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Antibodies
Cells of the vertebrate acquired
immune system produce antibodies with
an exquisite specificity for molecules
Biologists use antibodies to localize
molecules of interest in complex
preparations
Antibodies bind tightly and specifically to
their target molecules
Bound antibodies are visualized with
additional treatments that render the
bound antibody visible
CIL:10233 Hippocampal neurons
Actin (red); tubulin (green)
western blot of bacterial cell extracts
What is the relationship between antibodies, antigens and
epitopes?
What happens during an immune response?
How are monoclonal antibodies generated?
How are antibodies used to visualize molecules?
Antigens are foreign substances that stimulate the immune
system to produce antibodies
1
3
Antigen
bacteria
viruses
allergens
large molecules
Antigen with 3 epitopes
2
Epitopes refer to the portions of the antigen recognized by the
immune system
Lymphocytes produce antibodies capable of binding epitopes
Most antigens have multiple epitopes (1-3 in the figure above)
Antibodies are composed of two identical heavy chains (red and blue)
and two identical light chains (yellow and green)
Fab
Fab
Fc
Bi-functional molecules
Fab fragments bind antigen
Fc fragments are used by the
immune system to remove
antigen-antibody complexes
Lymphocytes can generate millions of different antigen binding sites by
DNA rearrangement and mutation - processes restricted to immune cells!!
Antigens bind hyper-variable regions at the tips of Fab fragments
antigen binding has been
compared to a lock-andkey fit (complementary
surfaces)
A limited number of (Fc) regions
determine how other cells will process
antibody-antigen complexes
Antibodies used in molecular biology
have a gamma heavy chain (IgG
immunoglobins)
Fab regions of antibodies bind specifically to
epitopes on antigens
1
3
Antigen
2
Antibody binding leads to
the removal of the antigen
from the system mediated
by the Fc fragment
What is the relationship between antibodies, antigens and
epitopes?
What happens during an immune response?
How are monoclonal antibodies generated?
How are antibodies used to visualize molecules?
When challenged with a foreign substance, or antigen, vertebrates
produce antibodies that help the system dispose of the antigen
Vaccination stimulates the production
of antibody-producing cells
Biologists use antibodies produced
in animals to localize molecules
Antigen stimulates the proliferation of
B lymphocyte clones that recognize
epitopes on the antigen.
1
3
Antigen
2
Each lymphocyte
secretes large amounts
of a SINGLE antibody
molecule.
Antibodies make their way to the animal’s bloodstream.
Serum contains antibodies that recognize many different epitopes.
Polyclonal antibodies are semi-purified
fractions derived from animal serum
(antiserum)
Polyclonal antibodies may recognize
multiple epitopes on the same antigen
Limitations of polyclonal antibodies:
A limited amount of serum can be obtained from an animal
It is often useful to have antibodies with a defined specificity
What is the relationship between antibodies, antigens and
epitopes?
What happens during an immune response?
How are monoclonal antibodies generated?
How are antibodies used to visualize molecules?
Monoclonal antibodies recognize a single, well-defined epitope
produced by cultured hybridoma cells
hybridoma cells are formed by fusing antibody-secreting
lymphocytes from an animal with myeloma cells
hybridoma cells can be maintained indefinitely in tissue culture
hybridoma cells secrete large amounts of antibody that can be
harvested from the culture medium
Lots of commercial interest in monoclonal antibodies!
Select hybridomas producing
antibody of interest
Culture cells
Comparison: polyclonal vs. monoclonal
Polyclonal antibodies
Monoclonal antibodies
Immunoglobin fraction from
animal serum
Purified from medium of
cultured hybridoma cells
Mixture of antibodies with
different specificities
Antibody recognizes a single
epitope
May provide greater
sensitivity by binding multiple
epitopes on antigen
May provide lower background
since less cross-reaction with
other proteins
Often less expensive
Usually more expensive
Supply is limited
Supply is theoretically limitless
What is the relationship between antibodies, antigens and
epitopes?
What happens during an immune response?
How are monoclonal antibodies generated?
How are antibodies used to visualize molecules?
Detection protocols often use a sequence of antibodies
Primary antibodies
often a mouse monoclonal antibody
for an epitope of interest
Secondary antibodies
animals are injected with Fc
fragments from a different species
polyclonal antibodies are common
enzyme or a chromochrome is often
covalently attached to the
secondary antibody
signal is amplified
Secondary antibodies amplify the signal
2. Secondary antibodies recognize multiple
sites on primary antibody
Enzyme or fluorochrome
amplifies the signal
1. Primary antibody (stoichiometric binding)
Antigen molecules
Western blot
Primary antibody:
mouse monoclonal antibody
Secondary antibody:
Goat anti-mouse IgG conjugated with
horseradish peroxidase (HRP)
HRP produces a colored reaction
product
western blot of bacterial cell extracts