Immunological
ApplicationsChapter 8: Background
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The Immune System
Types of Immunity
– Innate
• The natural immunity present at birth
– Acquired
• A specific response to foreign substances. Some
cells “remember” invaders and can respond quickly to
future infections
• Immunizations trigger this response so that recipients
are prepared for future infections
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The Immune System
Antigens
– Something that elicits an immune response
• Can be viruses, bacteria, toxins, drugs, DNA, RNA, and other
chemicals
Antibodies
– Proteins produced by white blood cells that bind to antigens
Epitope
– A single shape on the antigen that an antibody will bind to
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The Immune Response
Pathogen has antigen on surface, is contacted by
macrophage and engulfed through phagocytosis
The macrophage displays antigen on surface with
major histocompatibility complex (MHC)
T cells relay the message to B cells
B cells start producing antibodies and become
plasma cells
Some become memory B cells
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The Immune System
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Antibodies
Antibodies are also called
immunoglobulins (Ig)
– Types
• IgA: first line of defense with
invaders found in tears,
saliva, milk
• IgD: initiates B cells and
activates other immune cells
• IgE: allergic reactions
• IgG: most abundant,
produced when infected
• IgM: found in serum,
responsible for primary
immune response
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Immune Response
Primary response
– Initial production of antibodies
when infected
– Response is slow
Secondary response
– Response after an initial
exposure
– Much more rapid and robust
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Antibodies as Tools
Two main types of antibodies produced to be
used as tools in biotech
– Polyclonal antibodies are produced by injecting an
animal with an antigen and harvesting the serum that
contains the antibodies
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Antibodies as Tools
Monoclonal
antibody production
– Extract B cells from
an inoculated animal
– Fuse with a tumor
cell to make
immortal
– Clone the immortal
cells and collect
antibodies
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Antibodies as Tools
Antibodies to
deliver treatment
to specific cells
– Humanized
recombinant
antibodies
combine part of a
human and part of
a mouse antibody
sequence
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Immunoassays
Tests that use
antibodies as a
component
– Most common is
pregnancy test
• Urine containing hCG is
added to strip
• Mobile colored
antibodies bind and
move
• They bind to a stationary
set against hCG
• Or continue to bind to a
control set
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Other Types of Assays
Enzyme-linked immunosorbant assays (ELISA)
Ouchterlony or double diffusion assays
Western blots
Immunohistochemical and immunocytological assays
performed on slides or in tissues for microscopic
analysis
Multiplex assays that use beads and fluorescent
sorting also involve antibodies
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ELISA
Uses antibodies to detect an antigen or antibody of interest by
exploiting the tendency of proteins to adsorb (stick) to a plastic
microplate
Can detect either antigens or antibodies
Can be either direct (using only a primary antibody) or indirect
(using a secondary antibody to detect the first antibody)
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ELISA Types
Direct ELISA
– Antigens from sample coat the well
– Enzyme-linked antibodies bind to the antigen of interest if
present
– Colorimetric substrate is added
– Enzyme linked to the antibody cleaves the substrate, turning
the solution blue, which indicates a positive test
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ELISA Types
Indirect ELISA
– Antigens from sample coat the well
– Primary antibodies bind to the antigen of interest if present
– Enzyme-linked secondary antibody is added which binds to
the primary antibody
– Substrate color change indicates a positive test
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ELISA Types
Direct sandwich ELISA
– Primary antibodies are bound to wells
– Sample is added that may contain the antigen
– Enzyme-linked secondary antibody is added which binds to
the antigen if present
– Substrate color change indicates a positive test
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ELISA Types
Capture serum antibody
– Purified antigens are bound to wells
– Serum sample is added that may contain antibodies to the
purified antigen
– Enzyme-linked secondary antibody is added which binds to
serum antibodies if present
– Substrate color change indicates a positive test
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Instruments for ELISA
Microplate readers
– Use spectrophotometric methods to measure the
amount of absorbance by the developed substrate
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Ouchterlony or Double Diffusion Assay
Antibodies are placed into
the center well and diffuse
outward
Antigens placed in the
surrounding wells diffuse
inwards
Interactions form a
precipitation event
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Western Blotting
SDS-PAGE results can be
used to estimate the mass of
proteins, but cannot identify
them directly
Antibodies can be used in the
same way as an ELISA test
when the proteins from an
SDS- PAGE experiment are
transferred to a membrane
– Electroblotting is the general
method used
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Western Blotting
Once proteins have been transferred to the
membrane, the technique is similar to the indirect
ELISA
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Western Blot Detection
Colorimetric
– Visible color left after
substrate interaction
Chemiluminescent
– Substrate can emit light
when enzyme reacts
– Sometimes different
molecules linked to the
secondary antibody emit
different colors. This
allows for multiple
detections
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Other Applications for Western Blots
Viral protein detection
– Diagnosis of HIV infection
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Other Applications for Antibodies
Immunohistochemistry
and
immunocytochemistry
– When antibodies are
used to study tissues
and cells
• Immunofluorescence is
when an antibody is
labeled with a
fluorescent molecule
and will emit a different
wavelength of light than
the antibody being
stimulated
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Other Applications for Antibodies
Multiplex beads
– Beads that have a color themselves can be used
– If different antibodies are linked to various colors then
as they move past a detector, the color of the bead
showing an interaction can be used to determine what
was bound
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Other Applications for Antibodies
Fluorescent-activated
cell sorting (FACS)
– Different antibodies are
labeled with different
fluorescent molecules
enabling sorting due to a
charge that is applied to
the cell
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Chapter 8 Summary
Background
• The Immune System
• Types of Immunity
• Immune Response
Antibodies
• Structure
• Types
• Responses
Antibodies as Tools
• Treatment
• Immunoassays
• Other Applications that Utilize Antibodies