This presentation includes a brief description of hybridoma technology and detailed procedure of it along with some popular applications of it.

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PRESENTED BY: PRADDUM KUMAR NAMDEV
BSc (Hons) ZOOLOGY
Enrolment no: 17140007
IGNTU Amarkantak MP

2. 
 INTRODUCTION
 HISTORY
 WHAT IS MONOCLONAL ANTIBODY?
 HYBRIDOMA TECHNOLOGY
 PRNCIPLE
 PROCEDURE
 APPLICATIONS OF HYBRIDOMA
TECHNOLOGY
 REFERENCES
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SYNOPSIS

3. 
 Antibodies produced ordinarily by infection or
immunization are polyclonal because natural antigens
have multiple epitopes, each of which generates clones of
lymphocytes.
 This results in antisera containing antibodies from
different clones of lymphocytes with specificities against
different epitopes of the antigens.
 On the other hand, monoclonal antibodies (mAB) are
monospecific antibodies.
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Introduction

4. 
 The production of monoclonal antibodies was
invented by Cesar milstein and Georges J. kohler in
1975.
 And in 1984 they shared a noble prize for this
discovery.
 They make a hybrid cell that will make a numbers
of monoclonal antibodies against antigen.
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History

5. 
• Monoclonal antibodies are produced by hybridoma
technology.
• The term hybridoma is used to fused cells resulting due to
fusion of following two types of cells-a lymphocytes and
tumor cell.
• An antibody producing B- lymphocytes ( eg. Spleen cell of
mouse immunized with RBCs from sheep)
• A single myeloma cell (e.g. Bone marrow tumor cell) that
can adopted to grow for infinite time in culture
• The fused product derived the ability of two different types
of cells. i.e. Ability to produce large amount of pure
antibodies as lymphocytes and ability to grow or multiply
indefinitely like tumor cell.
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What is monoclonal antibody?

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 PRINCIPLE:
 The hybrid cell has the capacity of antibody
production derived from B-cells.
 At the same time it can divide continuously by the
quality derived from myeloma cells.
 By combining the desired qualities of both the cells,
the technology ensures large, antibody production of
single specificity.
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HYBRIDOMA TECHNOLOGY

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Rabbit or lab rat is immunized through repeated injection
(intravenously) of specific antigen.
Where in spleen it activate B-cell which produce plasma cell.
Plasma cell to produce monoclonal antibodies
Isolation of plasma cell from spleen of animal

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 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.
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Step 2. Isolation of myeloma cells

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 The extracted B-lymphocytes is added to a culture of
myeloma cell from bone marrow.
 The intended result is the formation of hybridoma cells
formed by fusion of B-cell and myeloma cell.
 The fusion is done by using Polyethylene glycol (PEG) or
by electrophoration or by using phages.
 This fusion will produce five types of cells:
1. Fused plasma cell
2. Fused myeloma
3. Hybridoma
4. Unfused plasma
5. Unfused myeloma
Step 3. Fusion of myeloma cells
with B-lymphocytes
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Step 4. Selection of HAT medium
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 (hypoxanthine, Aminopterin, Thymine)
 Before multiplication of antibody it has to synthesise
new copy of DNA and for that is require synthesis of
nucleotide.
 For synthesis of nucleotide mainly two pathways are
there:
1. Salvage pathway
2. De-Novo pathway
 In salvage pathway it requires degraded part of old
nucleotide to produce new nucleotide.
 In de-Novo pathway it synthesized completely new
nucleotide by small molecules (sugar, amino acids)

11. 
 So in HAT medium, cells not synthesized by De-
novo synthesis due to presence of Aminopterin in
HAT medium which blocks Di-hydro folate enzyme
which is necessary for these synthesis.
 For synthesis in salvage pathway it must require
HGPRT enzyme (Hypoxanthine Guanine phospho-
Ribosyl Transferase)
 Where hypoxanthine and thymidine are used as
precursers.
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Contd…

12. 
 The next step is selection of hybridoma cells.
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Step 5. Selection of hybridoms
cells
B-cells have HGPRT
enzyme
Myeloma cells doesn’t
have HGPRT enzyme
HGPRT
Fused plasma……………………………...(+)
Fused myeloma……….. ……………….....(-)
Hybridoma………………………………..(+)
Unfused plasma………………………….(+)
Unfused myeloma……………………......(-)

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 The myeloma cell fused with another myeloma cell or
those do not fused at all die in HAT medium since they
do not utilize Hypoxanthine.
 Similarly, B- cell that fuse with another B- cell or those do
not fuse at all die eventually because they do not have
capacity to divide indefinitely,
 So, only hybridoma cell ie. fused cell between myeloma
and B-cell can survive and divide in HAT medium.
 Screening is done to select hybridoma cells which are the
desired cell for monoclonal antibodies production.
Contd…
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14. 
 The selected hybridoma cells are cultured in suitable
culture medium, often supplemented with insulin,
transferon, ethanol, amine and other additional
hormones.
 Some commonly used culture media for hybridoma cell
for production of monoclonal antibodies are:
 DMEM (Dulbecco’s modified eagle medium)
 IMDM (Iscove’s Modified Dulbecco’s Medium)
 Ham’s F12
 RPMI 1640 medium (Roswell Park Memorial Institute 1640
medium)
Step 6. Culture of hybridoma cell
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15. 
 These hybridoma cells are then injected into lab
animal so that they starts to produce monoclonal
antibodies.
 These hybridoma cells may be frozen and store for
future use.
Step 7. Inoculation of hybridoma
cell into suitable host
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16. 
 Monoclonal antibodies from host animal is extracted
and purified by one of the following methods;
 Ion exchange chromatography
 Antigen affinity chromatography
 Radial immunoassay
 Immune precipitation
Step 6. extraction & purification of
monoclonal antibodies
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20. 
 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
 IMMUNOPURICATION
 Purification of individual interferon using monoclonal antibodies.
 Inactivation of T-lymphocytes responsible for rejection of organ
transplants
 Removal of tumor cells from bone marrow.
 Treatment of acute renal failure
 Treatment of malignant leukemic cells
Applications of hybridoma
technology
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21. 
 LIFE SCIENCES
 FUNDAMENTALS AND PRACTICES-1
 BY: PRANAV KUMAR
 Pathfinder publications, New Delhi ,India
 www.onlinebiologynotes.com
 www.biologydiscussion.com
 www.slideshare.com
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