Hybridoma Hybridomas are cells that have been engineered to produce a desired antibody in large amounts, to produce monoclonal antibodies. Monoclonal antibodies can be produced in specialized cells through a technique now popularly known as hybridoma technology. Hybridoma technology was discovered in 1975 by two scientists, G. Kohler and C. Milstein, were awarded Noble prize for physiology and medicine in 1984.

1. SJM College of Pharmacy,
Chitradurga
Prepare By,
Adarsh Patil
Ass Professor(Pharmacognosy)
SJM College of Pharmacy
1
PHARMACEUTICAL
BIOTECHNOLOGY

2. Hybridoma
• Hybridomas are cells that have been engineered to produce a
desired antibody in large amounts, to produce monoclonal
antibodies.
• Monoclonal antibodies can be produced in specialized cells through
a technique now popularly known as hybridoma technology.
• Hybridoma technology was discovered in 1975 by two scientists, G.
Kohler and C. Milstein, were awarded Noble prize for physiology
and medicine in 1984.

3. 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)

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 hybridomas(spleen cell and myeloma cell) obtain monoclonal
antibodies in artificial media, this technology called as HYBRIDOMA
TECHNOLOGY.

6. 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.
7. Purification of Antibodies

7. 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.

8. 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.

9. 3. Fusion of spleen cell and myeloma cell.
• It requires PEG (poly ethylene glycone) medium for fusion.
• It can also done by electro fusion.
• Fusion between spleen cell and myeloma cell produced five different types
of cells.
1. Fused plasma
2. Fused myeloma
3. Hybridoma
4. Unfused plasma
5. Unfused myeloma

10. 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 synthesized completely new nucleotide by small molecules
(sugar, amino-acid).

11. • 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.

12. 5. Isolation of hybridoma cell
Spleen cell have
HGPRT enzyme
Myeloma cell doesn’t
have HGPRT enzyme
HGPRT
1. Fused plasma present
2. Fused myeloma absent
3. Hybridoma present
4. Unfused plasma present
5. Unfused myeloma absent

14. • 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.

16. 6. 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

17. • 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.

18. 7. Purification of Antibodies:
• Monoclonal antibodies may need to be purified before
they are used for a variety of purposes.
• Before final purification, the cultures may be subjected
to cell fractionation for enrichment of the antibody
protein.
• Alternatively the antibodies may be purified from cell
homogenate or cell debris obtained from the medium.

19. • Antibodies can be purified by anyone of the following
techniques
(I) ion-exchange chromatography;
(ii) antigen affinity chromatography.
• Once a hybridoma colony is established, it will
continually grow in culture medium like RPMI-1640 and
produce antibodies.
• Storage: liquid nitrogen.

22. Applications of Monoclonal Antibodies
 Diagnostic Applications
Biosensors & Microarrays
 Therapeutic Applications
Transplant rejection Muronomab-CD3
Cardiovascular disease Abciximab
Cancer Rituximab
Infectious Diseases Palivizumab
Inflammatory disease Infliximab
 Clinical Applications
Purification of drugs, Imaging the target
 Future Applications
Fight against Bioterrorism

23. 1. A breakthrough in Diagnostics
 Antibodies are used in several diagnostic tests to detect small
amounts of drugs, toxins or hormones
 Human Monoclonal antibodies to Human chorionic
Gonadotropin (HCG) are used in pregnancy test kits
 Another diagnostic uses of antibodies is the diagnosis of AIDS by
the ELISA test

24. 2 Helps in critical diagnostic decisions
 Once monoclonal antibodies for a given substance have been
produced, they can be used to detect the presence of this substance
 The Western blot test and Immuno dot blot tests detect the protein on
a membrane
 Useful in immunohistochemistry, which detect antigen in fixed tissue
sections and
 Immunofluorescence test, which detect the substance in a frozen
tissue section or in live cells.

25. 3. Pregnancy Tests
 A pregnant woman has the hormone Human Chorionic Gonadotrophin (HCG)
in her urine.
 Monoclonal antibodies to HCG have been produced. These have been attached
to enzymes which can later interact with a dye molecule and produce a colour
change.

26. 4. Diagnosis of HIV infection
The test of HIV infection is based on detecting the presence of HIV
antibody in the patient’s blood serum.
5. Clinical Applications of Mabs
Imaging the target organ
• Monoclonal antibodies directed against tumour-associated
antigens labelled with radioisotopes localize specifically into
tumour after intravenous injection. This property is used for
diagnostic tumour imaging by immunoscintigraphy.

27. The radio-labeled antibody-
isotope conjugate is injected
into the patient and allowed to
localize to the target over a 2- to
7-day period. The patient then
undergoes imaging with a
nuclear medicine gamma
camera, and radioisotope
counts are analyzed.
used in colorectal & prostate
cancer

28. • ADEPT (Antibody Directed Enzyme Pro-drug
Therapy)
Involves the application of cancer associated
monoclonal antibodies which are linked to a drug-
activating enzyme
 Subsequent systemic administration of a non-toxic
agent results in its conversion to a toxic drug, and
resulting in a cytotoxic effect which can be targeted at
malignant cells

29. • Future use in Bioterrorism
• Raxibacumab
 It is a human monoclonal antibody
antibody against Bacillus anthracis protective antigen
Intended for the prophylaxis and treatment of
inhaled anthrax.
 Its efficacy has been proved in rabbits and monkeys.