Hybridoma cells are created by fusing B lymphocytes from immunized mice with myeloma cells. This fusion produces a hybrid cell that is selected and screened to produce monoclonal antibodies against a specific antigen. The hybridoma technique allows for large-scale, indefinite production of these identical monoclonal antibodies, which have various medical uses such as in treating cancer, rheumatoid arthritis, and cardiovascular diseases. Hybridoma cells can be cultured in vitro or grown in vivo in mice to produce monoclonal antibodies.
3. INTRODUCTION
• Hydridoma cell is fusion of 2 cells in order to obtain or express a specific monoclonal
antibodies.
B-cells Tumor
cell HYBRIDOMA
B-
lymphocytes
myeloma
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4. WHAT IS MONOCLONAL ANTIBODIES?
• Monoclonal antibodies artificially produced against a specific antigen in order to bind
to their target antigens.
• Uses of monoclonal antibodies are:
CANCER RHEUMATOID ARTHRITIS
CARDIOVASCULAR
DISEASES
ULCERATIVE COLITIS
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Method of culturing
hybridoma cells
1. Immunisation of mouse
2. Isolation of B cells from spleen
3. Cultivation of myeloma cells
4. Fusion of myeloma and B cells
5. Separation of cell lines
6. Screening suitable cell lines
7. In vitro / in vivo
8. Harvesting
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1. Immunisation of mouse
2-4 weeks old mice are immunized with antigen against
which to raised monoclonal antibodies by subcutaneous
injection.
Series of injections of the antigen over the course of
several weeks.
2.Isolation of B cells from
spleen
B cells are isolated from the spleen of immunized mice.
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3. Cultivation of myeloma cells
Myeloma cells are isolated from bone marrow.
The myeloma cells used are HGPRT ( Hypoxanthine-
guanine phosphoribosyl transferase) mutant cells which
raised by mutation using 8-azaguanine.
4. Fusion of myeloma and B
cells Using electrofusion: cells are allowed to fused with the
application of electric field.
By using PEG medium (Polyethylene Glycol)
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5. Separation of cell lines
HAT (Hyphoxanthine Aminopterine Thymidine)
medium is used for the selection of hybrid cells.
.B cells are HGPRT+ and can survive in the HAT
medium but the undergo normal cell death after some
division.
Myeloma cells are HGPRT deficient so the cells could
not survive in HAT medium since Aminopterin blocks
the pathway that allows for nucleotide synthesis.
Hybrid cells has HGPRT enzyme from B cells as well
as they have the ability to multiply repeatedly as
myeloma cells. So, only hybrid cells can survive in
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6. Screening suitable cell lines
Screening technique used is called ELISA.
The hybridoma culture supernatant, secondary enzyme labeled conjugate, and chromogenic
substrate.
Then it is incubated and the formation of a colored product indicates a positive hybridoma.
Alternatively, immunocytochemical screening can also be used.
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7.Methods multiplying hybridoma cell
Introduce of hybridoma cells into peritoneal
cavity of mice.
Ascetic fluid is isolated from the mice.
Isolate antibodies from ascetic fluid.
Culturing of hybridoma cells in suitable culture
media (
Antibodies is isolated and purified.
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8. Harvesting
Once a hybridoma colony is established, it will continually
grow in culture medium like RPMI-1640 (with antibiotics
and fetal bovine serum) and produce antibodies.
Multiwell plates are used initially to grow the hybridomas.
After selection, they are changed to tissue culture flasks to
provides enough cells for cryopreservation and supernatant
for subsequent investigations.
12. ADVANTAGES OF HYBRIDOMA
In vitro
• reduce the use of mice at the antibody-production stage
• ease of culture for production, compared with use of animals, and because of economic
considerations.
• decrease the need for laboratory personnel experienced in animal handling.
In Vivo
• produces very high mAb.
• high concentration of the desired mAb in mouse ascites fluid avoids the effects of contaminants
in in vitro batch-culture fluid when comparable quantities of mAb are used.
• reduce the need to teach the antibody producer tissue-culture methods.
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13. DISADVANTAGE OF HYBRIDOMA
• Some cell may do not grow well in culture or are lost in culture.
• Require the use of FBS, which limits some antibody uses.
• In batch tissue-culture methods, mAb concentration tends to be low in the
supernatant
In vitro
• involves the continued use of mice requiring daily observation.
• can be expensive if immunodeficient mice in a barrier facility must be used.
• can contain various mouse proteins and other contaminants that might require
purification.
In vivo
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14. CONCLUSION
• Hybridoma is fusion of two cell between tumor cell and Beta cell which specifically
beta lymphocytes cell from mice.
• The hydridoma is used to produce monoclonal antibodies to treat cancer disease such
as cardiovascular disease and ulcerative diseases
• Hybridoma can be cultured either in vitro or in vivo .
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15. REFERENCES
• National Research Council (US) Committee on Methods of Producing Monoclonal
Antibodies. Monoclonal Antibody Production. Washington (DC): National Academies
Press (US); 1999. 4, Summary of Advantages and Disadvantages of In Vitro and In Vivo
Methods.Available from: https://www.ncbi.nlm.nih.gov/books/NBK100200/
• Prospecs Protein Cloning, https://www.prospecbio.com/monoclonal_antibodies
• Arya George. 2018. Hybridoma Technology. Slide. Mar Ivanios College
Trivandrum.Linkedln
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