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Keynotes

Nobel Prize Presented


Principle of monoclonal antibodies
Development
Uses
Recent market antibodies
Nobel Prize 1984

Georges J.F. Khler Csar Milstein Niels K. Jerne


Antibodies

Also known as immunoglobulins

Functions as a protective mechanism


for the body

Specific antigen-binding site

Each antibody produced by


specific B cells
Mechanism of
Antibodies

Specific Antigen-Binding Site:
Each tip of the Y of an antibody contains a paratope
specific to a particular epitope on an antigen

Mechanism 1: Tagging
Mechanism 2: Neutralization
Mechanism of
Antibodies (Tagging)

3) Stimulates
2) Antibody
other immune
binds to the 4)Antigen is
1) Antigen is responses
antigen removed
detected such as
(Antigen is from the body
complement
tagged)
pathway
Mechanism of
Antibodies
http://www.youtube.com
/watch?v=KpNFAEbLcvk
&feature=related
Development of

Monoclonal Antibodies
ANTIGENS

IMMUNISATION
HOST
The first step in making a
ANIMAL hybridoma is to generate antibody-
(MOUSE) producing B lymphocytes
(immune cells of a mouse).
Multiple immunizations are
performed until an appropriate
level of antibodies are achieved.
TEST
BLEEDS

To determine if the mouse is


producing antibodies of interest,
test bleeds are performed. (Use of
ELISA method)
Some B cells will produce
antibodies that bind to epitopes on
the antigen of interest whilst others
produce antibodies that do not.
HGPRT+ HGPRT-

Myeloma cells

Spleen of mouse

Polyethylene glycol
(PEG) is added to culture
to promote membrane
fusion.

Hybridomas

Separate hybridomas from unfused


cells.
Success rate of fusion of cells is low so a selective medium is used
HAT
Hypoxanthine
Medium

Aminopterin

Thymidine

Unfused myeloma
cells
THEREFORE, only the
Unfused spleen cells hybridomas survive as
the myeloma cells cannot
Hybridomas
replicate their DNA (
because they lack
HGPRT) and unfused
spleen cells have limited
lifespan.
Surviving
Hence the
hybridomas
name,
are separated
monoclonal,
and
as the cells
individually
are derived
cultured. (one
from one
cell per wall)
single cell.
After a few weeks of culture,
the culture fluid is screened
for presence of desirable
antibodies.

Antibodies secreted by the different clones are


assayed for their ability to bind to the antigen.

This is done by ELISA (Enzyme-linked


Immunosorbent Assay)
The most stable and productive clone is then
selected for future use.
PURIFICATION STEP

Protein A/G affinity chromatography
Antibody selectively binds to protein A/G so a high level
of purity is obtained.
But harsh conditions are used and may damage some
antibodies. (eg low pH)
Hence they immediately neutralize it against a buffer of
pH 7
Recombinant versions of protein-A and protein-G
developed that elute at milder conditions (pH 6.0)
CHIMERIC
ANTIBODIES
Methods used to produce monoclonal
antibodies yielded mouse antibodies,
not human.
This led to an immune response.

Using recombinant DNA, the


expression of this chimeric DNA
through cell culture yielded partially-
mouse, partially-human monoclonal
antibody
Human Monoclonal
Antibodies
Generate in-vitro.

1) Phage display-generated
antibodies
Use of bacteriophage and E.
coli
2) Transgenic Mice
Insert human antibody
gene loci
knocking out genes for
making antibodies
Uses of monoclonal

antibodies
Diagnostic tests

Used in several diagnostic tests to detect small amounts
of drugs, toxins, hormones or infectious diseases.
1) Immunoassay procedures
2) Radioimmunodetection - attachment of MAbs to
radioactive atoms
3) Fluorescent molecules or metal atoms such as copper
and gold coupled to the antibody to assist in imaging
the target.
Diagnostic tests

Monoclonal antibodies to human chorionic
gonadotropin (HCG) are used in pregnancy test kits.
Diagnosis of AIDS by the ELISA (enzyme-linked
immunosorbent assay) test
Monoclonal antibody
therapy

Use of monoclonal antibodies to specifically bind to
antigens of target cells or proteins. This may then
stimulate the patient's immune system to attack
those cells.

Used in the treatment of cancer.


Cancer treatment

Naked monoclonal antibodies are those without any
drug or radioactive material attached to them.
Conjugated monoclonal antibodies are those joined to a
chemotherapy drug, radioactive particle, or a toxin
Naked monoclonal antibodies

Markers for destruction
act as a marker for the body's immune system to destroy
them.
Rituximab (Rituxan) - treat B-cell non-Hodgkin
lymphoma
Activation blockers
attach to the specific antigens that are working parts of
cancer cells or other cells that help cancer cells grow, and
stop them from working.
Cetuximab (Erbitux)- blocks the activation of EGFR. It is
used to treat some advanced colorectal cancers as well as
some head and neck cancers
Conjugated monoclonal
antibodies

Monoclonal antibodies that are attached to drugs,
toxins, or radioactive substances.
Used as homing devices to take these substances
directly to the cancer cells.
It then delivers the toxic substance where it is
needed most. This lessens the damage to normal
cells in other parts of the body.
Conjugated monoclonal
antibodies

MAbs with radioactive particles attached are
referred to as radiolabeled, and therapy with this type
of antibody is known as radioimmunotherapy (RIT).
MAbs with chemotherapy drugs attached are often
referred to as chemolabeled.
MAbs attached to toxins are called immunotoxins.
Other uses

OKT3, an antibody to the T3 antigen of T cells, is
used to alleviate the problem of organ rejection

The Future of Monoclonal Antibodies


Monoclonal antibodies may soon be extended to the
therapeutic treatment of diseases such as infectious
diseases and ophthamology.
With sales exceeding 32 billion dollars in 2008, MAbs
are set to become an important sector of the
pharmaceutical industry.

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