Antibodies Cells of the vertebrate acquired immune system produce antibodies with an exquisite specificity for molecules CIL10233 Hippocampal neurons Actin red tubulin green western blot of bacterial cell extracts ID: 914549
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Slide1
Biologists use antibodies to localize molecules of interest in complex preparations
Antibodies
Cells of the vertebrate acquired immune system produce antibodies with an exquisite specificity for molecules
Slide2CIL:10233 Hippocampal neurons
Actin (red); tubulin (green)
western blot of bacterial cell extracts
Antibodies bind tightly and specifically to their target molecules
Bound antibodies are visualized with additional treatments that render the bound antibody visible
Slide3What 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?
Slide4Antigen
213
Antigens are foreign substances that stimulate the immune system to produce antibodies
bacteria
viruses
allergens
large molecules
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)
Antigen with 3 epitopes
Slide5F
ab
F
ab
F
c
Antibodies are composed of two
identical
heavy chains (red and blue) and two
identical
light chains (yellow and green)
Bi-functional molecules
F
ab
fragments bind antigen
F
c
fragments are used by the immune system to remove antigen-antibody complexes
Slide6A
limited number
of (F
c
) regions determine how other cells will process antibody-antigen complexes
Antibodies used in molecular biology have a gamma heavy chain (IgG immunoglobins)
Antigens bind hyper-variable regions at the tips of F
ab
fragments
antigen binding has been compared to a lock-and-key fit (complementary surfaces)
Lymphocytes can generate millions of different antigen binding sites by DNA rearrangement and mutation -
processes restricted to immune cells!!
Slide7Antigen
213
F
ab
regions of antibodies bind specifically to epitopes on antigens
Antibody binding leads to the removal of the antigen from the system mediated by the F
c
fragment
Slide8What 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?
Slide9When challenged with a foreign substance, or
antigen, vertebrates produce antibodies that help the system dispose of the antigen
Biologists use antibodies produced in animals to localize molecules
Vaccination stimulates the production of antibody-producing cells
Slide10Antigen
213
Antigen stimulates the proliferation of B lymphocyte clones that recognize epitopes on the antigen.
Each lymphocyte secretes large amounts of a SINGLE antibody molecule.
Slide11Antibodies 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
Slide12What 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?
Slide13Monoclonal 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 cellshybridoma 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!
Slide14Select hybridomas producing antibody of interest
Culture cells
Slide15Comparison: polyclonal vs. monoclonal
Polyclonal antibodies
Immunoglobin fraction from animal serumMixture of antibodies with different specificities
May provide greater sensitivity by binding multiple epitopes on antigen
Often less expensive
Supply is limited
Monoclonal antibodies
Purified from medium of cultured hybridoma cells
Antibody recognizes a single epitope
May provide lower background since less cross-reaction with other proteins
Usually more expensive
Supply is theoretically limitless
Slide16What 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?
Slide17Detection 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 F
c
fragments from a different species
polyclonal antibodies are common
enzyme or a chromochrome is often covalently attached to the secondary antibody
signal is amplified
Slide18Antigen molecules
1. Primary antibody (stoichiometric binding)
2. Secondary antibodies recognize multiple sites on primary antibody
Enzyme or fluorochrome amplifies the signal
Secondary antibodies amplify the signal
Slide19western blot of bacterial cell extracts
Western blot
Primary antibody:
mouse monoclonal antibody
Secondary antibody:
Goat anti-mouse IgG conjugated with horseradish peroxidase (HRP)
HRP produces a colored reaction product