Hawassa University
Department of Biotechnology
MEDICAL BIOTECHNOLOGY
(Biot-3113)
Eshet Lakew Tesfaye
(MSc. in Industrial Biotechnology)
Email:
[email protected]CHAPTER-3 We strive for wisdom
What are antibodies?
An antibody is a protein used by immune system to identify
and neutralize foreign objects like bacteria and viruses.
Each antibody recognizes a specific antigen unique to its
target.
The high specificity of antibodies makes them an excellent
tool for detecting and quantifying a broad array of targets,
from drugs to serum proteins to microorganisms.
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Cont…
With in vitro assays, antibodies can be used to;
precipitate soluble antigens,
agglutinate (clump) cells,
opsonize and kill bacteria with the assistance of
complement, and neutralize drugs, toxins, and viruses.
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Monoclonal Antibodies
Monoclonal antibodies are identical immunoglobulins,
generated from a single B-cell clone.
These antibodies recognize unique epitopes, or binding
sites, on a single antigen.
Derivation from a single B-cell clones and subsequent
targeting of a single epitope is what;
differentiates monoclonal antibodies from polyclonal
antibodies.
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Cont…
Polyclonal antibodies are antibodies that are derived from
different cell lines.
They differ in amino acid sequences.
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Characters of monoclonal Antibodies
• Monoclonal antibodies (mAB) are single type of antibody
that are;
identical and are directed against a specific epitope
(antigen, antigenic determinant) and;
are produced by B-cell clones of a single parent or a single
hybridoma cell line.
• A hybridoma cell line is formed by the fusion of one B-cell
lymphocyte with a myeloma cell.
• Some myeloma cell synthesize single mAB naturally.
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Advantages of Monoclonal Antibodies
They are cheaper to develop than conventional drugs,
because it is based on tested technology.
• Side effects can be treated and reduced by using mice-
human hybrid cells or by using fractions of antibodies.
• They bind to specific diseased or damaged cells needing
treatment.
• They treat a wide range of conditions.
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Disadvantages of Monoclonal Antibodies
• Time consuming project - 6 -9 months.
• Very expensive and needs considerable effort to produce them.
• Small peptide and fragment antigens may not be good antigens,
monoclonal antibody may not recognize the original
antigen.
• Hybridoma culture may be subject to contamination.
• System is only well developed for limited animal and not for
other animals.
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Cont…
• More than 99% of the cells do not survive during the
fusion process –
reducing the range of useful antibodies that can be
produced against an antigen
possibility of generating immunogenicity.
Q= What is immunogenicity?
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Cont…
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Hybridoma Technology
It is technique of producing hybrid cell lines called
“hybridomas” by the fusion of;
a specific antibody-producing lymphocyte B cell with,
a myeloma cell that has an ability to grow in tissue culture
Hybridoma produce antibodies produced that have single
specificity and are called monoclonal antibodies.
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Cont…
This is based on fusion between,
Myeloma 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
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Cont…
Myeloma cells cannot synthesize antibodies as they lack
HGPRT gene required for synthesize the enzyme,
hypoxanthine guanine phosphoribosyl transferase
(azaquinine resistant) or
thymidine kinase (bromodeoxy uridine resistant)
Mutantmyeloma cell can not grow in HAT (Hypoxanthine
Aminopterin Thymidine) medium
Onlyhybrid cell can grow in HAT medium and produce
monoclonal antibody
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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
Q=
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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
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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.
Major Applications of mAbs
(1) Diagnostic Applications
• Biochemical analysis
• Diagnostic Imaging
(2) Therapeutic Applications
• Direct use of MAbs as therapeutic agents
• MAbs as targeting agents.
(3) Protein Purification
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Biochemical analysis
• Routinely used in radioimmunoassay (RIA) and enzyme-
linked immunosorbent assays (ELISA) in the laboratory.
These assays measure the circulating concentrations of
hormones,
(insulin, human chorionic gonadotropin, growth hormone,
progesterone, thyroxine, thyroid stimulating hormone) and;
other tissue and cell products
(blood group antigens, blood clotting factors, interferon’s,
interleukins, tumor markers).
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Cont…
Example:
Pregnancy by detecting the urinary levels of human chorionic
gonadotropin.
Hormonal disorders analysis of thyroxine, etc.,
Cancers estimation of plasma carcinoembryonic antigen in
colorectal cancer, and prostate specific antigen for prostate
cancer
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Diagnostic imaging
• Radiolabeled—MAbs are used in the diagnostic imaging of
diseases and;
this technique is referred to as immunoscintigraphy.
The radioisotopes commonly used for labeling MAb are iodine
—131 and technetium—99.
The MAb tagged with radioisotope are injected intravenously
into the patients.
• These MAbs localize at specific sites (say a tumor) which can
be detected by imaging the radioactivity.
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Cont…
In recent years, single photon emission computed tomography
(SPECT) cameras are used;
to give a more sensitive three dimensional appearance of the
spots localized by radiolabeled— MAbs.
• Myocardial infarction, DVT, atherosclorosis etc.
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Direct use of MAbs as therapeutic agents
In destroying disease-causing organisms:
MAbs promote efficient opsonization of pathogenic organisms
(by coating with antibody) and enhance phagocytosis.
In the immunosuppression of organ transplantation:
In the normal medical practice, immunosuppressive drugs such
as cyclosporin and prednisone are administered to overcome the
rejection of organ transplantation.
In recent years, MAbs specific to T-lymphocyte surface
antigens are being used for this purpose
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Protein Purification
• Monoclonal antibodies can be produced for any protein.
And the so produced Mab can be conveniently used for the
purification of the protein against which it was
raised.
• MAbs columns can be prepared by coupling them to
cyanogen bromide activated Sepharose (chromatographic
matrix).
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Cont…
The immobilized MAbs in this manner are very useful for the
purification of proteins by;
immunoaffinity method.
• There are certain advantages of using MAbs for protein
purification.
These include:
the specificity of the MAb to bind to the desired protein,
very efficient elution from the chromatographic column
and,
high degree of purification.
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