaining antibody
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Serum is fluid portion of blood with no clotting
factors
Plasma is fluid portion with clotting factors
Laboratory animals are used to produce known
antibodies
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Animal is immunized with antigen and produces
specific antibodies
Antibodies are retrieved by harvesting animal’s
serum
Principles of Immunological Testing
 Obtaining antibody
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Certain serological tests bind human IgG
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Antibodies are termed anti-human IgG
These can be produced in animals immunized
with IgG from human serum
Principles of Immunological Testing
 Quantifying antigen-antibody reactions
 Concentrations of antibody usually determined through
dilution
 Antigen added to dilution
 Titer is taken from last dilution to give detectable
reaction
Observing Antigen-Antibody
Aggregations
 Antigen-antibody complexes form aggregates
 Antigen-antibody binding can be seen in
precipitation and agglutination reactions
Observing Antigen-Antibody
Aggregations
 Precipitation reactions
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Antibodies binding to
soluble antigen form
insoluble complexes
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Complexes precipitate
out of solution
Complete aggregate
formation occurs at
certain concentrations
To achieve
concentrations place
separate antigen and
antibody suspensions
side by side
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Diffuse together to create
zone of optimal proportion
Agglutination - Precipitation
Antibody
Antigen
When there is an excess of antibodies
each antigen is individually bound and
no agglutination occurs
If there is an appropriate ratio When there is an excess of antigen
each antibody is individually bound
of antigen / antibody
agglutination and precipitation and no agglutination occurs
occur
Observing Antigen-Antibody
Aggregations
 Immunodiffusion tests
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Most widely know is Ouchterlony
Antigen and antibody placed in separate wells
cut in gel
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Solutions diffuse and meet between the wells
 Results in line of precipitation at zone of optimal
proportion
Ouchterlony Double Diffusion
Note: A line of precipitation has formed
between the center well and wells 3 & 5.
This indicates there is antigen/antibody
specificity between the center well and
these two wells.
Usually a known antigen or known
antibody is placed in the center and test
serum is placed in the peripherial wells.
Antigens and antibodies will diffuse and
at some point optimal concentrations
will occur and if the antigen is specific
for the antibody a precipitate line will
form.
Example: Has this patient ever had rubella, rubeolla, or diptheria? If they have their
serum will contain antibodies against the disease.
Put patient serum in the center. Put the disease agents (antigens) in wells 1 – 5,
and allow to diffuse. A precipitation line between wells indicates that the patient
has had that disease
Observing Antigen-Antibody
Aggregations
 Immunodiffusion tests
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Radial immunodiffusion test is
quantitative
Antibody is added to liquid agar
that is allowed to harden
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Antigen added to wells cut in gel
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Created a uniform antibody
concentration
Diffusion outward forms
concentration gradient
Ring forms at antigen-antibody
precipitation
Standards can be used to
construct standard curve to
establish concentration
Observing Antigen-Antibody
Aggregations
 Immunoelectrophoresis
 Proteins separated using gel
electrophoresis
 Antibodies are placed in wells
and allowed to diffuse towards
separated proteins
 Line of precipitation forms at
antibody-protein recognition
 Used to determine patient
antibody levels
 High levels of certain antibody
classes can indicate disease
Observing Antigen-Antibody
Aggregations
 Agglutination reactions
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Large insoluble particles are involved
Obvious aggregations are formed
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Makes the easier to see
Direct agglutination
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Specific antibody mixed with insoluble antigen
 Readily visible clumping indication of positive result
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Indirect agglutination
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Amplifies aggregation formation
 Antibody attached to latex bead
 Agglutination of these beads much easier to see
Using Labeled Antibodies to
Detect Interactions
 Detectable markers can be attached to specific
antibodies
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Marked antibodies used to detect presence of
given antigen
 Test include
 Fluorescent Antibody (FA) test
 Enzyme Linked Immunosorbant Assay (ELISA)
 Western blotting
 Fluorescence Activated Cell Sorter (FACS)
Using Labeled Antibodies to
Detect Interactions
 Fluorescent antibody test
 Relies on fluorescence microscopy to
locate labeled antibodies fixed to a
microscope slide
 Fluorescent polarized immunoassay
uses beam of polarized light to rate
spin of labeled antibodies
 Works under principle that bound
antibodies are heavier then
unbound and will spin more slowly
Using Labeled Antibodies to
Detect Interactions
 Enzyme Linked Immunosorbant
Assay
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Employs antibody that has been
labeled with detectable enzyme
 Commonly horseradish
peroxidase
Labeled antibody binds to
antigen
 Binding can be direct or
indirect
Antigen location is determined
using colormetric assay
Using Labeled Antibodies to
Detect Interactions
 Enzyme Linked Immunosorbant Assay
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Direct ELISA
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Looks for specific antigen
 Specimen placed in wells of microtiter plate
 Wells treated with antibody for antigen
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Indirect ELISA
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Looks of antibody in patient serum
 Human IgG
 Wells of plate treated with known antigen
Using Labeled Antibodies to
Detect Interactions
 Western blotting
 Technique used detect
antigenic proteins
 Proteins are separated by size
before reacting with antibody
 Proteins separated by
special gel electrophoresis
 SDS PAGE
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Makes it possible to establish
which proteins are recognized
by antibodies
Using Labeled Antibodies to
Detect Interactions
 Fluorescence Activated Cell Sorter (FACS)
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Special version of flow cytometry counts cells
labeled with fluorescent antibodies
Used to count subsets of T cells
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CD4 and CD8 cell especially
Antibodies are attached to the CD4 and CD8 markers
 Cells with fluorescently labeled markers are counted