Monoclonal antibodies are identical antibodies produced by a single clone of cells that bind to a specific epitope. They are produced through the fusion of antibody-producing B cells with myeloma cells, generating hybridoma cells that can produce antibodies indefinitely. Monoclonal antibodies have applications in therapy and diagnosis, including detecting antigens through techniques like ELISA, purifying proteins, and treating cancers by delivering toxins or radioisotopes to tumor cells. Advances in engineering have reduced issues like human anti-mouse antibody responses by creating chimeric or humanized antibodies.

1. Production of Monoclonal Antibodies and Applications in
therapy and diagnosis
Introduction
Antibodies are proteins produced by the B lymphocytes of the immune system in
response to foreign proteins, called antigens. Antibodies function as markers, binding to
the antigen so that the antigen molecules can be recognized and destroyed by phagocytes.
The part of the antigen that the antibody binds to is called the epitope. The epitope is thus
a short amino acid sequence that the antibody is able to recognize.
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.
Characteristics are
(i) Mono-specific - recognize only one epitope (antigenic determinant)
(ii) Homogenous - identical immunoglobulin molecule or Display identical binding
strengths (affinity)
(iii) Produced in unlimited quantities
(iv)Usually derived from mice
Polyclonal antibodies are antibodies that are derived from different cell lines. They
differ in amino acid sequence.
The procedure of production
In 1975, Kohler and Milstein discovered that murine (mouse) antibody-secreting plasma
cells and immortal murine myeloma cells could be fused with the benefits of each
retained. This discovery propelled science and medicine into the modern monoclonal
antibody era.
The basic strategy includes
(i) Purification and characterization of the desired antigen in adequate quantity
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(i) Purification and characterization of the desired antigen in adequate quantity
(ii) Immunization of mice with the purified antigen
(iii) Culture of myeloma cells which are unable to synthesize hypoxanthine-guanine-
phosphoribosyl transferase (HGPRT) enzyme necessary for the salvage pathway
of nucleic acids

2. (iv) Removal of spleen cells from mice and its fusion with the myeloma cells
(v) Following fusion, the hybridomas were grown in hypoxanthine aminopterin
thymidine (HAT) medium.
Unfused myeloma cells cannot grow because they lack HGPRT
Unfused normal spleen cells cannot grow indefinitely because of their limited
life span.
However, Hybridoma cells (produced by successful fusions) are able to grow
indefinitely because the spleen cell partner supplies HGPRT and the myeloma
partner is immortal.
(vi) The hybrid cell clones are generated from single host cells
(vii) The antibodies secreted by the different clones are then tested for their ability to
bind to the antigen using an enzyme-linked immunosorbent assay (ELISA)
(viii) The clone is then selected for future use
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3. Applications in therapy and Diagnosis
Monoclonal antibodies are widely used as diagnostic and research reagents as well as in
human therapy. In some in vivo applications, the antibody itself is sufficient. Once bound
to its target, it triggers the normal effector mechanisms of the body.
In other cases, the monoclonal antibody is coupled to another molecule, for example
a fluorescent molecule to aid in imaging the target
a strongly-radioactive atom, such as Iodine-131 to aid in killing the target
Diagnostic application
The diagnostic applications of monoclonal antibodies are by far the most advanced,
especially for tests that are performed on body fluids such as blood and urine sample.
MAb is used to detect pregnancy as early as a week or two after conception by reacting
with human chorionic gonadotrophin, a hormone secreted by the placenta and found in
the urine of pregnant women.
To detect the presence of the substance/antigen, MAbs can be used. Different
technologies in which MAbs are used include Western blot, immunodot blot, ELISA,
radioimmuno assay (RIA), flow cytometry, immunohistochemistry, fluorescence
microscopy, electron microscopy, confocal microscopy, as well as other biotechnologicalmicroscopy, electron microscopy, confocal microscopy, as well as other biotechnological
applications. Such as
Gene cloning
One of the difficulties of gene cloning is identifying the cells that contain the desired
gene. If MAb that recognizes that the gene product is available, it can be used as a probe
for detecting those cells that make the product and therapy to detect the gene.
To identify cell types
MAbs contribute to the identification of many different types of cells that participate in
the immune response and to unravel interactions occurring during this process. For
example, in the lymphocytes with B, T helper (TH) cells and suppressor T, the use of
MAbs has established that the various types of T-cells carry cell surface antigens on their
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MAbs has established that the various types of T-cells carry cell surface antigens on their
surfaces that allow one type to be distinguished from another. The MAbs were also
helpful in defining changes in T and B-cells during development.
Protein purification
MAb affinity columns are readily prepared by coupling MAbs to a cyanogen bromide-
activated chromatography matrix, eg, Sepharose. Since the MAbs have unique specificity

