Hybridoma technology

 INTRODUCTION: Monoclonal antibodies can be produced through a technique known as hybridoma technology.

 HISTORY: The production of monoclonal antibodies was invented by Niels K.J. Georges, J.F. Kohler and Cesar Milstein in 1975.

 PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS: HAT (hypoxanthine aminopterin and thymidine) medium – Only hybridoma cells can proliferate in HAT medium.

 PRODUCTION OF MONOCLONAL ANTIBODIES (HYBRIDOMA TECHNOLOGY): The establishment of hybridomas and production of monoclonal antibodies involves the following steps-
 Immunization (ii) Cell fusion (iii) Selection of hybridomas (iv) Screening the products (v) Cloning and propagation (vi) Characterization and storage.

 ADVANTAGES AND DISADVANTAGES OF MONOCLONAL ANTIBODIES:
 Advantages- Monoclonal antibodies is specific to a given antigenic determinant.
 Disadvantages- There is no guarantee that monoclonal antibodies produced is totally virus-free, despite the purification.



 APPLICATIONS OF MONOCLONAL ANTIBODIES: Diagnostic applications, therapeutic applications, protein purification and miscellaneous applications.

 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., Stranford, 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.

1. Department of Microbiology (Ch. Charan Singh University Campus, Meerut) Presented By: Manisha Sirohi M.Sc. Microbiology 3 rd Semester

2. CONTENTS INTRODUCTION HISTORY PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS PRODUCTION OF MONOCLONAL ANTIBODIES (HYBRIDOMA TECHNOLOGY) ADVANTAGES AND DISADVANTAGES OF MONOCLONAL ANTIBODIES APPLICATIONS OF MONOCLONAL ANTIBODIES REFERENCES

3. INTRODUCTION Hybridoma technology is a method of forming hybrid cell lines (called hybridomas ) by fusing a specific antibody-producing B-cell with a myeloma cell (cancerous cell). The antibodies produced by the hybridoma are of a single specificity and are therefore monoclonal antibodies .

4. HISTORY The production of monoclonal antibodies was invented by Niels K.J. Georges, J.F. Kohler and Cesar Milstein in 1975 . They shared the Nobel Prize in 1984 for Medicine and Physiology .

5. PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS 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 ( h ypoxanthine a minopterin and t hymidine) medium – Only hybridoma cells can proliferate in HAT medium.

6. Pathways for the synthesis of nucleotides. (HGPRT-Hypoxanthine guanine phosphoribosyl transferase; TK- Thymidine kinase)

7. PRODUCTION OF MONOCLONAL ANTIBODIES (HYBRIDOMA TECHNOLOGY) Immunization – Immunization of an animal (usually a mouse) and the removal of spleen for further process. Cell fusion – Hybridomas (fused cells) + free myeloma cells + free lymphocytes. Selection of hybridomas – Only the hybridoma cells grow in HAT medium.

8. iv. Screening the products – Hybridoma cells producing the desired antibody can be identified by screening. (ELISA and RIA) The antibody secreted by the hybrid cells is referred to as monoclonal antibody. v. Cloning and propagation – The single hybrid cells producing the desired antibody are isolated and cloned. (Limiting dilution method and Soft agar method). vi. Characterization and storage – Characterization and stability of MAbs are important for their ability to withstand freezing.

9. 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 .

10. APPLICATIONS OF MONOCLONAL ANTIBODIES 1. Diagnostic applications Biochemical analysis for the diagnosis of pregnancy, cancers, hormonal disorders, infectious diseases. 2. Therapeutic applications (A) Direct use as therapeutic agents to destroy disease-causing organisms, in the treatment of cancers, in the immunosuppression of organ transplantation, in the treatment of AIDS, and autoimmune diseases. (B) As targeting agents in therapy as immunotoxins (for treatment of cancers), in drug delivery, for dissolving blood clots, in radioimmunotherapy (for tumors). 3. Protein purification by immunoaffinity techniques. 4. Miscellaneous applications as catalytic agents (abzymes).

11. REFERENCES Satyanarayana, U. 2016. Biotechnology. Books and Allied (P) Ltd, Kolkata. p p. 213-226. Gupta, P.K. 2016. Biotechnology and Genomics. Rastogi Publications, Meerut. pp. 299-311. Owen, J.A., Punt J., Stranford, S.A. and Patricia, P.J. 2013. Kuby Immunology . 7 th 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.