Monoclonal Antibodies in Animal Biotechnology
Monoclonal antibodies
VBC-321
Animal Biotechnology
antibody
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.
Polyclonal antibodies
are antibodies that are derived from
different cell lines.
Isotypes
According to differences in their heavy chain constant
domains, immunoglobulins are grouped into five classes, or
isotypes:
IgG
,
IgA
,
IgM
,
IgD
, and
IgE
.
Monoclonal Antibodies
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
Given (almost) any substance, it is possible to create
monoclonal antibodies that specifically bind to that
substance; they can then serve to detect or purify that
substance
This has become an important tool in
biochemistry
,
molecular biology
and
medicine
The structure of antibodies
http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
History of Mab development
1890 Von Behring and Kitasato discovered the serum of vaccinated persons
contained certain substances, termed antibodies
1900 Ehrlich proposed the “ side-chain theory”
1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet
expended.
Almost the same time, Porter isolated fragment of antigen binding (Fab) and
fragment crystalline (Fc) from rabbit y-globulin.
1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines
using the hypoxanthine-aminopterin-thymidine (HAT) selection media.
1975 Kohler and Milstein provided the most outstanding proof of the clonal
selection theory by fusion of normal and malignant cells. This resulted in the
first monoclonal antibodies, for which they received the Nobel Prize in 1984.
Hybridoma technology
It is technique of producing hybrid cell lines called
“
hybridomas
”
by the fusion of fusing a specific
antibody-producing lymphocyte B cell
with a
myeloma
cell
that has an ability to grow in tissue culture
Hydridoma produce
antibodies
produced that have
single
specificity and
are called
monoclonal antibodies
.
This technique was discovered
Georges Kohler
of West
Germany,
Cesal Milstein
of Argentina and
Niels Jerne
of
Denmark in 1975.
They were awarded Nobel Prize for Physiology and
Medicine the 1984
Principal
This is based on fusion between myeoma cell (malignant plasma
cell) and spleen cell from suitable immunised animal.
Spleen can produce antibody but die in short period under culture
condition
Myeloma cell are adapted to grow permanently in cell culture
Myeloma cells cannot synthesize antibodies as they lack HGPRT
gene required for the synthesize the enzyme, hypoxanthine
guanine phosphoribosyl transferase (azaquinine resistant) or
thymidine kinase (bromodeoxy uridine resistant)
Mutant myeloma cell can not grow in HAT (Hypoxanthine
Aminopterin Thymidine) medium
Only hybrid cell can grow in HAT medium and produce
monoclonal antibody
Method
1.
Immunization of a mouse
2.
Isolation of B cells from the spleen
3.
Cultivation of myeloma cells
4.
Fusion of myeloma and B cells
5.
Separation of cell lines
6.
Screening of suitable cell lines
7.
in vitro
(a) or
in vivo
(b) multiplication
8.
Harvesting
Immunization of a mouse
Immunize a rabbit through repeated injection of a specific
antigen for the production of specific antibody, facilitated
due to proliferation of the desired B cells
Produce tumors in a mouse or a rabbit
Culture separately the spleen cells that produce specific
antibodies and the myeloma cells that produce tumors
Fusion
Fusion of spleen
cells to myeloma cells is
induced using
polyethylene glycol (PEG),
to
produce
hybridoma
Hybridomas are grown in selective
hypoxanthine aminopterin thymidine (HAT)
medium.
HAT medium contains a
drug,
aminopterin
that blocks one pathway for nucleotide
synthesis, making the cells dependent on
another pathway that needs HGPRT enzyme,
which is absent in myeloma cells.
Myeloma cells that do not fuse with B cells
will die.
B cells that do not fuse will also die because
they lack tumorigenic property of immortal
growth.
Select desired hybridoma for cloning and
antibody production
Prepare
single cell colonies
that can grow
and use them to screen of antibody
producing hybridomas
Only one in several hundred cell hybrids will
produce antibodies of the desired specificity
Culture selected hybridoma cells for the
production of monoclonal antibodies in large
quantities
Hybridoma cells can be
frozen
for future use
or can be
injected
in the body of an animal,
so that antibodies will be produced in the
body and recovered later from the body fluid.
