Shreya Talreja et al /J. Pharm. Sci. & Res. Vol.

12(1), 2020, 49-53

A review on Monoclonal antibody and its application in

biotechnology
Shreya Talreja*
(Lecturer , JP College of Pharmacy, Mohanlalganj , Lucknow)

Swarnima Pandey
(Asst Professor , Goel Institute of Pharmacy & Sciences , Lucknow)

Sushant Kumar
(Asst Professor, Uttar Pradesh University of Medical Sciences, Saifai, Etawah)

Abstract
The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Kohler in 1975 and they got a
Nobel Prize for this work in 1984.Hybridoma technology is the method useful for the production of large quantity of
identical antibodies these antibodies are known as monoclonal antibodies. The production of monoclonal antibody is done by
the administration of antigen in mouse which produces an immune response. The B-cells producing antibodies are then
harvested from the injected mouse. The harvested B-cells are then fused with B cancer cells which remain immortal. This
produces hybrid cell line called hybridoma which possesses the antibody-producing ability of the B-cell. The hybridomas
can be grown in culture with one viable cell which produces cultures having genetically identical hybridomas. It produces
monoclonal antibodies. It retains the ability to grow in tissue culture and do not possess antibody producing capability.
Monoclonal antibodies are used to track cancer antigens and, alone or linked to anticancer agents, to attack cancer
metastases. The monoclonal antibody known as OKT3 is saving organ transplants threatened with rejection, and preventing
bone marrow transplants. The advantage of this process it combine the qualities of the two different types of cells; the
ability to grow continually, and to produce large amounts of pure antibody. Monoclonal antibody is also useful for the
treatment of non-infectious diseases such as immune disease, arthritis, cancer etc.
Key Words- Monoclonal antibody production, hybridoma technology, application

INTRODUCTION blood cells known as B-lymphocytes. Each antibody

Hybridomas are cells that have been engineered to produced is specific to that particular antigen which has
produce a desired antibody in large amounts, to produce stimulated its production.Antibodies may be classified five
monoclonal antibodies. (1,2) Monoclonal antibodies can major classes such as – IgG (monomer), IgA (Dimer),
be produced in specialized cells through a technique now IgM (Pentamer), IgD (monomer), IgE (monomer).
popularly known as hybridoma technology.(1) Antibodies are also called as Immunoglobulin (Ig) and
Hybridoma technology was discovered in 1975 by two the structure of antibody contains two chain such as –
scientists, Georges Kohler of West Germany and Cesar Heavy chain,Light chain and constant region ,variable
Milstein of Argentina and they got the noble prize for this region and they also contain di sulphide bond etc (6).
work in 1984.
The production of one MAb, using the hybridoma
technology, costs between $8,000 and $12,000.
Monoclonal antibodies is valuable for the analysis of
parasites antigen Monoclonal antibodies are being used to
track cancer antigens and, alone or linked to anticancer
agents, to attack cancer metastases. The monoclonal
antibody known as OKT3 is saving organ transplants
threatened with rejection, and preventing bone marrow
transplants from setting off graft-versus-host disease. The
mAbs have various applications in the fields of cell
biology, immunology, biotechnology and medicines. They
are also being used in vivo imaging techniques of different
kinds of diseases (3,4). mAbs are important diagnostic
reagents used in biomedical research, microbiological
research in the diagnosis of Hepatitis, AIDS, influenza,
herpes simplex, infections and in the treatment of such
diseases and cancer (5). Diag. Structure of antibody
Antibodies are glycoprotein molecule present in the
serum. They are produced in response to antigen which are Procedure for the Production of monoclonal antibody
either protein or polysaccharide molecules which may be Basic steps involved in the production of a monoclonal
foreign to the body. Antibodies are secreted by a class of antibody:

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1. Immunization (Immunize the animal) • 40% PEG (1000dal); PEG should be protested for cell
2. Cell fusion process toxicity
3. Selection of hybridomas • Fusion at 37∙c, pH 7.5 -8.0, 3 minute
4. Screening the products 3. Selection of Hybridomas:
5. Cloning and propagation When the cells are cultured in HAT medium only the
6. Characterization and storage 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.

