The document summarizes the process of producing monoclonal antibodies (mAbs) through hybridoma technology. It involves immunizing an animal, usually a mouse, to elicit an immune response. B cells from the animal's spleen are then fused with myeloma cells to generate immortal hybridoma cells. These hybridoma cells are screened and selected in HAT medium to identify clones that produce the desired mAb. The selected clones are then subjected to further characterization and mass production methods.
Production and applications of monoclonal antibodiesKaayathri Devi
production and applications of monoclonal antibodies, monoclonal antibodies ,applications of monoclonal antibodies, production of monoclonal antibodies,
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
Production and applications of monoclonal antibodiesKaayathri Devi
production and applications of monoclonal antibodies, monoclonal antibodies ,applications of monoclonal antibodies, production of monoclonal antibodies,
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Students of medical and allied subjects must be exposed to the concept of monoclonal antibodies for the efficient practice of clinical and laboratory medicine.
Various diagnostic tools now a days relied on the Hybriodoma technology and monoclonal antibodies,so this presentation will give some basic information about mAb and Hybridoma technology.
HYBRIDOMA TECHNOLOGY IT IS DEFINED AS THE PROCESS WERE THERE IS A FUSION OF SPLLEN CELL AND MYELOMA CELLS IN THE PRESENCE OF POLYETHYLENE GLYCOL OR SENDAI VIRUS AND LEADS TO THE PRODUCTION OF MONOCLONL ANTIBODY.
Hybridoma Technology ( Production , Purification , and Application ) Sakshi Ghasle
Hybridoma technology revolutionized the field of immunology by enabling the production of monoclonal antibodies with high specificity and affinity. This presentation delves into the principles of DNA hybridoma technology, highlighting its significance in antibody production, therapeutic applications, and biomedical research. Learn about the key steps involved in generating hybridomas, from immunization to antibody screening, and discover the potential of recombinant DNA techniques in enhancing antibody engineering. Whether you're a student, researcher, or industry professional, this overview will provide valuable insights into the innovative world of hybridoma technology."
Uncover the wide-ranging applications of monoclonal antibodies in areas such as cancer therapy, autoimmune diseases, infectious diseases, and beyond. Learn about the latest advancements in antibody engineering and the development of novel therapeutic modalities, including bispecific antibodies, antibody-drug conjugates, and immune checkpoint inhibitors.
Whether you're a seasoned researcher or a newcomer to the field, this SlideShare presentation serves as a valuable resource for understanding the principles, techniques, and applications of hybridoma technology in modern biomedicine. Join a journey through the fascinating world of monoclonal antibodies and the groundbreaking science behind their creation.
Unlock the potential of hybridoma technology and propel your research to new heights. Dive into this SlideShare presentation now and explore the limitless possibilities of monoclonal antibody production with hybridoma technology.
BIOTECHNOLOGY IS
CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ......
ITS A VERY INTERESTING TO LEARN ABOUT HYBRIDOMA TECHNOLOGY .. THEIR PRODUCTION AND
APPLICATION ALSO ....
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3. The monoclonal antibody (mAb) production process
usually starts with generation of mAb-producing cells
(i.e. hybridomas) by fusing myeloma cells with
desired antibody-producing splenocytes (e.g. B cells).
These B cells are typically sourced from animals,
usually mice. After cell fusion, large numbers of
clones are screened and selected on the basis of
antigen specificity and immunoglobulin class. Once
candidate hybridoma cell lines are identified, each
"hit" is confirmed, validated, and characterized using
a variety of downstream functional assays.
Upon completion, the clones are scaled up where
additional downstream bioprocesses occur.
4. Monoclonal antibody (MAb) is a single type of
antibody that is directed against a specific
antigenic determinant (epitope).
George Kohler and Cesar Milstein (Nobel
Prize, 1984) achieved large scale production
of MAbs. They could successfully hybridize
antibody—producing B-lymphocytes with
myeloma cells in vitro and create a
hybridoma.
5. 1. Immunization
2. Cell fusion
3. Selection of hybridomas
4. Screening the products
5. Cloning and propagation
6. Characterization and storage.
6.
7. The very first step in hybridoma technology is to
immunize an animal (usually a mouse), with
appropriate antigen.
The antigen, along with an adjuvant like
Freund’s complete or incomplete adjuvant is
injected subcutaneously (adjuvants are non-
specific potentiators of specific immune
responses).
This enables increased stimulation of B-
lymphocytes which are responding to the
antigen. Three days prior to killing of the
animal, a final dose of antigen is intravenously
administered.
8. The concentration of the desired antibodies is
assayed in the serum of the animal at frequent
intervals during the course of immunization.