Selection and Storage
Monoclonal antibodies (mAb) play a crucial role in identifying and neutralizing foreign substances in the body. This article delves into the significance of monoclonal antibodies, their structure, development history, and the revolutionary hybridoma technology. Explore the world of antibodies and their impact on biochemistry, molecular biology, and medicine.
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Presentation Transcript
VBC-321 Animal Biotechnology Monoclonal antibodies ?
antibody 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. Polyclonal antibodies are antibodies that are derived from different cell lines. Isotypes According to differences in their heavy chain constant domains, immunoglobulins are grouped into five classes, or isotypes: IgG, IgA, IgM, IgD, and IgE.
Monoclonal Antibodies 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 Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance This has become an important tool in biochemistry, molecular biology and medicine
The structure of antibodies http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html
History of Mab development 1890 Von Behring and Kitasato discovered the serum of vaccinated persons contained certain substances, termed antibodies 1900 Ehrlich proposed the side-chain theory 1955 Jerne postulated natural selection theory. Frank Macfarlane Burnet expended. Almost the same time, Porter isolated fragment of antigen binding (Fab) and fragment crystalline (Fc) from rabbit y-globulin. 1964 Littlefield developed a way to isolate hybrid cells from 2 parent cell lines using the hypoxanthine-aminopterin-thymidine (HAT) selection media. 1975 Kohler and Milstein provided the most outstanding proof of the clonal selection theory by fusion of normal and malignant cells. This resulted in the first monoclonal antibodies, for which they received the Nobel Prize in 1984.
Hybridoma technology It is technique of producing hybrid cell lines called hybridomas by the fusion of fusing a specific antibody-producing lymphocyte B cell with a myeloma cell that has an ability to grow in tissue culture Hydridoma produce antibodies produced that have single specificity and are called monoclonal antibodies. This technique was discovered Georges Kohler of West Germany, Cesal Milstein of Argentina and Niels Jerne of Denmark in 1975. They were awarded Nobel Prize for Physiology and Medicine the 1984
Principal This is based on fusion between myeoma cell (malignant plasma cell) and spleen cell from suitable immunised animal. Spleen can produce antibody but die in short period under culture condition Myeloma cell are adapted to grow permanently in cell culture Myeloma cells cannot synthesize antibodies as they lack HGPRT gene required for the synthesize the enzyme, hypoxanthine guanine phosphoribosyl transferase (azaquinine resistant) or thymidine kinase (bromodeoxy uridine resistant) Mutant myeloma cell can not grow in HAT (Hypoxanthine Aminopterin Thymidine) medium Only hybrid cell can grow in HAT medium and produce monoclonal antibody
Method Immunization of a mouse Isolation of B cells from the spleen Cultivation of myeloma cells Fusion of myeloma and B cells Separation of cell lines Screening of suitable cell lines in vitro (a) or in vivo (b) multiplication Harvesting 1. 2. 3. 4. 5. 6. 7. 8.
Immunization of a mouse Immunize a rabbit through repeated injection of a specific antigen for the production of specific antibody, facilitated due to proliferation of the desired B cells Produce tumors in a mouse or a rabbit Culture separately the spleen cells that produce specific antibodies and the myeloma cells that produce tumors
Fusion Fusion of spleen cells to myeloma cells is induced using polyethylene glycol (PEG), to produce hybridoma Hybridomas are grown in selective hypoxanthine aminopterin thymidine (HAT) medium. HAT medium contains a drug, aminopterin that blocks one pathway for nucleotide synthesis, making the cells dependent on another pathway that needs HGPRT enzyme, which is absent in myeloma cells. Myeloma cells that do not fuse with B cells will die. B cells that do not fuse will also die because they lack tumorigenic property of immortal growth.
Selection and Storage Select desired hybridoma for cloning and antibody production Prepare single cell colonies that can grow and use them to screen of antibody producing hybridomas Only one in several hundred cell hybrids will produce antibodies of the desired specificity Culture selected hybridoma cells for the production of monoclonal antibodies in large quantities Hybridoma cells can be frozen for future use or can be injected in the body of an animal, so that antibodies will be produced in the body and recovered later from the body fluid.
