 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
3rd 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 (hypoxanthine aminopterin and thymidine)
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)
i. Immunization –
Immunization of an animal
(usually a mouse) and the
removal of spleen for
further process.
ii. Cell fusion – Hybridomas
(fused cells) + free
myeloma cells + free
lymphocytes.
iii. 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.

10. ADVANTAGES AND DISADVANTAGES
OF MONOCLONALANTIBODIES
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.

11. 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).

13. 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.