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
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
Weakened form of pathogen
Generally unable to cause disease
Strain replicates in vaccine recipient
Causes infection with undetectable or mild
symptoms
Results in long lasting immunity
Vaccines and Immunization
Attenuated vaccines Disadvantages
Advantages Have potential to
Single dose usually
cause disease in
sufficient to induce long- immunocompromised
lasting immunity individuals
Due to multiplication of Pregnant women
microbe in body should also avoid
Continued stimulation immunization with
of immune system attenuated vaccine
Vaccine as added potential Attenuated vaccines in
for being spread use include
“Disease” after Sabin polio vaccine
immunization could be
spread to un-immunized MMR
individuals inadvertently 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
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
Person not yet exposed to antigen and has no
specific antibodies
Seropositive
Person with exposure and actively producing
antibody
Titer
Concentration of antibody in serum
Indicates previous exposure
Principles of Immunological Testing
Obtaining antibody
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
Animal is immunized with antigen and produces
specific antibodies
Antibodies are retrieved by harvesting animal’s
serum
Principles of Immunological Testing
Obtaining antibody
Certain serological tests bind human IgG
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
Antibodies binding to
soluble antigen form
insoluble complexes
Complexes precipitate
out of solution
Complete aggregate
formation occurs at
certain concentrations
To achieve
concentrations place
separate antigen and Diffuse together to create
antibody suspensions zone of optimal proportion
side by side
Agglutination - Precipitation
Antibody
Antigen
When there is an excess of antibodies If there is an appropriate ratio When there is an excess of antigen
each antigen is individually bound and of antigen / antibody each antibody is individually bound
no agglutination occurs agglutination and precipitation and no agglutination occurs
occur
Observing Antigen-Antibody
Aggregations
Immunodiffusion tests
Most widely know is Ouchterlony
Antigen and antibody placed in separate wells
cut in gel
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
Radial immunodiffusion test is
quantitative
Antibody is added to liquid agar
that is allowed to harden
Created a uniform antibody
concentration
Antigen added to wells cut in gel
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
Large insoluble particles are involved
Obvious aggregations are formed
Makes the easier to see
Direct agglutination
Specific antibody mixed with insoluble antigen
Readily visible clumping indication of positive result
Indirect agglutination
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
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
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
Direct ELISA
Looks for specific antigen
Specimen placed in wells of microtiter plate
Wells treated with antibody for antigen
Indirect ELISA
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
Makes it possible to establish
which proteins are recognized
by antibodies
Using Labeled Antibodies to
Detect Interactions
Fluorescence Activated Cell Sorter (FACS)
Special version of flow cytometry counts cells
labeled with fluorescent antibodies
Used to count subsets of T cells
CD4 and CD8 cell especially
Antibodies are attached to the CD4 and CD8 markers
Cells with fluorescently labeled markers are counted