Hybridoma Technology and cloning of cells-1.pptx

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HYBRIDOMA TECHNOLOGY
Muhammad Hassam
Immunology and Serology
MLT 4th
Semester

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 .
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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 hybridomas(spleen cell and myeloma cell)
obtain monoclonal antibodies in artificial media, this
technology called as HYBRIDOMA TECHNOLOGY.
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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.
 Is used for treatment of cancerous cells and as
anti-venom( anti snake venom)
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PROCEDURE:
1. Immunization of specific animal which
generate hybridoma cell with spleen cell.
2. Isolation of myeloma cells.
3. Fusion between spleen cell and myeloma
cell.
4. Selection of HAT medium.
5. Isolation of hybridoma cell.
6. Screening of hybridoma cell.
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1. 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.
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2. 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.
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3. Fusion of spleen cell and myeloma cell.
 It requires PEG (poly ethylene glycol) medium
for fusion
 It can also be 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
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4. 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 is synthesized completely new
nucleotide by small molecules (sugar, amino-acid).
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 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.
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5. Isolation of hybridoma cell
Myeloma cell doesn’t
have HGPRT enzyme
Spleen cell have
HGPRT enzyme
1. Fused plasma present
2. Fused myeloma absent
3. Hybridoma present
4. Unfused plasma present
5. Unfused myeloma absent
HGPRT
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 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 it is only
cell which survives in HAT medium.
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6. Screening of hybridoma cell.
 ELISA screening method which is 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
o In-vivo
o In-vitro
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 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.
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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.
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Hybridoma Technology and cloning of cells-1.pptx

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