4. for the desired protein, the level of contamination by unwanted protein species usually is
very low. Since the MAb-antigen complex has a single binding affinity it is possible to
elute the required protein in a single, sharp peak. The concentration of the relative protein
relative to total protein in a mixture can ever be very low. Therefore Monoclonal
antibodies can also be used to purify a substance with techniques called Immuno-
precipitation and affinity chromatography.
This method also has limitations. Achieving 100% pure protein is difficult because there
is always a tendency for small amounts of immunoglobulin to leak off the immune-is always a tendency for small amounts of immunoglobulin to leak off the immune-
affinity column. Additionally, MAb do not distinguish between intact protein molecules
and fragments containing the antigenic site.
Therapeutic application
Possible treatment for cancer involves monoclonal antibodies that bind only to cancer
cells specific antigen and induce immunological response on the target cancer cell (naked
antibodies). MAb can be modificated for delivery of toxin, radioisotope, cytokine. Toxins
or radioactive isotopes are bound to the constant region of the MAbs. When the MAb
binds to the surface cells of a tumor the toxin or radioactivity will kill the cancer cells and
all cells within a certain radius (a killing zone). In this way cancer cells within the tumor
will be killed.
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5. Mouse antibodies are "seen" by the human immune system as foreign, and the human
patient mounts an immune response against them, producing HAMA "human anti-mouse
antibodies". These not only cause the therapeutic antibodies to be quickly eliminated
from the host, but also form immune complexes that cause damage to the kidneys.
However, using genetic engineering it is possible to make mouse-human hybrid
antibodies to reduce the problem of HAMA. Antibodies with increased efficiency were
engineered for which the murine immunogenic content was removed
Chimeric antibodies: The antibody combines the antigen-binding parts (variableChimeric antibodies: The antibody combines the antigen-binding parts (variable
regions) of the mouse antibody with the effector parts (constant regions) of a
human antibody. Infliximab, rituximab, and abciximab are examples.
Humanized antibodies: The antibody combines only the amino acids responsible
for making the antigen binding site (the hypervariable regions) of a mouse (or rat)
antibody with the rest of a human antibody molecule thus replacing its own
hypervariable regions. Daclizumab, Vitaxin, Mylotarg, Herceptin, and Xolair
are examples.
In both cases, the new gene is expressed in mammalian cells grown in tissue culture
(i) To suppress the immune system
Muromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals. Bind toMuromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals. Bind to
the CD3 molecule on the surface of T cells. Used to prevent acute rejection of
organ, e.g., kidney, transplants. The humanized versions show promise in
inhibiting the autoimmune destruction of beta cells in Type 1 diabetes mellitus.
Infliximab (Remicade) and adalimumab (Humira). Bind to tumor necrosis factor-
alpha (TNF- promise against some inflammatory diseases such as
rheumatoid arthritis (by blunting the activity of Th1 cells). Side-effects: can
convert a latent case of tuberculosis into active disease; can induce the formation
of autoantibodies (by promoting the development of Th2 cells).
(ii) To kill or inhibit malignant cells
Rituximab (trade name = Rituxan). Binds to the CD20 molecule found on most B-
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cells and is used to treat B-cell lymphomas.
(iii) As Angiogenesis Inhibitors
Cancer cells (probably like all tissues) secrete substances that promote the formation of
new blood vessels, a process called angiogenesis.

6. Vitaxin binds to a vascular integrin (alpha-v/beta-3) found on the blood vessels of
tumors but not on the blood vessels supplying normal tissues. In Phase II clinical
trials, Vitaxin has shown some promise in shrinking solid tumors without harmful
side effects.
(iv) Abciximab (ReoPro). Inhibits the clumping of platelets by binding the receptors on
their surface that normally are linked by fibrinogen. Helpful in preventing reclogging
of the coronary arteries in patients who have undergone angioplasty.
Conclusion
Monoclonal antibodies are monospecific antibodies that are made by identical immune
cells that are all clones of a unique parent cell. Monoclonal antibodies are typically made
by fusing myeloma cells with the spleen cells from a mouse that has been immunized
with the desired antigen. However, recent advances have allowed the use of rabbit B-cells
to form a rabbit hybridoma. Hybridoma a hybrid cell used as the basis for the production
of antibodies in large amounts for diagnostic or therapeutic use. Once monoclonal
antibodies for a given substance have been produced, they can be used to detect the
presence of the substance. The Western blot test and immune-dot blot tests detect the
protein on a membrane. They are also very 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. Therapeutic monoclonal antibodies act through ain a frozen tissue section or in live cells. Therapeutic monoclonal antibodies act through a
number of mechanisms, such as blocking of targeted molecule functions, inducing
apoptosis of cells which express the target, or by controlling signaling pathways.
References
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Monoclonals.html
http://www.bio.davidson.edu/molecular/MolStudents/01rakarnik/mab.html
http://www.sumanasinc.com/webcontent/animations/content/monoclonalantibodies.ht
ml
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