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  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. Isolation of myeloma cells • Myeloma cells are cancerous cells which is isolated from bone-marrow. • Generally immortal in nature and has multiplication property.
  7. 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
  8. Fusion of spleen cell & myeloma cell
  9. 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
  10. Dihydrofolate Tetrahydrofolate Nucleoside DNA Denovo synthesis Salvage pathway Aminopterin Hypoxanthine Thymine
  11. 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.
  12. 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.
  14. 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. •
  15. 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.
  16. 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
  17. 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.