BSE492A Presentation

Hybridoma Technology and

Applications of Monoclonal Antibody
in Medical Sciences
Group 1:-
Abhishek Yadav (170036)
Ankur Bajaj (170128)
Sahil Badal (170608)
Sanket Patil (170628)
Antibodies
What Are Antibodies?
Antibodies, also known as immunoglobulins,
are Y-shaped proteins that are produced by the
immune system to help stop intruders from
harming the body. When an intruder enters the
body, the immune system springs into action.
These invaders, which are called antigens, can
be viruses, bacteria, or other chemicals.

Function:- The antibodies act sort of like the

immune system's scouts. They find antigens,
stick to them, and identify for the immune
system the exact type of antigen so that it can
be destroyed. Each antibody is made for one
and only one antigen, and it's fitted with special
receptors that will only bind to that antigen.
Antibodies
1) Polyclonal antibodies (pAbs):- are
antibodies that are secreted by different B
cell lineages within the body (whereas
monoclonal antibodies come from a single
cell lineage). They are a collection of
immunoglobulin molecules that react
against a specific antigen, each identifying
a different epitope.
2) Monoclonal antibodies (mAb or
moAb):- are antibodies that are made by
identical immune cells that are all clones
of a unique parent cell. Monoclonal
antibodies can have monovalent affinity,
in that they bind to the same epitope (the
part of an antigen that is recognized by
the antibody).
Monoclonal Antibodies
Monoclonal antibodies (mAb or moAb) 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.

Synthesis of Monoclonal Antibodies:-

Monoclonal antibodies (MAbs) are produced by
introducing an antigen to a mouse and then fusing
polyclonal B cells from the mouse's spleen to myeloma cells.
The resulting hybridoma cells are cultured and continue to
produce antibodies to the antigen.

The major production of antibodies (Monoclonal) is carried by the HYBRIDOMA TECHNOLOGY.

Hybridoma Technology
Hybridoma technology was invented by Cesar Milstein and Georges
J. F. Kohler in 1975. For this invention, they received the Nobel Prize
for Medicine and Physiology with Niels Kaj Jerne in 1984.

Hybridoma: A hybrid cell used as the basis for the production of

antibodies in large amounts for diagnostic or therapeutic use.
Hybridomas are produced by injecting a specific antigen into a
mouse, collecting an antibody-producing cell from the mouse's
spleen, and fusing it with a tumor cell called a myeloma cell.

Myeloma Cells: Multiple myeloma cells are abnormal plasma cells (a

type of white blood cell) that build up in the bone marrow and form
tumors in many bones of the body. Normal plasma cells make
antibodies to help the body fight infection and disease.
Applications of Monoclonal Antibody

The four major types of applications are:

(1) Diagnostic Applications : Monoclonal antibodies have revolutionized the laboratory

diagnosis of various diseases.

(2) Therapeutic Applications : Over 40 MAbs are approved worldwide which have
helped improve the treatment of over 50 major diseases.

(3) Protein Purification : Monoclonal antibodies can be produced for any protein and
the so produced MAbs can be conveniently used for the purification of the protein
against which it was raised.

(4) Miscellaneous Applications

 Diagnostic Applications
MAbs in Biochemical Analysis
Diagnostic tests : These are based on the use of MAbs as reagents in radioimmunoassay (RIA)
and enzyme-linked immunosorbent assays (ELISA) in the laboratory. These assays measure
the circulating concentrations of hormones and several other tissue and cell products.

Cancer : Cancerous cells have antigens. Monoclonal antibodies can be designed to bind specifically
with these antigens. When injected into a person's body, the monoclonal antibodies will bind with these
cancer cells and clump them together. This makes it easier to identify a cancerous tumour, which can
then be treated or removed.

Pregnancy Test Kits : MAbs present at the end of kit binds with a hormone called HCG which
is found only in the urine of pregnant women. This will cause a change in colour or pattern
which will indicate pregnancy.

Infectious diseases : By detecting the circulatory levels of antigens specific to the infectious
agent e.g., antigens of Neisseria gonorrhoeae & herpes simplex virus for the diagnosis of STDs.
 Diagnostic Applications
MAbs in 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 MAbs are iodine-131 and technetium-99. The
MAb tagged with radioisotope are injected intravenously into the patients.

These MAbs localize at specific sites which can be detected by imaging the radioactivity.
Immunoscintigraphy is a better diagnostic tool than the other imaging techniques as it can
differentiate between cancerous and non-cancerous growth, since radiolabeled-MAbs are
tumor specific.

