MONOCLONAL ANTIBODY

What are antibodies?

Antibody:An antibody is a protein produced by the immune system that is capable of binding
with high specificity to an antigen.These antigens are typically other proteins, but may be
carbohydrates, small molecules or even nucleotides.

 The specific region on an antigen that an antibody recognizes and binds to is called the
epitope, or antigenic determinant.
 Serum containing antigen-specific antibodies is called antiserum. There are five classes
of immunoglobulins including IgM, IgG, IgA, IgD, and IgE

Monoclonal Antibody:A monoclonal antibody refers to an antibody normally recognizing only

a single antigen (e.g. a single protein) and within which only a single common epitope is
recognized
Polyclonal antibody: A polyclonal antibody refers to an antibody normally recognizing only a
single antigen but within which a number of different epitopes are recognized

What is the difference between polyclonal and monoclonal antibodies?

Antibodies, also known as immunoglobulins, are secreted by B cells to neutralize antigens such
as bacteria and viruses. The classical representation of an antibody is a Y-shaped molecule
composed of four polypeptides-two heavy chains and two light chains. Each tip of the “Y”
contains a paratope (a structure analogous to a lock) that is specific for one particular epitope
(similarly analogous to a key) on an antigen, allowing these two structures to bind together with
precision. The ability of binding to an antigen has led to their ubiquitous use in a variety of life
science and medical science. These antibodies can be classified into two primary types
(monoclonal and polyclonal) by the means in which they are created from lymphocytes. Each of
them has important role in the immune system, diagnostic exams, and treatment.
Polyclonal vs Monoclonal Antibodies: differences in production

Fig: The process to generate the polyclonal antibody

Polyclonal antibodies (pAbs) are mixture of heterogeneous which are usually produced by
different B cell clones in the body. They can recognize and bind to many different epitopes of a
single antigen.

Polyclonal antibodies are produced by injecting an immunogen into an animal. After being
injected with a specific antigen to elicit a primary immune response, the animal is given a
secondary even tertiary immunization to produce higher titers of antibodies against the particular
antigen. After immunization, polyclonal antibodies can be obtained straight from the serum
(blood which has had clotting proteins and red blood cells removed) or purified to obtain a
solution which is free from other serum proteins.

Monoclonal antibodies (mAbs) are generated by identical B cells which are clones from a single
parent cell. This means that the monoclonal antibodies have monovalent affinity and only
recognize the same epitope of an antigen.

Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are
produced ex vivo using tissue-culture techniques. The process begins with an injection of the
desired antigen into an animal, often a mouse, multiple times. Once the animal develops an
immune response, the B-lymphocytes are isolated from the animal’s spleen and fused with a
myeloma cell line, creating immortalized B cell-myeloma hybridomas. The hybridomas, which
are able to grow continuously in culture while producing antibodies, are then screened for
desired mAb.
History of mAb development

 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
 1990 Milstein produced the first monoclonal antibodies.
 Paul Ehrlich at the beginning of the 20th century theorized that a cell under threat grew
additional side-chains to bind the toxin, and that these additional side chains broke off to
become the antibodies that are circulated through the body. It was these antibodies that
Ehrlich first described as "magic bullets" in search of toxins.

Characters of Monoclonal Antibodies

 Monoclonal antibodies (mAb) are a 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 a one Bcell lymphocyte with a myeloma
cell.
 Some myeloma cells synthesize single mAb antibodies Naturally

Monoclonal Antibody Production Method

 Monoclonal Antibody Production technology was developed in 1975. Since its

development it has been very important in the modern medical science with the
diagnosis, therapy, research and even basic science today. It is still largely dependent
upon animal testing however. Because it requires immunization of mice in order for them
to create the antibodies to be grown.
 Monoclonal Antibody Production or mAb is produced by cell lines or clones obtained
from the immunized animals with the substance to be studied. Cell lines are produced by
fusing B cells from the immunized animal with myeloma cells. To produce the desired
mAb, the cells must be grown in either of two ways: by injection into the peritoneal
cavity of a suitably prepared mouse (the in vivo, or mouse ascites, method) or by in vitro
tissue culture.
 The vitro tissue culture is the method used when the cells are places in culture outside the
mouse's body in a flask.

 The previous picture is showing a mouse being immunized against a target cell “X”. This
will allow the mouse to produce antibodies for that will target against the “X” antigen.
 Once the mouse has formed antibodies to the “X” antigen the cells are then isolated in the
mouse’s spleen. Monoclonal antibodies are produced by fusing single antibody-forming
cells to tumor cells grown in culture. The resulting cell is called a hybridoma. Hybridoma
cells are continuously growing cell line generated by the fusion of a myeloma cell and a
normal cell that are capable of producing antibodies.
 Each hybridoma will produce relatively large quantities of identical antibody molecules.
Because the hybridoma is multiplying in culture, it is possible to produce a population of
cells, each is producing identical antibody molecules. These antibodies are called
"monoclonal antibodies" because they are produced by the identical offspring of a single,
cloned antibody producing cell.
Practical steps in monoclonal antibody production:

 Immunize animal
 Isolate spleen cells (containing antibody-producing B cells)
 Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol)
 Allow unfused B cells to die
 Add aminopterin to culture to kill unfused myeloma cells
 Clone remaining cells (place 1 cell/well and allow each cell to grow into a clone of cells)
 Screen supernatant of each clone for presence of the desired antibody
 Grow the chosen clone of cells in tissue culture indefinitely.
 Harvest antibody from the culture supernatant.
 (If you’re a biotech company) charge about $1,000-$2,000 per mg.
Why this method is used!!
Diagnostic applications
 A monoclonal antibody can be used to detect pregnancy only 14 days after conception.
Other monoclonal antibodies allow rapid diagnosis of hepatitis, influenza, herpes,
streptococcal, and Chlamydia infections.
 They can be used to detect for the presence and quantity of this substance, for instance in
a Western blot test (to detect a substance in a solution) or an immunofluorescence test.
 Monoclonal antibodies can also be used to purify a substance with techniques called
immunoprecipitation and affinity chromatography.

1. Detects protein of interest by immune florescence or blotting.

2. Cardiovascular diseases.
3. Deep vein thrombosis.
4. Location of primary and secondary metastatic tumors.
5. Immunosuppressive therapy.
6. Pregnancy testing kits.
Therapeutic applications
 Radioisotope immune conjugates.
 Toxin and drug immune conjugates.
 Immunoliposome based kits.
 In cancer.
 Inflammatory disease

Possible side effects of monoclonal antibodies

 Monoclonal antibodies are given intravenously (injected into a vein). Compared with the
side effects of chemotherapy, the side effects of naked mAbs are like an allergic reaction.
These are more common while the drug is first being given.
 Some mAbs can also have other side effects that are related to the antigens they target.
For example, bevacizumab (Avastin®), an mAb that targets tumor blood vessel growth,
can cause side effects such as high blood pressure,bleeding, poor wound healing, blood
clots, and kidney damage. Fever  Chills  Weakness  Headache  Nausea 
Vomiting  Diarrhe  Low blood pressure Rashes