4. Screening the Products:

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
Diag. Production of monoclonal antibody antibody and other components of the medium can be
washed off. Thus, the hybridoma cells producing the
1. Immunize the animal desired antibody can be identified by screening. The
The first step in hybridoma technology is to immunize an antibody secreted by the hybrid cells is referred to as
animal (mouse), with appropriate antigen. The antigen, monoclonal antibody.
along with an adjuvant like Freund’s complete or
incomplete adjuvant is injected subcutaneously. The 5. Cloning and Propagation:
injections at multiple sites are repeated several times. The single hybrid cells producing the desired antibody are
This enables increased stimulation of B-lymphocytes isolated and cloned. Two techniques are commonly
which are responding to the antigen. Three days prior to employed for cloning hybrid cells:
killing of the animal, a final dose of antigen is • limiting dilution method
intravenously administered. The immune-stimulated cells • soft agar method.
for synthesis of antibodies have grown maximally by this
approach. The concentration of the desired antibodies is Limiting dilution method: In this procedure, the
assayed in the serum of the animal at frequent intervals suspension of hybridoma cells is serially diluted and the
during the course of immunization. aliquots of each dilution are put into micro culture wells.
When the serum concentration of the antibodies is optimal, The dilutions are so made that each aliquot in a well
the animal is sacrificed. The spleen is aseptically removed contains only a single hybrid cell. This ensures that the
and disrupted by mechanical or enzymatic methods to antibody produced is monoclonal.
release the cells. The lymphocytes of the spleen are Soft agar method: In this technique, the hybridoma cells
separated from the rest of the cells by density gradient are cultured in soft agar. It is possible to simultaneously
centrifugation. grow many cells in semisolid medium to form colonies.
These colonies will be monoclonal in nature. In actual
2. Cell Fusion process: practice, both the above techniques are combined and used
The thoroughly washed lymphocytes (spleen cell) are for maximal production of MAbs.
mixed with HGPRT negative myeloma cells. The mixture
of cells is exposed to polyethylene glycol (PEG) for a 6. Characterization and Storage:
short period (a few minutes), since it is toxic. PEG is The monoclonal antibody has to be subjected to
removed by washing and the cells are kept in a fresh biochemical and biophysical characterization for the
medium. These cells are composed of a mixture of desired specificity. It is also important to elucidate the
hybridomas (fused cells), free myeloma cells and free MAb for the immunoglobulin class or sub-class, the
lymphocytes. epitope for which it is specific and the number of binding
Condition for Fusion Procedure: sites it possesses.
Before carrying out the fusion the following condition The stability of the cell lines and the MAbs are important.
should be met for the mouse system: The cells (and MAbs) must be characterized for their
• Myeloma cells in the logarithmic growth phase ability to withstand freezing, and thawing. The desired cell
• Ratio of 2-5 lymphocyte per myeloma cell lines are frozen in liquid nitrogen at several stages of
cloning and culture.

