Monoclonal and Polyclonal antibodies

Monoclonal antibodies (mAbs) are a type of antibody produced by identical immune cells that
are all clones of a single parent cell. These antibodies are highly specific to a single antigen and
have a wide range of therapeutic, diagnostic, and research applications.
The production of monoclonal antibodies using hybridoma technology involves the fusion of an
antibody-producing B cell with an immortalized myeloma cell to create a hybrid cell called a
hybridoma. The hybridoma can then be used to produce large quantities of identical monoclonal
antibodies. The process can be broken down into several steps:

Hybridoma technology steps:

✓ Immunization: The first step in producing monoclonal antibodies is to immunize an
animal with the desired antigen. The animal's immune system will produce B cells that
generate antibodies against the antigen. In order to isolate B-lymphocyte producing certain
antibodies, rabbit or lab rat is immunized through repeated injection of specific antigen
(sheep RBCs)
A sample of B-cells is extracted from spleen of rabbit or rat
✓ Cell fusion: B cells are then isolated from the animal's spleen and fused with myeloma
cells in the presence of a fusogen or the fusion is done by using Polyethylene glycol (PEG)
or by electrophoration or by using phages. This creates hybridomas that have the ability to
produce antibodies against the antigen and can proliferate indefinitely.
✓ Selection: The hybridomas are then cultured in a selective medium that allows only
hybridomas to survive, and not the parent cells. The selective medium usually contains
hypoxanthine, aminopterin, and thymidine (HAT). HAT medium selects for
hybridomas because the myeloma cells are unable to synthesize nucleotides without the
hypoxanthine and thymidine, while the B cells cannot survive without the aminopterin.
The B-lymphocytes contains HPRT1 gene which codes for enzyme Hypoxanthine-guanine
phosphoribosyltransferase (HGPRT). The enzyme HGPRT involved in synthesis of
nucleotides from Hypoxanthine present in culture medium. Therefore B- cells can grow in
medium containing Hypoxanthine aminopterin thymine (HAT media).
But myeloma cell lack HPRT1 gene so, it does not produce HGPTR enzyme and it does
not grow in HAT medium.
 The myeloma cell fused with another myeloma cell or those do not fused at all die in HAT
medium since they do not utilize Hypoxanthine.
Similarly, B- cell that fuse with another B- cell or those do not fuse at all die eventually
because they do not have capacity to divide indefinitely,
So, only hybridoma cell ie. fused cell between myeloma and B-cell can survive and divide
in HAT medium.
Screening is done to select hybridoma cells which are the desired cell for monoclonal
antibodies production.
✓ Culture of Hybridoma cell:
The selected hybridoma cells are cultured in suitable culture medium, often supplemented
with insulin, transferon, ethanol, amine and other additional hormones.
Some commonly used culture media for hybridoma cell for production of monoclonal
antibodies are:
DMEM (Dulbecco’s modified eagle medium)
IMDM (Iscove’s Modified Dulbecco’s Medium)
Ham’s F12
RPMI 1640 medium (Roswell Park Memorial Institute 1640 medium)
✓ Inoculation of hybridoma cell into suitable host
These hybridoma cells are then injected into lab animal so that they starts to produce
monoclonal antibodies.
These hybridoma cells may be frozen and store for future use.
✓ Step VI: extraction and purification of Monoclonal antibodies:
Monoclonal antibodies from host animal is extracted and purified by one of the following
methods;
Ion exchange chromatography
Antigen affinity chromatography
Radial immunoassay
Immune precipitation
Applications of monoclonal antibodies
Monoclonal antibodies (mAbs) are laboratory-produced antibodies that mimic the immune
system's ability to fight off harmful pathogens, such as viruses or cancer cells. The unique
specificity and high affinity of mAbs make them a powerful tool in various fields of medicine and
research. Here are some applications of monoclonal antibodies:
✓ Therapeutic uses: mAbs have been used to treat a range of diseases, including cancer,
autoimmune disorders, infectious diseases, and cardiovascular diseases. For example,
trastuzumab is a mAb used to treat breast cancer, and rituximab is a mAb used to treat
lymphoma.
✓ Diagnostic tests: mAbs can be used to detect the presence of specific antigens, such as
viral or bacterial proteins. This allows for the development of highly sensitive and specific
diagnostic tests, such as enzyme-linked immunosorbent assays (ELISAs) and lateral flow
assays.
✓ Research tools: mAbs are valuable tools for studying the structure and function of
proteins. They can be used to isolate and purify proteins, as well as to detect and measure
their expression levels.
✓ Imaging agents: mAbs can be labeled with radioactive isotopes or fluorescent dyes, which
allow them to be used as imaging agents in medical diagnostics. This is known as molecular
imaging, which provides highly specific and sensitive information about the location and
extent of disease.
✓ Theranostics: mAbs can be used both as diagnostics and therapeutics. For example, some
mAbs have been labeled with radioactive isotopes to enable their use in imaging and
subsequent delivery of targeted radiation therapy.
✓ Biodefense: mAbs have been used as potential countermeasures against bioterrorism
agents, such as anthrax, botulinum toxin, and Ebola virus.
✓ Agriculture: mAbs have been used in agricultural applications, such as in the detection
and control of plant diseases.
✓ Environmental monitoring: mAbs have been used for environmental monitoring, such as
in the detection of contaminants in water or soil.
✓ Food safety: mAbs have been used to detect and quantify food allergens, such as peanuts
and gluten, in food products
 ✓ Industrial applications: mAbs have been used in various industrial applications, such as
in the production of biofuels and the removal of impurities from pharmaceuticals.

