Hybridoma technology allows scientists to produce monoclonal antibodies at scale. It involves fusing antibody-producing plasma cells from immunized mice with myeloma cancer cells, creating a hybridoma cell line that is immortal and continuously produces identical monoclonal antibodies. This overcomes the short lifespan of plasma cells. The hybridoma cells are selected and isolated using HAT media, which only the hybridomas can survive in due to possessing a key enzyme. This technique has generated monoclonal antibodies useful for diagnosing and treating various diseases.

1. HYBRIDOMA TECHNOLOGY
DONE BY : LAMYA ALSADHAN
SUBMITTED TO : DR. ELSAYED AHMED ELSAYED

2. INTRODUCTION
 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,
Georges Kohler and Cesar Milstein, who jointly with Niels Jerne of
Denmark (now working in Germany) were awarded the 1984 noble
prize for physiology and medicine.
 The term hybridoma was coined by Leonard Herzenberg during his

3. MONOCLONAL ANTIBODIES
 An antibody is a protein used by the immune system to identify and
neutralize foreign objects like bacteria and viruses. Each antibody
recognizes a specific antigen unique to its target.
 Monoclonal antibodies (mab) are antibodies that are identical because
they were produced by one type of immune cell, all clones of a single
parent cell.
 A monoclonal antibody can be used to detect pregnancy only 14 days
after conception. Other monoclonal antibodies allow rapid diagnosis of
hepatitis, influenza, herpes, streptococcal, and chlamydia infections.
 Monoclonal antibodies have a wide range of therapeutic applications.
MAbs are used in the treatment of cancer, transplantation of bone
marrow and organs, autoimmune diseases, cardiovascular diseases
and infectious diseases.

4. HYBRIDOMA TECHNOLOGY
 Hybridoma technology is a method for producing large numbers of
identical antibodies (also called monoclonal antibodies).
 This process starts by injecting a mouse (or other mammal) with an
antigen that provokes an immune response.
 A type of white blood cell, the B cell will be converted to Plasma cell
by the presence of the antigen.
 The plasma cell will produce antibodies that bind to the antigen are
then harvested from the mouse. These isolated plasma cells are in
turn fused with immortal B cell cancer cells, a myeloma, to produce a
hybrid cell line called a hybridoma.

5. The conversion of the B cell to plasma
cell that secretes antibodies

6. Why do we even bother?
 You might wonder.. Why we even bother making the hybridoma
cells when we could just isolate the plasma cells, culture them
and have them produce the antibodies for us?
 The idea is right and possible, but the only problem is the
plasma cells life span is very short, ranging from few weeks to
few months maximum.
 In hybridoma technology, this problem is over comes by fusing
the plasma cells with immortal cancerous B cells known as
myeloma cells.

7. Hybridoma Technology Idea

8. PROCEDURE
1. Mouse is immunized by giving
antigen injection intravenously,
meaning directly into the blood, so
the antigen will be dropped into the
spleen.
2. Inside the spleen, the antigen will
interact with B cells and will convert
them to plasma cells, once the
plasma cells are produced they will
secrete a large number or
monoclonal antibodies

10. PROCEDURE
4. After the purification of the plasma cells, it is then fused with
the myeloma cells in the presence of polyethylenglycol.
5. Since the fusion procedure is random, five different types of
cells will appear :

11. How to isolate the Hybridoma
between all those cells?
6. Between all those five cells,
we need the hybridoma only,
to isolate it we have to use
the HAT Media.
7. The HAT stands for
(Hypoxanthine +
Aminopterin + Thymidine).
8. When ever the cell divides, it
must synthesize nucleotides,
Salvage Pathway
De novo
Pathway
The cells use the
degraded parts of
old nucleotides to
make new
nucleotides
The cell completely
make new
nucleotides using
small metabolites
like sugar and
amino acids
AminopterinHGPRT

12. HGPRT
+
HGPRT
+
HGPRT
+
Short life
span
Short life
span
Immortal
HA
T
HA
T
HA
T
9. only the hybridoma will remain because its immortal and have
the HGPRT enzyme, in this way the hybridoma is selected and
used for continues MAb production.

13. Purification of
Antibodies
 Monoclonal antibodies may
need to be purified before they
are used for a variety of
purposes.
 Antibodies can be purified by
any of the following techniques
:
 Ion-exchange
chromatography.

14. ADVANTAGES OF SERUM FREE MEDIA IN HYBRIDOMA CELL
CULTURE AND PREPARATION OF MONOCLONAL
ANTIBODIES
1. Greatly simplified purification of antibodies due to increased Initial purity
and absence of contaminating immunoglobulin.
2. Decreased variability of culture medium.
3. Reduced risk of infectious agents.
4. Fewer variables for quality control/quality assurance.
5. Increased control over bioreactor conditions.
6. Potential for increased antibody secretion.
7. Low or no dependence on animals.
8. Cost effective.
9. Overall enhanced efficiency

15. DISADVANTAGES OF SERUM FREE MEDIA IN HYBRIDOMA
CELL
CULTURE AND PREPARATION OF MONOCLONAL
ANTIBODIES
1. Not all serum free media are applicable to all cell lines.
2. Cells may not grow to as high densities and may be more
fragile than cells in serum
3. Media may take longer to prepare

16. CONCLUSION
Hybridoma technology is the valuable for preparing
antibody in vitro condition
 Monoclonal antibodies, as they are known, have
opened remarkable new approaches to preventing,
diagnosing, and treating disease.
It is helpful in various aspects .