A specific and single type of antibody that is produced by hybridoma cells. This technique was achieved by George Kohler and Cesar Milstein in 1975. (Nobel Prize, 1984) Antibodies (or) immunoglobulins is a protein - produced by B- lymphocytes (Plasma cell) in mammals. Antibodies used by immune system to identify and neutralize foreign objects like bacteria and viruses. Each antibody recognizes a specific antigen unique to its target.

1. Production of Monoclonal Antibodies (MAbs)
and their Applications
(Hybridoma Technology)
By
Dr. S.SELVARAJ
Assistant professor
Dept. of Zoology
Vivekananda College
Tiruvedakam West
1

2. Synopsis
 Introduction
 Principle
 Production of MAbs
 Immunization
 Cell fusion
 Selection of hybridomas
 Screening the products
 Cloning and propagation
 Characterization and storage
 Applications of MAbs
Diagnostic Applications
Therapeutic Applications
Protein purification
Miscellaneous Applications
 References
2

3.  A specific and single type of antibody that is produced
by hybridoma cells.
This technique was achieved by George Kohler and
Cesar Milstein in 1975. (Nobel Prize, 1984)
 Antibodies (or) immunoglobulins is a protein -
produced by B- lymphocytes (Plasma cell) in
mammals.
 Antibodies used by immune system to identify and
neutralize foreign objects like bacteria and viruses.
Each antibody recognizes a specific antigen unique to
its target.
3
Introduction

4. • Fusion between myeloma cell (malignant plasma cell)
and spleen cell from suitable immunised animal.
• Myeloma cells - Growth and multiplying
• B- lymphocytes-Secrete their own antibodies
• Myeloma cells cannot synthesize their own antibodies
because, lack of HGPRT enzyme.
• Mutant myeloma cell can not grow in HAT medium.
• Only hybrid cell can grow in HAT medium and produce
monoclonal antibody.
Principle
4
Hypoxanthine
Guanine
phosphoribosyl
Transferase

5. 5
Production of Monoclonal Antibodies (MAbs)

6. Production of MAbs
1. Immunization
Immunize the animal with appropriate antigen
Inject multiple site and repeated several times
B-lymphocytes stimulation increased against antigen
Antibodies synthesized against specific antigen
Spleen is aseptically removed
Spleen cells are removed using mechanical (or) enzymatic
B-lymphocytes of spleen cells are separated
6

7. 2. Cell fusion
Fusion of B-lymphocytes to myeloma cells is induced using
polyethylene glycol (PEG)
After few minutes
1. Mixture of Hybridoma (Fused cells)
2. Free myeloma cells
3. Free lymphocytes
(The production of hybrid cells is referred to as
Hybridoma technology)
7

8. 3. Selection of Hybridoma
Cells are cultured in HAT medium (Specific)
Hybridoma cells only grow, other cells slowly disappear
(7-10 days of culture)
Select the single antibody producing hybrid cells
Hybridomas are isolated and grown individually
8
Hyphoxanthine
Aminopterin
Thymidine that
blocks one
pathway for
nucleotide
synthesis
Cells lacking
HGPRT
enzyme,
grown in HAT
medium die

9. Screened the desired antibody producing cells
Two techniques namely ELISA and RIA
In both the assays, the antibody binds to the specific antigen
and other components can be washed off
Hybridoma cells producing the desired antibody can be
identified by screening.
Antibody secreted by the hybrid cells is referred to as
monoclonal antibody.
4. Screening the products
9

10. Desired antibody are isolated and cloned
Two techniques are commonly used for cloning hybrid cells
1. Limiting dilution method – Serial dilution method
2. Soft agar method – Semisolid medium- to form colonies
5.Cloning and propagation
10

11.  Biochemical and biophysical characterization for
desired specificity
 MAbs of the immunoglobulin class or sub-class, the
epitope for which it is specific and the number of
binding sites it possesses.
 Desired cell lines are frozen in liquid nitrogen for
future uses.
6.Characterization and storage
11

12. Applications of MAbs
1. Diagnostic applications
• Biochemical analysis for pregnancy, cancer, hormonal
disorder.
• Diagnostic imaging for myocardial infarction, bacterial
infections.
2. Theraputic applications
Treatment of cancer, Asthma, Viral infections.
3. Protein purification by immunoaffinity techniques.
4. Miscellaneous applications as catalytic agents, in
autoantibody fingerprinting.
12

13. https://www.slideshare.net/YashikaSaini7/monoclonal-
antibody-146466667.
https://www.slideshare.net/ArchanaSoni3/hybridoma-
technology-93942798.
Dr. U. Satyanarayana (2011) Biotechnology, Books and
Allied (P) Ltd. Kolkata, India.
References
13