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Wild type Mutant

Methods in cell biology and functional genomics

Applications Using Monoclonal and Polyclonal Antibodies

• 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.

• 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.

Why Induce on Purpose?

Phagocyte

Plasma Cell

Several line of defense offer protection from invaders

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 Aand 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 weekswait 2 weeks

rabbit bled several times over several weeks and blood containing Ab is tested

Y

Y

Y

Y

Y

Ab purified from blood

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y

YY

Y Y

Y

YY

Y Y

Y

+

Monoclonal Antibody Production

1

Select for hybridomas.

2

3

4

5

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 hypoxanthine-aminopterin-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.

Monoclonal Antibody Production

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.

antigen

antibody

antigenepitope epitope

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– 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

Monoclonal v. Polyclonal Antibodies

Antibodies production

Choice of antigen

Whole proteinpeptidesmall molecule - need to be conjugated or coupledcoupling is also done for non-immunogenic proteinscoupling agents - BSA, PPD, ovalbumin,

keyhole limpet hemacyanin (KLH)

Use peptide, if:Protein is conservedSite-directed antibodies are neededBudget is not a problem

Use protein, if:Budget is a problemAntibodies for native protein is required

Adjuvantsnon-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)

Antibodies production