Transcript MCB 181 (Nov 4 – Dec 4) Information and Heredity

Methods in cell biology and functional genomics
Individualized
Cells
Tissues
DNA
RNA
Genomics
Markers
Transcriptomics
Protein
Organs
Metabolite
Organisms
FACS
High throughput
Structure
Wild type
In situ hyb
Immuno
techniques
Microscopy
Mutant
Proteomics
Metabolomics
Applications Using Monoclonal and
Polyclonal Antibodies
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Western Blotting (WB)
Immunoprecipitation (IP)
Immunocytochemistry (ICC)
Immuno electron microscopy (IEM)
Immunofluorescence
Enzyme-Linked Immunosorbent Assay (ELISA)
Flow Cytometry (FC)
Antibody Production
• Immune response when foreign molecule enters
system.
• Binds antigen with high specificity and affinity.
Why Induce on Purpose?
• Same response can be induced on purpose for
specific selection or detection of Your Favorite
Antigen (YFA).
• Can produce millions of antibodies that
recognize YFA.
Several line of defense offer protection from invaders
Plasma Cell
Phagocyte
Blood Cells (Part 1)
Blood Cells (Part 2)
Blood Cells (Part 3)
Third line of defense consists of a specific response from
lymphocytes and antibodies of the immune system.
• There are two main types of lymphocytes: B lymphocytes (B
cells) and T lymphocytes (T cells)
• Both are types of white blood cells produced in bone marrow.
They circulate in blood and lymph, and are concentrated in
spleen, lymph nodes, and other lymphatic tissues.
• Lymphocytes respond to and recognize specific microbes and
foreign molecules or antigens (antibody generator)
• Antigens include molecules belonging to viruses, bacteria,
fungi, protozoa, and parasitic worms, as well as molecules on
the surface of pollen and transplanted tissue/organs.
• Antibody proteins provide highly specific recognition of
foreign invaders.
The immune system has two types of responses: the humoral immune response
and the cell-mediated immune response.
Antigens interact with lymphocytes to initiate the
immune response.
• Bone marrow produces a large
and diverse pool of B and T
lymphocytes each with antigen
receptor of unique specificity.
• The specificity of antigen
receptors is so enormous that
there will be, at least, one B
cell and T cell line capable of
dealing with almost any
antigen.
• Binding of an antigen to the
receptor triggers proliferation
(clonal selection) of antibody
producing effector B (plasma)
and T cells, and long-lived
memory cells ready to rapidly
respond upon future antigen
exposure.
• The graph below shows the time required for antibody
production after initial exposure to an antigen (e.g. flu shot).
• Notice that upon second exposure (second peak of the blue
line) to the same antigen the production of antibodies is both
faster and dramatically larger (log scale).
• The reason for the rapid and dramatic response upon second
exposure to the same antigen is because of the presence of
memory B and T cells produced during the first exposure.
Second exposure to A
and first exposure to B
First exposure to A
Each B cell produces a unique antibody expressed on its surface.
• Antibodies are proteins, immunoglobulins, of two “light” and two
“heavy” polypeptides held together by disulfide bonds.
• Each polypeptide has a “constant region” and a unique “variable
region” composing the antigen binding site.
• The range of antibody specificity is accomplished by rearrangements
and mutation in genes coding for variable regions.
• The “constant region” determines the type, location, and action of
the antibody in the immune response.
Variable
Constant
- Four types of non-covalent forces operates over a very short distance
( generally 1 angstrom )
- The interaction depends on a very close fit between the Ab & Ag.
Antibody affinity
- is a quantitative measure of binding strength
- combined strength of the noncovalent interactions
between a binding site on an Ab & monovalent Ag
Antibody avidity
- Incorporates affinity of multiple binding sites
- True strength of the Ab-Ag interaction within biological systems
- The interaction at one site will increase the possibility
of reaction at a second site
- High avidity can compensate for low affinity
( secreted pentameric IgM has a higher avidity than IgG )
Polyclonal Antibody Production
antigen
boost for 4-8 weeks
wait 2 weeks
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Ab purified from blood
rabbit bled several
times over several
weeks and blood
containing Ab is tested
Monoclonal Antibody Production
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Select for hybridomas.
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5
Monoclonal Antibody Production
Immunization with antigen A and a booster immunization
three days before they are killed, in order to produce a large
population of spleen cells secreting specific antibody.
Spleen cells die after a few days in culture.
Fusion with immortal myeloma cells to produce a hybridoma.
The myeloma cells are sensitive to the hypoxanthineaminopterin-thymidine (HAT) medium because they lack the
enzyme hypoxanthine:guanine phosphoribosyl transferase
(HGPRT). The HGPRT gene contributed by the spleen cell
allows hybrid cells to survive in the HAT medium, and only
hybrid cells can grow continuously in culture because of the
malignant potential contributed by the myeloma cells.
Individual hybridomas are then screened for antibody
production.
The cloned hybridoma cells are grown in bulk culture to
produce large amounts of antibody.
HAT medium contains aminopterin, which blocks
DNA de novo synthesis by blocking
tetrahydrofolate production.
If tetrahydrofolate is not produced, IMP, a
precursor for GTP is not produced and therefore
blocks DNA synthesis.
Hypoxanthiine and Phosphoribosyl pyrophosphate
are components of the HAT medium. HGPRT
reacts hypoxanthine absorbed from the medium
with PRPP, liberating pyrophosphate, to produce
IMP by a salvage pathway.
So, cells containing HGPRT can grow in the
presence of medium containing aminopterin.
Monoclonal v. Polyclonal Antibodies
• Monoclonal
– Specific target
– Single antibody
recognizes single
epitope of antigen.
• Polyclonal
– Specific target
– Several antibodies
recognize several
epitopes of antigen.
antibody
epitope
epitope
antigen
antigen
Monoclonal v. Polyclonal Antibodies
• Monoclonal
– Typically produced in mice
and rats
– Antibodies ultimately
produced and recovered in
cell culture
– Virtually unlimited resource
• Polyclonal
– Produced in larger
animals such as
rabbits, sheep, goats,
donkeys
– Antibodies recovered
from blood at a certain
time-point
– Finite resource
Monoclonal v. Polyclonal Antibodies
• Monoclonal
– Very specific
– Can bind antigen
within mix of related
molecules
• Polyclonal
– Tolerant of small
changes in antigen
– Can recognize native
and denatured
proteins
– More robust detection
because multiple
epitopes recognized
Antibodies production
Choice of antigen
Whole protein
peptide
small molecule - need to be conjugated or coupled
coupling is also done for non-immunogenic proteins
coupling agents - BSA, PPD, ovalbumin,
keyhole limpet hemacyanin (KLH)
Use peptide, if:
Protein is conserved
Site-directed antibodies are needed
Budget is not a problem
Use protein, if:
Budget is a problem
Antibodies for native protein is required
Antibodies production
Adjuvants
non-specific stimulators of immune response
Need to satisfy following two properties:
(I) prevent rapid catabolism of antigen
-mineral oil or aluminium hydroxide precipitates
(ii) substance to stimulate immune response by inducing
lymphokines production and a local inflammatory response
Heat killed bacteria or lipo poly saccharide (LPS)
Freund’s adjuvants (FA), (FCA) (FIA)