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Hybridoma Technology

Monoclonal antibodies (mAb) are identical antibodies produced by a single type of immune cell, derived from one parent cell. Hybridoma technology, discovered in 1975, involves fusing B-cells with myeloma cells to create hybrid cells that produce specific antibodies. The process includes immunization, cell fusion, selection, screening, cloning, and characterization of the hybridoma cells to obtain and purify the desired monoclonal antibodies.

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21 views7 pages

Hybridoma Technology

Monoclonal antibodies (mAb) are identical antibodies produced by a single type of immune cell, derived from one parent cell. Hybridoma technology, discovered in 1975, involves fusing B-cells with myeloma cells to create hybrid cells that produce specific antibodies. The process includes immunization, cell fusion, selection, screening, cloning, and characterization of the hybridoma cells to obtain and purify the desired monoclonal antibodies.

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biswajit182001
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© © All Rights Reserved
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MONOCLONAL ANTIBODIES (mAb)

MONOCLONAL

Single Group of cells which


are genetically identical
and derived from single
parent cell

Monoclonal antibodies are antibodies that are identical because they are
produced by one type of immune cell, all clones of a single parent cell.
HYBRIDOMA TECHNOLOGY
• Hybridoma technology is a method in biotechnology that
is used to produce large quantities of monoclonal
antibodies
• The cells are produced by the fusion of B-cell(B-
lymphocytes)
with myeloma cells to produce hybrid cell which is called
hybridoma (produces a single type of antibody)
B-cell + myeloma cell

hybrid cells(HYBRIDOMA)
• Hybridoma technology was discovered in1975 by
Georges Kohler and Cesar Milstein
PRINCIPLE
STEPS:
a)Immunization
In hydridoma technology the very first step is to immunize an
animal(eg: mouse) with appropriate antigen. Isolate B-cell from spleen
of animal.

b)Isolation of myeloma cells


Myeloma cells(having multiplication property) are isolated from bone
marrow. Myeloma cells are generally immortal in nature(that which
never dies).

c)Cell fusion
In this step B-cells are fused with myeloma cells and form hybrid
cells.The cells are exposed to PEG(polyethylene glycol)for a short
period and then removed by washing.
d)Selection of Hybridoma
When the cells are cultured in HAT (hypoxanthine aminopterin
thymidine) only the hybridoma cells grow, while the rest will
slowly disappear.
Selection of a single antibody producing hybrid cell is very
important which is possible if the hybridomas are isolated and
are grown individually.

e)Screening of the hybridoma cell


The culture medium from each hybridoma culture is periodically
tested for the desired antibody specificity. ELISA(Enzyme Linked
Immunosorbent Assay) and RIA(Radioimmunoassay) are used for
this purpose.
In both the assay ,the antibody binds to specific antigen and the
unbound antibody and other components are washed off.
f)Cloning and propagation:
The single hybrid cells producing the desired antibody are isolated and cloned
commonly by two techniques
a)Limiting dilution method
b)Soft agar method

g)Characterization and storage :


The monoclonal antibody has to be subjected to biochemical and biophysical
characterization for the desired specificity.
Cloned cells

cells

frozen

MONOCLONAL
ANTIBODIES ARE
PURIFIED

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