Presentation 0 n hybridoma technology

HYBRIDOMA TECHNOLOGY IT IS DEFINED AS THE PROCESS WERE THERE IS A FUSION OF SPLLEN CELL AND MYELOMA CELLS IN THE PRESENCE OF POLYETHYLENE GLYCOL OR SENDAI VIRUS AND LEADS TO THE PRODUCTION OF MONOCLONL ANTIBODY.

1. PREPARED BY: VIPIN KUMAR SHUKLA ASSISTANT PROFESSOR DEPARTMENT OF BIOTECHNOLOGY PRESENTATION 0N HYBRIDOMA TECHNOLOGY

2. Introduction: Hybridoma technology is a method of forming hybrid cell lines ( called Hybridoma) by fusing a specific Antibody-producing B-cell with a myeloma cell (cancerous cell). The antibodies produced by the Hybridoma are of a single specific ity and are therefore monoclonal antibodies .

3. HISTORY: In 1975, these technology developed by Georges J.F.Kohler and Cesar Milstein . And in 1984, they shared a Nobel prize for this discovery. They make a hybrid cell that will make a numbers of monoclonal antibodies against antigen .

4. PRINCIPLE: The hybrid cell has the capacity of antibody production derived from B-cells (spleen cell ). At the same time it can divide continuously by the quality derived from myeloma cell. By combining the desired qualities of both the cells , the technology ensures large, antibody production of single specificity. Specific Hybridoma(spleen cell and myeloma cell ) obtain monoclonal antibodies in artificial media , this technology called as HYBRIDOMA TECHNOLOGY .

5. Continued…… The selection of Hybridoma cells is based on inhibiting the nucleotide (consequently the DNA) synthesizing machinery . De novo synthesis and salvage pathway are the two pathways through which mammalian cells can synthesize nucleotides. HAT (hypoxanthine Aminopterin and Thymidine) medium – Only Hybridoma cells can proliferate in HAT medium .

6. General Flow Diagram of Hybridoma Technology

7. MONOCLONAL ANTIBODY: Monoclonal antibodies (mab) are antibodies that are identical because they are produced by one type of immune cell, all clones of a single parent cell. Basically produced by white blood cell which is called as plasma cell. Is used for treatment of cancerous cells and as anti-venom( anti snake venom ).

8. PROCEDURE: Immunization of specific animal which generate Hybridoma cell with spleen cell. Isolation of myeloma cells. Fusion between spleen cell and myeloma cell . Selection of HAT medium. Isolation of Hybridoma cell. Screening of Hybridoma cell

9. Immunization of specific animal: An antigen immunized to an animal (like mice) via intravenously(directly to blood ) by injection . Where in spleen it activate B-cell which produce plasma cell (spleen cell ). Plasma cell to produce monoclonal antibodies . Isolation of plasma cell from spleen of animal.

10. Isolation of myeloma cells: Myeloma cells are cancerous cells which is isolated from bone-marrow. Myeloma cells are generally immortal in nature (that which never dies) and has multiplication property .

11. Fusion of spleen cell and myeloma cell: It requires PEG (poly ethylene glycol) medium for fusion. It can also done by electro fusion. Fusion between spleen cell and myeloma cell produced five different types of cells. Fused plasma Fused myeloma Hybridoma Unfused plasma Unfused myeloma

12. Selection of HAT medium. ( Hypoxanthine, Aminopterin, Thymidine) Before multiplication of Anti-body, it has to synthesize new copy of DNA and for that it require synthesis of nucleotide. For synthesis of nucleotide mainly two pathways are there : 1. Salvage pathway 2. De-novo Synthesis In 1 , Salvage pathway it requires degraded part of old nucleotide to produce new nucleotide . In 2, De-novo synthesis it synthesized completely new nucleotide by small molecules (sugar , amino-acid ).

13. Continued……. So in HAT medium, Cells not synthesized by De-novo synthesis due to presence of Aminopterin in HAT medium which blocks Di-hydro follate enzyme which is necessary for these synthesis . For synthesis in salvage pathway it must requires HGPRT enzyme ( Hypoxanthine Guanine Phospho-Ribosyl Transferase). Where hypoxanthine and Thymidine are used as precursors.

14. Isolation of Hybridoma cell:

15. Continued…… Fused myeloma and unfused myeloma didn’t have HGPRT enzyme so, can’t survive in HAT medium. Fused plasma and unfused plasma have HGPRT enzyme but didn’t have long-life. Hybrid cell has HGPRT enzyme from spleen cell as well as they have the ability to multiply repeatedly as myeloma cell . So, isolation of hybrid cell because is only cell which survive in HAT medium .

16. Screening of Hybridoma cell: ELISA screening method which done by incubating Hybridoma culture in which secondary enzyme gets conjugate and formation of colored product shows positive Hybridoma. Used for multiplying the Hybridoma cells In-vivo In-vitro

17. Continued….. In-vivo procedure involves introduction of Hybridoma cells into the peritoneal cavity of the animal , then from ascetic fluid antibodies are isolated. In-vitro method involves culturing of Hybridoma cells in suitable culture media and then antibodies are isolated and purified. Once a Hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 and produce antibodies . Storage : liquid nitrogen.

18. APPLICATION OF HYBRIDOMA TECHNOLOGY Serological: Identification of ABO blood group Diagnosis: Detection of pregnancy by assaying of hormones with monoclonal . Separation of one substance from a mixture of very similar molecules. Immunopurification: Purification of individual interferon using monoclonal. Inactivation of T-lymphocytes responsible for rejection of organ transplants . Therapy : Removal of tumor cell from bone marrow. Treatment of acute renal failure. Treatment malignant leukemic cells, B cell lymphomas, and a variety of allograft rejections after transplantatio n .

19. ADVANTAGES AND DISADVANTAGES OF MONOCLONAL ANTIBODIES Advantages- Represent a homogeneous state of a single molecular species. Each Mab is specific to a given antigenic determinant. Disadvantages- Hybridoma technology is laborious and time consuming. There is no guarantee that Mab produced is totally virus-free, despite the purification. For this reason, US Food and Drug Administration insists that Mab for human use should be totally free from all pathogenic organisms, including viruses.

20. REFERENCES Satyanarayana, U. 2016. Biotechnology. Books and Allied (P) Ltd, Kolkata . pp. 213-226. Gupta , P.K. 2016. Biotechnology and Genomics. Rastogi Publications, Meerut . pp. 299-311. Owen, J.A., Punt J., Stanford, S.A. and Patricia, P.J. 2013. Kuby Immunology . 7th Ed. W.H. Freeman and Company, New York. pp.645- 655 . Singh , B.D. 2017. Biotechnology Expanding Horizons. Kalyani Publishers , New Delhi. pp. 172-174 . Dubey, R.C. and Maheshwari, D.K. 2018. A Textbook of Microbiology. S Chand and Company Limited, New Delhi. pp. 662-663.