HYBRIDOMA
TECHNOLOGY
Presented by,
ASWINI SASIDHARAN
FIRST SEMESTER M.PHARM
PHARMACEUTICAL CHEMISTRY

HYBRIDOMA TECHNOLOGY
• Hybridoma technology is a method of forming hybrid cell lines
(hybridomas) by fusing a specific antibody-producing B-cell
with a myeloma cell (cancerous cell).
HISTORY
• The production of monoclonal antibodies was first done by
Niels K.J. Georges, J.F. Kohler and Cesar Milstein in 1975.

PRINCIPLE
• It is based on the fusion between myeloma cells (malignant plasma cells)
and spleen cells from a suitably immunized animal.
• Spleen cells die in a short period under ordinary tissue culture conditions
while myeloma cells are adopted to grow permanently in culture.
• From the growing hybrids, individual clones can be chosen that secrete
the desired antibodies.

PROCEDURE
1. Immunization.
2. Isolation of myeloma cells.
3. Fusion between spleen cell and myeloma cell.
4. Selection of HAT medium.
5. Isolation of hybridoma cell.
6. Screening of hybridoma cell.

Immunization of specific animal
Antigen immunized to animal (like mice) via intravenously.
In spleen, activates B-cell in which it produces plasma cell
Plasma cell produces monoclonal antibodies
Isolation of plasma cell from spleen of animal.

Isolation of myeloma cells
• Myeloma cells are cancerous cells which is isolated from
bone-marrow.
• Generally immortal in nature and has multiplication
property.

Fusion of spleen cell and myeloma cell
• It requires PEG medium for fusion
• It can also done by electro fusion.
• Fusion between spleen cell and myeloma cell produces five
different types of cells;
● Fused plasma
● Fused myeloma
● Hybridoma
● Unfused plasma
● Unfused myeloma

Fusion of spleen cell & myeloma cell

Selection of HAT medium
• Before multiplication of Anti-body, it has to synthesize new
copy of DNA and for that it require synthesis of nucleotide.
• Synthesis of nucleotide have two pathways:
•
1. Salvage pathway
2. De-novo Synthesis

Dihydrofolate
Tetrahydrofolate
Nucleoside DNA
Denovo synthesis
Salvage pathway
Aminopterin
Hypoxanthine
Thymine

Isolation Of Hybridoma Cells
Only the hybridoma cells survive in HGPRT Medium.
HGPRT
● Fused plasma -present
● Fused myeloma -absent
● Hybridoma -present
● Unfused plasma -present
● Unfused myeloma -absent
Spleen cells have HGPRT Enzyme while myeloma doesn’t have it.

Screening of hybridoma cell
• ELISA screening method which done by incubating hybridoma
culture in which secondary enzyme gets conjugate and
formation of coloured product shows positive hybridoma.
• Hybridoma cells producing desired antibody was identified by
screening.
• Cloning, Propagation, Production & Purification has been done.

PRODUCTION OF MONOCLONALANTIBODIES BY HYBRIDOMA TECHNOLOGY

MERITS
• It produces highly specific antibodies in abundant amounts.
• The clones developed are far cheaper than the traditionally
• employed animals.
• It produces high quality products, to avoid the batch to batch
variation.
•

DEMERITS
• Many patients develop immune resistance to monoclonal
antibodies produced by mice, as these are foreign proteins.
• Hybridoma culture may be subjected to contamination.
• System is only well developed for mouse and rat, not for the
other animals.
• More than 99% of the cells do not survive during the fusion
process.

APPLICATIONS
1. Serological - Identification of ABO blood group.
2. Diagnosis - Early detection of Pregnancy
HIV
Cancer
3. Immunopurification - Purification of individual interferones.
Inactivation of T lymphocytes
4. Therapy - Removal of tumour cell.
Acute renal failure
Leukemia

References
• Current Trends in Biotechnology By Dr S satyalakshmi, pageno; 294-
302.
• A Textbook of Pharmaceutical Biotechnology By Mr Akshay Shashikant
Patil, Dr Md Rageeb Md Usman, Dr Vijay Mishra, Dr Bhushankumar S
Sathe, pageno; 126-134.
• Textbook of Biotechnology By Sathyanarayanan U, pageno; 213-226.

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