What is monoclonal antibody 03

Animal Biotechnology
Group 1
05/03/2011


Outline content
• Introduction
• Production of Monoclonal Antibodies
• Advantage and Disadvantages of MAbs
• Application


Beginning of Monoclonal Era
Georg Kohler and Cesar Milstein fuse mouse
lymphocytes with neoplastic mouse plasma cells
to yield hybridomas that produce specific
antibodies. This offers a limitless supply of
monoclonal antibodies. Monoclonal antibodies
permit diagnostic tests that are specific, and
function as probes.


Discovery of Monoclonal Antibodies
Monoclonal antibodies
were produced in mice
using a technique
described by Köhler and
Milstein et al.. They were
awarded the Nobel Prize in
1984 (along with Jerne) for
their work.

What is monoclonal antibody
Antibodies are proteins produced by the B lymphocytes of the immune system
in response to foreign proteins, called antigens.
Monoclonal antibodies are proteins that are made in a laboratory.
These proteins are designed to attach to areas on the surface of cancer cells and
interfere with their growth and spread.
Monoclonal antibodies are monospecific antibodies that are identical because they
are produced by one type of immune cell that are all clones of a single parent cell.
Given (almost) any substance, it is possible to create monoclonal antibodies that
specifically bind to that substance


Monoclonal antibodies are similar to the antibodies your body naturally
produces when you are exposed to bacteria or viruses, such as a cold or the
flu (influenza).
Two features of the antibody-epitope relationship are key to the use of
monoclonal antibodies as a molecular tool.
•specificity -- the antibody binds only to its particular epitope
•sufficiency -- the epitope can bind to the antibody on its own


Production of monoclonal antibodies
• Monoclonal antibodies are antibodies produced by one cell
line which are specific to one target (antigen)
• Antibodies derived from mouse hybridomas are of limited use
as human therapeutics, since they produce an adverse
immune reaction with repeated use.

• The term hybridoma is myeloma cell culture applied to fused
cells resulting due to fusion of following two types of cells:
1. An antibody producing lymphocyte cell, and
2. A single myeloma cell which is capable of multiplying
indefinitely. These fused hybrid cells or hybridoma have the
antibody producing capability inherited from lymphocytes and
have the ability to grow continuously (immortal) like
malignant cancer cells.


Production of monoclonal antibodies
• Steps in the production of monoclonal antibodies using
hybridoma technology:
1. Immunize a rabbit or mouse through repeated injection of a
specific antigen for the production of specific antibody,
facilitated due to proliferation of the desired B cells.
2. Produce tumors in a mouse or a rabbit.
3. From the above two types of animals, culture separately
spleen cells that produce specific antibodies, and myeloma
cells that produce tumors.


Production of monoclonal antibodies
4. Induce fusion of spleen cells to myeloma cells, using
polyethylene glycol (PEG), to produce hybridoma; the hybrid
cells are grown in selective hypoxanthine aminopterine
thymidine (HAT) medium.

Production of monoclonal antibodies
5. Select the desired hybridoma for cloning and antibody
production.
6. Culture selected hybridoma cells for the production of
monoclonal antibodies in large quantity.


Screening and propagating
• The hybridomas now are ready to be diluted and grown, thus
obtaining a number of different colonies, each producing only
one type of antibody.
• The desired antibodies from the different colonies are then
tested for their ability to bind to the antigen (ELISA), and the
most effective one is picked out.


ELISA to test Mab production
• The dark blue spot represents a positive clone.
• The light blue spots are positive controls, the next two wells to
the right are negative controls.


Purification of Antibodies
• Before final purification, the cultures may be subjected
to cell fractionation for enrichment of the antibody
protein.
• In E.coli, the antibodies may be secreted in the
periplasm, used for enrichment of antibody, so that
further purification is simplified.
• Alternatively the antibodies may be purified from cell

homogenate or cell debris obtained from the medium.
• Antibodies can be purified by techniques:
1. Ion-exchange chromatography
2. Antigen affinity chromatography


Monoclonal antibodies are produced by
Hybridoma technique


Advantages of Monoclonal Ab
• Single Mabs are chemically defined, are specific
for a particular type of antigen and can be used
for standardization of specific assays.
• The Ab created are 100% active antibodies =>
Possible of high specific activity of labelling in
radioimmunoassay, enzyme linked
immunosorbent assay…
• MAbs can be used as immunosorbents for antigen
purification.


Advantages of Monoclonal Ab
• MAb can be mass produced, easy to manipulate and mostly
inexpensive.
• Distinct antigenic cross reactivities can be defined and very
useful in diagnosis.


Disadvantages of Monoclonal Ab

• MAbs are too specific. Limited amounts of MAb migh miss
important cross-reactive determinant.
• Any physical or chemical treatment that affect one molecule
will affect MAbs, causing deactivation of MAbs activities.
• Time consuming to produce MAbs, 3-4 months for each fusion
experiment.


Monoclonal antibodies’s
applications


Diagnostic tests
• Detecting small amounts
of drugs, toxins or
hormones
Ex : monoclonal antibodies
to human chorionic
gonadotropin (HCG) are
used in pregnancy test kits
(Biotech, 1989)
Another diagnostic uses of antibodies
is the diagnosis of AIDS by the ELISA
test, diagnosis of hepatitis, influenza,
herpes, streptococcal, and Chlamydia
infections ...


- Can be used to detect for the presence and quantity of
substances by The Western blot test and immuno dot blot

tests.


Therapeutic treatment
• Limitations : The rejection of monoclonal antibodies by the
human immune system


• Solution :
Mice have been genetically engineered to produce antibodies
that have human constant regions. By using these hybrid, the
immune system only "sees" a human protein and does not
react against them.


• Cancer treatment :
Monoclonal antibodies bind to
cancer cell-specific antigens and
induce an immunological
response against the target
cancer cell.


Monoclonal antibodies can also be used as delivery
vehicles, guiding radioactive molecules or toxins to the
cancer cell.
Antibodies attached to a cell can trigger an immune
response that destroys the cell.