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Hybridoma Technique

In 1975, Kohler and Milstein first

fused lymphocytes to produce a cell line
which was both immortal and a producer
of specific antibodies. The two scientists
were awarded the Nobel Prize for
Medicine in 1984 for the development of
this "hybridoma." The value of
hybridomas to the field was not truly
appreciated until about 1987, when MAbs
were regularly produced in rodents for
In a specific immune response, only those T and B cells that can bind to the antigens
of the pathogen are selected to participate in the response.

Proliferation of cells
Mixture of T and B cells with receptors capable
with different antigen of binding epitopes of
specificities An antigen with 2 epitopes
- red epitope, blue epitope the antigen

Clonal selection of lymphocytes during the specific immune response

Polyclonal antibodies:

If an animal is immunized with a protein, a wide array of B cells will be stimulated to

produce anti-protein antibodies.

Antibodies may be made to a number of different epitopes of the protein.

Even antibodies that bind to the same epitope may have different antigen-binding
sites and bind the epitope with different affinity.

The mixture of antibodies produced in response to an antigen are referred to as

polyclonal antibodies (they are produced by many different clones of B cells).

Immune Response


A mixture of antibodies - all bind to epitopes of

the original antigen. Some bind with higher
affinity than others.

Polyclonal antibodies
Polyclonal antibodies:

Polyclonal antibodies are a mixture of antibodies with different antigen binding sites
that may bind to different epitopes or antigens of the immunizing agent with varying
affinities. They may be of different antibody classes.

The serum obtained from an immunized animal is referred to as a polyclonal


A polyclonal antiserum contains antibody to different epitopes and different antigens

that were present in the immunizing inoculum.
Affinity chromatography - antibody purification.

Antigen can be bound to the support matrix in

order to purify antigen-specific antibody from a
polyclonal antiserum.

Polyclonal antibodies
(Polyclonal antiserum)

Harvest Ab
Monoclonal antibodies

Hybridomas Technique

- B lymphocytes can mutate into tumor

cells that result in a type of cancer termed
- Myeloma cells become “immortal” and
will grow indefinitely in culture.
- Fusion of a single activated B cell and a
myeloma cell will create a hybridoma
that can grow indefinitely in culture.
Secrete antibody but don't
in tissue culture

Grow indefinitely in
Myeloma cells cell culture but don't
secrete the desired

Grow indefinitely in
FUSE Hybridoma cells cell culture AND
secrete antibody

Harvest Ab
Monoclonal antibodies
Monoclonal antibodies:

Antibodies produced from a single clone of B cells.

Produced by fusing a B cell secreting the desired antibody with a myeloma

cell capable of growing indefinitely in tissue culture.

Monoclonal antibodies all have identical antigen-binding sites. Thus they all
bind to the same epitope with the same affinity. They are all of the same
antibody class (isotype).
Inverted microscope
Hybridoma Selection
The “HAT Trick”

Myeloma cells have been genetically

engineered such that they can not use
hypoxanthine, aminopterin, and thymidine
(HAT medium) as a source for nucleic acid
biosynthesis and will die in culture.
Only B cells that have fused with the
engineered myeloma cells will survive in
culture when grown in HAT medium.
Practical steps in monoclonal antibody production:

1) Immunize animal

2) Isolate spleen cells (containing antibody-producing B cells)

3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol)

4) Allow unfused B cells to die

5) Add aminopterin to culture to kill unfused myeloma cells

6) Clone remaining cells (place 1 cell/well and allow each cell to grow into a
clone of cells)

7) Screen supernatant of each clone for presence of the desired antibody.

8) Grow the chosen clone of cells in tissue culture indefinitely.

9) Harvest antibody from the culture supernatant.

10) (If you’re a biotech company) charge about $1,000-$2,000 per mg.
Kuby Figure 4-22
Rasing ascitic fluid
In vitro cultivation
Polyclonal antibodies Monoclonal Antibodies

Produced by: Many B cell clones A single B cell clone

Bind to: Multiple epitopes of all A single epitope of a single

antigens used in the antigen

Antibody class: A mixture of different All of a single Ab class

Ab classes (isotypes)

Ag-binding sites: A mixture of Abs with All Abs have the same antigen
different antigen-binding binding site

Potential for cross-reactivity: High Low


Measuring protein and drug levels in serum

Typing tissue and blood
Identifying infectious agents
Identifying clusters of differentiation for the
classification and follow-up therapy of leukemias
and lymphomas
Identifying tumor metastasis
Identifying and quantifying hormones
Immunoaffinity Purification
Tumor diagnosis

in vitro in vivo

Affinity chromatography:

1. Bind antibody to a support matrix (e.g. sepharose gel)

2. Add protein mixture - antigen binds to antibody on support

3. Wash to remove unbound material

4. Lower pH - antibody releases the antigen - which is now free of contaminants

Figure 6.24
Affintity chromatography - protein elution profile

Potential Future Uses:

The remarkable specificity of antibodies

makes them promising agents for human

Imagine, for example, being

able to make an antibody that will bind only
to the cancer cells in a patient coupling a
cytotoxic agent (e.g. a strong radioactive
isotope) to that antibody, and then giving
the complex to the patient so it can seek
out and destroy the cancer cells (and no
normal cells).
Humanized Monoclonal Antibodies

Mouse monoclonal antibodies have been

genetically engineered to replace all of the
antibody molecule with human counterparts
except the hypervariable regions directly
involved with antigen binding.
Humanized monoclonal antibodies are
currently be tested in human clinical trials.
Genetic engineering methods can be used to modify monoclonal antibodies

e.g. creation of “humanized” monoclonal antibodies.

Figure 6.21
Antibodies of single specificity (monoclonal) can be obtained by cloning the
appropriate heavy and light chain genes.

Figure 6.22
Engineering to create “immunotoxins”

Figure 6.23d