Hybridoma technology is a method for producing monoclonal antibodies by fusing antibody-producing B cells with myeloma cells. This results in a hybrid cell, or hybridoma, that is immortal and produces antibodies of a single specificity. Georges J.F. Kohler and Cesar Milstein developed this technique in 1975 and were later awarded the Nobel Prize. The key steps are immunizing an animal, isolating spleen cells and myeloma cells, fusing them to generate hybridomas, selecting hybridomas using HAT medium, and screening for antibody production. Monoclonal antibodies have applications in research, diagnostics, and therapy.

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
specificity 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:
Spleen cell have HGPRT enzyme
Myeloma cell doesn’t have HGPRT enzyme
Fused plasma present
2. Fused myeloma absent
3. Hybridoma present
4. Unfused plasma present
5. Unfused myeloma absent

16. 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.

17. 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

19. 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.

21. 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 transplantation.

22. 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.

24. REFERENCES
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 Gupta, P.K. 2016. Biotechnology and Genomics. Rastogi
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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
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