When the serum concentration of the antibodies
is optimal, the animal is sacrificed.
The spleen is aseptically removed and disrupted
by mechanical or enzymatic methods to release
the cells. The lymphocytes of the spleen are
separated from the rest of the cells by density
gradient centrifugation.
9. The thoroughly washed lymphocytes are
mixed with HGPRT defective myeloma cells.
The mixture of cells is exposed to
polyethylene glycol (PEG) for a short period
(a few minutes), since it is toxic.
PEG is removed by washing and the cells are
kept in a fresh medium. These cells are
composed of a mixture of hybridomas (fused
cells), free myeloma cells and free
lymphocytes.
10. When the cells are cultured in HAT medium (the
principle described above), only the hybridoma
cells grow, while the rest will slowly disappear.
This happens in 7-10 days of culture.
Selection of a single antibody producing hybrid
cells is very important. This is possible if the
hybridomas are isolated and grown individually.
The suspension of hybridoma cells is so diluted
that the individual aliquots contain on an
average one cell each. These cells, when grown
in a regular culture medium, produce the
desired antibody.
11. The myeloma cells used in hybridoma
technology must not be capable of
synthesizing their own antibodies.
The selection of hybridoma cells is based on
inhibiting the nucleotide (consequently the
DNA) synthesizing machinery.
The mammalian cells can synthesize
nucleotides by two pathways—de novo
synthesis and salvage pathway
12.
13. The de novo synthesis of nucleotides requires
tetrahydrofolate which is formed from
dihydrofolate.
The formation of tetrahydrofolate (and
therefore nucleotides) can be blocked by the
inhibitor aminopterin.
The salvage pathway involves the direct
conversion of purines and pyrimidine’s into
the corresponding nucleotides. Hypoxanthine
guanine phosphoribosyl transferase (HGPRT)
is a key enzyme in the salvage pathway of
purines.
14. When cells deficient (mutated cells) in HGPRT
are grown in a medium containing hypoxanthine
aminopterin and Thymidine (HAT medium), they
cannot survive due to inhibition of de novo
synthesis of purine nucleotides (Note : Salvage
pathway is not operative due to lack of HGPRT).
Thus, cells lacking HGPRT, grown in HAT medium
die.
The hybridoma cells possess the ability of
myeloma cells to grow in vitro with a functional
HGPRT gene obtained from lymphocytes (with
which myeloma cells are fused).
Thus, only the hybridoma cells can proliferate in
HAT medium, and this procedure is successfully
used for their selection.
15. The hybridomas must be screened for the
secretion of the antibody of desired specificity.
The culture medium from each hybridoma
culture is periodically tested for the desired
antibody specificity.
The two techniques namely ELISA and RIA are
commonly used for this purpose.
In both the assays, the antibody binds to the
specific antigen (usually coated to plastic plates)
and the unbound antibody and other components
of the medium can be washed off.
Thus, the hybridoma cells producing the desired
antibody can be identified by screening. The
antibody secreted by the hybrid cells is referred
to as monoclonal antibody.
16. The single hybrid cells producing the desired antibody are
isolated and cloned. Two techniques are commonly
employed for cloning hybrid cells-limiting dilution method
and soft agar method.
Limiting dilution method:
In this procedure, the suspension of hybridoma cells is
serially diluted and the aliquots of each dilution are put
into micro culture wells. The dilutions are so made that
each aliquot in a well contains only a single hybrid cell.
This ensures that the antibody produced is monoclonal.
Soft agar method:
In this technique, the hybridoma cells are cultured in soft
agar. It is possible to simultaneously grow many cells in
semisolid medium to form colonies. These colonies will be
monoclonal in nature. In actual practice, both the above
techniques are combined and used for maximal production
of MAbs.
17. The monoclonal antibody has to be subjected
to biochemical and biophysical
characterization for the desired specificity. It
is also important to elucidate the MAb for
the immunoglobulin class or sub-class, the
epitope for which it is specific and the
number of binding sites it possesses.
The stability of the cell lines and the MAbs
are important. The cells (and MAbs) must be
characterized for their ability to withstand
freezing, and thawing. The desired cell lines
are frozen in liquid nitrogen at several stages
of cloning and culture.
18. The production MAbs in the culture bottles is
rather low (5-10 (ig/ml). The yield can be
increased by growing the hybrid cells as
ascites in the peritoneal cavity of mice. The
ascitic fluid contains about 5-20 mg of
MAb/ml. This is far superior than the in vitro
cultivation techniques.
But collection of MAb from ascitic fluid is
associated with the heavy risk of
contamination by pathogenic organisms of
the animal. In addition, several animals have
to be sacrificed to produce MAb.
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