Polyclonal antibodies
They are a group of antibodies that are produced by different B cells in response to an antigen.
Each B cell produces a unique antibody with a slightly different amino acid sequence, resulting in
a mixture of antibodies that recognize different epitopes of the same antigen.
The production of polyclonal antibodies typically involves immunizing an animal, such as a rabbit
or mouse, with the antigen of interest. The animal's immune system will recognize the antigen as
foreign and respond by producing a wide range of antibodies. After a period of time, blood is
collected from the animal and the serum, which contains the polyclonal antibodies, is isolated.

The following steps can be followed to produce polyclonal antibodies:

✓ Antigen selection: The antigen of interest is selected based on its ability to stimulate an
immune response and its specificity for the target protein.
✓ Animal selection: Animals, such as rabbits or mice, are selected based on their ability to
produce high levels of polyclonal antibodies.
✓ Immunization: The antigen is injected into the animal, usually multiple times, to stimulate
an immune response. The animal's immune system will produce a wide range of antibodies
against the antigen.
✓ Serum collection: Blood is collected from the animal and the serum, which contains the
polyclonal antibodies, is isolated. The serum can be stored for long periods of time and
used for various applications.
✓ Antibody purification: The polyclonal antibodies can be purified from the serum using
various techniques, such as protein A/G affinity chromatography or ammonium sulfate
precipitation.
✓ Antibody characterization: The purified antibodies can be characterized for their
specificity and affinity to the antigen using techniques such as ELISA, Western blotting,
or immunohistochemistry.
Polyclonal antibodies have several advantages over monoclonal antibodies, such as their ability to
recognize multiple epitopes and their relatively low cost of production. They are commonly used
in research, diagnostic assays, and therapeutics.

Polyclonal Antibody Applications.

Polyclonal antibodies are a mixture of antibodies produced by different B cells in response to an
antigen. Here are some applications of polyclonal antibodies:
Diagnostic Tests: Polyclonal antibodies can be used in diagnostic tests to detect the presence of
specific antigens in patient samples. For example, polyclonal antibodies are commonly used in
ELISA (enzyme-linked immunosorbent assay) tests to detect the presence of infectious agents or
tumor markers in blood or other bodily fluids.
Therapeutics: Polyclonal antibodies can be used as therapeutics to treat a variety of diseases. For
example, polyclonal antibodies can be used to treat snake bites, rabies, and some viral infections.
Research: Polyclonal antibodies are widely used in research to study protein expression and
function. They can be used to identify and isolate proteins of interest from complex biological
samples, such as tissue extracts or cell lysates.
Immunoassays: Polyclonal antibodies can be used in immunoassays to measure the concentration
of a specific antigen. These assays can be used in medical research and diagnosis, as well as in the
food and environmental industries to detect contaminants or pollutants.
Immunohistochemistry: Polyclonal antibodies can be used in immunohistochemistry to detect
specific proteins in tissue samples. This technique is commonly used in pathology to diagnose
diseases, such as cancer.

Differences between monoclonal antibodies and polyclonal antibodies

Monoclonal antibodies are antibodies Polyclonal antibodies, on the other hand, are
that are derived from a single type of antibodies that are derived from multiple types
immune cell, known as a B cell clone. of B cells, each of which recognizes a different
antigen.
Monoclonal antibodies are produced In contrast, polyclonal antibodies are produced
by isolating a single B cell clone and by injecting an animal (such as a rabbit or goat)
then generating large quantities of with an antigen, and then harvesting the
identical antibodies in the laboratory. antibodies from the animal's serum.
Monoclonal antibodies are highly Polyclonal antibodies, in contrast, are less
specific and recognize only one specific and can recognize multiple antigens.
particular antigen or epitope.
Monoclonal antibodies are Polyclonal antibodies, in contrast, are
homogeneous, meaning that they are heterogeneous, meaning that they consist of a
all identical in structure and function. mixture of different antibodies that vary in their
structure and function.
Monoclonal antibodies are highly Polyclonal antibodies, in contrast, are less
reproducible, meaning that they can reproducible, and the quality of the antibodies
be produced in large quantities with can vary between batches.
consistent quality.
Monoclonal antibodies are typically They are less expensive
more expensive to produce due to the
complex production process involved.
Monoclonal antibodies are widely Polyclonal antibodies are also used in similar
used in various applications, such as applications, but are more commonly used in
diagnostic assays, therapeutic drugs, research applications.
and research tools.
Monoclonal have longer lifespan Polyclonal antibodies have a shorter lifespan as
they are eliminated from the body more quickly
compared to monoclonal antibodies.
Monoclonal are less likely to be Polyclonal antibodies are more likely to have
contaminated non-specific binding and be contaminated with
other antibodies and substances compared to
monoclonal antibodies.
Monoclonal antibodies can be The production of polyclonal antibodies
produced without animal use using involves the use of animals, which raises ethical
hybridoma technology or may use concerns
animals