• 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).
• The production of monoclonal antibodies was first done by
Niels K.J. Georges, J.F. Kohler and Cesar Milstein in 1975.
• 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.
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
• Generally immortal in nature and has multiplication
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
● Unfused plasma
● Unfused myeloma
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
Isolation Of Hybridoma Cells
Only the hybridoma cells survive in HGPRT Medium.
● 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
• Cloning, Propagation, Production & Purification has been done.
• 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
• 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
• More than 99% of the cells do not survive during the fusion
1. Serological - Identification of ABO blood group.
2. Diagnosis - Early detection of Pregnancy
3. Immunopurification - Purification of individual interferones.
Inactivation of T lymphocytes
4. Therapy - Removal of tumour cell.
Acute renal failure
• Current Trends in Biotechnology By Dr S satyalakshmi, pageno; 294-
• 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.