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Applications ( 1,7)
 Diagnostic Applications:
Monoclonal antibodies have revolutionized the laboratory
diagnosis of various diseases. For this purpose, MAbs may
be employed as diagnostic reagents for biochemical
analysis or as tools for diagnostic imaging of diseases.
MAbs in Biochemical Analysis: Diagnostic tests based
on the use of MAbs as reagents are 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, triiodothyronine, thyroid
stimulating hormone, gastrin, renin), and several other
tissue and cell products (blood group antigens, blood
clotting factors, interferon’s, tumor markers).
In recent years, a number of diagnostic kits using MAbs
have become commercially available.Now it is useful for Diag. Monoclonal antibody bound to cancer cell
diagnosis the various disease: protein
• Pregnancy: Pregnancy by detecting the urinary
levels of human chorionic gonadotropin. The patients suffering from leukemia, colorectal cancer,
• Cancers: Cancers estimation of plasma lymphoma and melanoma have been treated with MAbs.
carcinoembryonic antigen in colorectal cancer, and However, there was a wide variation in the success rate. A
prostate specific antigen for prostate cancer. Besides monoclonal antibody specific to the cells of leukemia is
diagnosis, estimation of tumor markers is also useful for used to destroy the residual leukemia cells without
the prognosis of cancers. That is a gradual fall in a specific affecting other cells. MAbs are used in vitro to remove the
tumor marker is observed with a reduction in tumor size, residual tumor cells prior to autologous bone marrow
following treatment. transplantation (transplantation of the patient’s own bone
• Hormonal disorders: Hormonal disorders marrow cells, due to non-availability of a suitable donor).
analysis of thyroxine, triiodothyronine and thyroid Limitations for direct use of MAbs in cancer:
stimulating hormone for thyroid disorders. 1. The MAbs produced in mice and directly used for
therapeutic purposes may lead to the development of anti-
 Therapeutic Applications: mouse antibodies and hypersensitivity reactions.
Monoclonal antibodies have a wide range of therapeutic 2. All the cancer cells may not carry the same antigen for
applications. MAbs are used in the treatment of cancer, which MAb has been produced. Thus, MAbs may not be
transplantation of bone marrow and organs, autoimmune attached to some cancer cells at all.
diseases, cardiovascular diseases and infectious diseases. 3. The free antigens (of target cells) present in the
The therapeutic applications of MAbs are broadly circulation may bind to MAbs and prevent them from their
grouped into 2 types: action on the target cells
• Direct use of MAbs as therapeutic agents In the treatment of AIDS: Immunosuppression is the hall
• MAbs as targeting agents mark of AIDS. This is caused by reduction in CD4 (cluster
determinant antigen 4) cells of T-lymphocytes. The human
Direct use of MAbs as therapeutic agents immunodeficiency virus (HIV) binds to specific receptors
Monoclonal antibodies can be directly used for enhancing on CD4 cells by using surface membrane glycoprotein
the immune function of the host. Direct use of MAbs (gp120).
causes minimal toxicity to the target tissues or the host. Genetic engineers have been successful to attach Fc
In destroying disease-causing organisms: MAbs portion of mouse monoclonal antibody to human CD4
promote efficient opsonization of pathogenic organisms molecule. This complex has high affinity to bind to
(by coating with antibody) and enhance phagocytosis. In membrane glycoprotein gp120 of virus infected cells. The
fact, MAbs were found to protect chimpanzees against Fc fragment induces cell-mediated destruction of HIV
certain viral (hepatitis B-virus) and bacterial (E. coli infected cells (Fig. 17.7).
Haemophilus influenza, Streptococcus sp and In the treatment of autoimmune diseases: Autoimmune
Pseudomonas sp) infections. diseases like rheumatoid arthritis and multiple sclerosis are
In the treatment of cancer: MAbs, against the antigens of great concern. Some success has been reported in the
on the surface of cancer cells, are useful for the treatment clinical trials of rheumatoid arthritis patients by using
of cancer. The antibodies bind to the cancer cells and MAbs directed against T-lymphocytes and B-
destroy them. This is brought out by antibody—dependent lymphocytes.
cell-mediated cytotoxicity, complement-mediated MAbs as Targeting Agents in Therapy
cytotoxicity and phagocytosis of cancer cells (coated with Toxins, drugs, radioisotopes etc., can be attached or
MAbs) by reticuloendothelial system. conjugated to the tissue-specific monoclonal antibodies

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and carried to target tissues for efficient action. This is mainly due to the fact that sufficient quantity of the drug
allows higher concentration of drugs to reach the desired does not reach the target tissue. This problem can be -
site with minimal toxicity. In this way, MAbs are used for solved by using tissue-specific MAbs. The drugs can be
the appropriate delivery of drugs or isotopes. coupled with MAb (directed against a cell surface antigen
MAbs in drug delivery: In general, the drugs are less of the cells, say a tumor) and specifically targeted to reach
effective in vivo (in the living body) when compared to in the site of action (Fig. 17.9A).
vitro (in laboratory when tested with cultured cells). This

In the treatment of certain diseases, a pro-drug (an inactive The some examples of enzymes that have been used in
form of the drug) can be used. This can be enzymatically ADEPT:
converted to active drug in the target tissues. For this i. Alkaline phosphatase for the conversion of phosphate
purpose, the enzyme (that converts pro-drug to drug) is pro-drugs.
coupled with MAb that is directed against a specific cell ii. Carboxy peptidase for converting inactive carboxyl pro-
surface antigen (Fig. 17.9A). This approach, referred to as drugs to active drugs.
antibody-directed enzyme pro-drug therapy (ADEPT), iii. Lactamase for hydrolyzing β-lactam ring containing
allows an effective delivery of the drug to the cells where antibiotics.
it is required.

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