Monoclonal antibodies are successfully used in the diagnostic imaging of cardiovascular

diseases such as Myocardial Infarction, Deep vein thrombosis (DVT), Atherosclerosis, etc.
Therapeutic Applications
(A) MAbs as Direct Therapeutic Agents: Monoclonal antibodies can be directly used for enhancing
the immune function of the host. Direct use of MAbs causes minimal toxicity to the target tissues or
the host.

It is used in destroying disease causing organisms. MAbs promote efficient opsonization of

pathogenic organisms (by coating with antibody) and enhance phagocytosis.

It is used in the treatment of AIDS as immunosuppression is the hallmark of AIDS. This is caused by
reduction in CD4 (cluster determinant antigen 4) cells of T-lymphocytes.
Therapeutic Applications
(B) MAbs as Targeting Agents in Therapy:

MAbs in drug delivery:

In general, the drugs are less effective in vivo (in the living body) when compared to in vitro
(in laboratory when tested with cultured cells). This is mainly due to the fact that sufficient
quantity of the drug does not reach the target tissue. This problem can be solved by using
tissue-specific MAbs. The drugs can be coupled with MAb (directed against a cell surface
antigen of the cells, say a tumor) and specifically targeted to reach the site of action.

MAbs in the dissolution of blood clots:

The blockage of arteries occurs due to inadequate dissolution

of blood clots.

Tissue plasminogen activator (tPA) can be used as a therapeutic

agent to remove the blood clots.

Therapeutic Applications
A TNF inhibitor is a pharmaceutical drug that suppresses the physiologic response to tumor
necrosis factor (TNF). TNF is involved in autoimmune and immune-mediated disorders such
as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis,
hidradenitis suppurativa and refractory asthma, so TNF inhibitors may be used in their
treatment.

Inhibition of TNF effects can be achieved with a monoclonal antibody such

as infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),
and golimumab (Simponi), or with a circulating receptor fusion protein such
as etanercept (Enbrel).

Therapy which uses anti-TNF are found to be more effective at restoring quality of life to
sufferers of rheumatoid arthritis than using either drug alone.

Anti-TNF therapy has shown only modest effects in cancer therapy. Treatment of renal cell
carcinoma with infliximab resulted in prolonged disease stabilization in certain patients.
 Protein Purification:
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). The immobilized
MAbs in this manner are very useful for the
purification of proteins by immunoaffinity
method.

In protein purification monoclonal

antibodies are used to purify proteins and
are especially useful for the purification of
recombinant proteins (immunoaffinity
chromatography).
Miscellaneous Applications:
(A) Catalytic MAbs (ABZYMES):
Certain similarities between enzyme-substrate interaction and antibody-antigen
interaction have tempted researchers to explore the possibility of using antibodies in
catalysis. The antibody enzymes, appropriately regarded as abzymes, are the catalytic
antibodies. There is a difference in the antibody recognition of an antigen and enzyme
recognition of a substrate. While the antibodies recognize in ground state, the
enzymes recognize in a transition state (associated with a conformational change of
protein). Applications (cutting of peptides and DNAs, dissolution of blood clots, killing
of viruses etc.)

(B) Autoantibody Fingerprinting:

The occurrence of autoantibodies and their involvement in certain diseases is well
known (e.g. rheumatic arthritis).

Monoclonal antibodies produced against IS-autoantibodies can be used for their

detection, and identification of individuals. This technique referred to as autoantibody
fingerprinting, is particularly useful for the detection of criminals.
Advantages & Disadvantages
● Hybridoma serves as an immortal
source of monoclonal antibody.
● Highly reproducible and scalable,
unlimited production source.
● Speed and sensitivity and specificity
of assays.
● Antigen or immunogen need not be
pure.
● Once stabilized, hybridomas offer
unlimited quantities of cost-
effective, consistent, homogenized
antibody production.
● It is perfect for research, assay
development and downstream
research, diagnostic and therapeutic
uses.
● It’s a time consuming project, it
takes between 6 to 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.
● System is only well developed for
mouse and rat and not for other
animals.
● 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
References

1. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883214/
2. http://www.biologydiscussion.com/biotechnology/applications-of-monoclonal-
antibodies-4-applications/10045
3. https://www.ncbi.nlm.nih.gov/pubmed/29060935
4. https://alzres.biomedcentral.com/articles/10.1186/alzrt220
5. https://alzheimersanddementiajournal.com/article/S1552-5260(18)33270-9/abstract
6. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2883214/