PRODUCTION OF MONOCLONAL AND POLYCLONAL ANTIBODIES PRESENTED BY SANJAY D.pptx

PRODUCTION OF
MONOCLONAL AND
POLYCLONAL ANTIBODIES
PRESENTED BY
SANJAY .D
22MP0215
MPHE-RMRIMS
2022-2024

INTRODUCTION
MONOCLONAL ANTIBODY
Antibodies which are derived from a single clone of plasma cells, all
antibodies having the same antigen specificity, i.e. produced against a single
epitope of an antigen is called monoclonal antibody
POLYCONAL ANTIBODY
Antibodies which are derived from a multiple clone of plasma cells, all
antibodies having the different antigen specificity, i.e. produced against a
various epitope of an antigen is called polyclonal antibody

Monoclonal antibodies have revolutionized the field of medicine, as they
can be designed to target specific cells or proteins in the body. This
specificity makes them incredibly useful for diagnosing and treating
diseases such as cancer, autoimmune disorders, and infectious diseases.
In addition to their specificity, monoclonal antibodies also have low
toxicity, making them ideal for use in humans. They can be used to deliver
drugs directly to cancer cells they are also called as Magic bullets.

HOW ARE MONOCLONALANTIBODIES PRODUCED?
• Monoclonal antibodies are produced by hybridoma technology.
• This technology was discovered in 1975 by George Kohler of west
Germany and Cesal Milstein of Argentina (Nobel prize, 1984).
Kohler experiment
• Kohler (1974) successfully produced hybridoma by fusing a P3
myeloma cell and a lymphocyte (from the spleen of a mouse)
immunized against the sheep red blood cells.
• These hybridomas retained the property of immortality of the
myeloma cells as well as that of secreting an antibody specific for
an sheep red blood cells antigen.

MONOCLONALANTIBODY PRODUCTION PROCESS
1. Purity form of immunogen
2. Choice of animals
3. Immunization
4. Separation of lymphocytes
5. Selection of myeloma cell line
6. Immortalization by fusion with tumor cells
7. Selection of hybridoma cells
8. Isolation of a monoclonal antibody producing hybridoma cell
9. Purification of desired antibodies

Purity and Form of Immunogen
 The purity of the antigen to be used as immunogen is crucial for generation
of monoclonal antibodies.
 Molecules of low molecular weight (1000 Daltons) are poor immunogens
and have to be coupled to larger immunogenic molecules.
 Aggregated and particulate antigens elicit stronger responses.
 Adjuvants help to achieve stronger responses. The most common carriers are
albumins, keyhole limpet hemocyanin, fowl gamma globulin and
synthetic polypeptides.
Selection of Myeloma Cell Line
 The myeloma cell line used must itself not be capable of synthesizing
antibody otherwise hybridoma cell line will produce a mixture of antibodies.
 HPRT-negative myeloma cell line should be selected

Immunization procedures
 Generally the antigen is injected subcutaneously or into the peritoneal cavity of
the animal along with an adjuvant to stimulate the immune system.
Sacrifice of animal and separation of lymphocytes
 Three days after the final dose of antigen has been given intravenously to
immunize the animal, the latter is killed.
 The spleen of the killed animal is removed aseptically and gently disrupted to
release the spleen fluid containing lymphocytes and red blood cells.
 The lymphocytes are separated from the spleen fluid (and red blood cells) by
density gradient centrifugation, and washed.

Immortalization by Fusion with Tumor Cells
 The cell fusion process between myeloma cells and spleen lymphocytes is
usually induced in the presence of polyethylene glycol (PEG-1500)
 The first fusion experiments were performed with Sendai virus as a
fusogenic agent.
 Both regents aggregate the cells which eventually lead to fusion of cells.
Selection of Hybridoma Cells
 The basis of hybridoma technology was the development of suitable
myeloma mutant cell lines that are non-antibody secreting and deficient in
the enzyme Hypoxanthine Guanine Phosphoribosyl Transferase
(HGPRT) and are not able to grow in toxic tissue culture.
 The enzyme HGPRT is essential for DNA synthesis (by purine salvage
pathway) after fusion of these myeloma cells with lymphocytes.

 The HAT (Hypoxanthine-Aminopterin-Thymidine) Medium is used for metabolic
selection of fused cells.
 The medium containing HAT will eliminate the unfused myeloma cells and allow the
growth of hybridomas which harbour the gene(s) for HGRPT from the parent lymphocytes.
Principle of HAT Medium
 The HGPRT myeloma cells die off as aminopterin blocks the main pathway of DNA
synthesis, i.e., de novo pathway of DNA synthesis by inhibiting the activity of
dihydrofolate reductase. In hybrid cells, spleen cells contribute the functional HGPRT
enzyme necessary to overcome the aminopterin block.
 Unfused Lymphocyte cells are eliminated due to poor growth in vitro.

Isolation of a Monoclonal antibody Producing Hybridoma cell
 If all the hybridoma cells that have been selected using HAT-medium, a polyclonal antibody mixture
would be obtained.
 Consequently, a single antibody (monoclonal antibody) producing hybridoma cells need to be isolated
and grown individually.
 This is done by diluting a suspension of hybridoma cells to such an extent that individual aliquots
contain, on an average, only one cell.
 Such cells are transferred to separate fresh media for growth.
 Each mass of hybridoma cells (clone) produced from a single parent hybridoma cell is now examined
to determine whether it produces the desired monoclonal antibody thought the affinity
chromatography or precipitation techniques.

Isolation of a
monoclonal antibody
producing hybridoma
cell
Selection of
hybridoma cells
Immortalizati
on by fusion
with tumor
cells
Sacrifice of
animal and
separation of
lymphocytes
Immunization
MONOCLONAL
ANTIBODY
PRODUCTION
PROCESS

APPLICATION
Therapeutic Applications. Monoclonal antibodies are used as therapeutic agents
to treat a wide range of diseases, including: Cancer Treatment, Autoimmune
Disorders, Infectious Diseases and Transplant Medicine.
Diagnosis and Monitoring. Monoclonal antibodies are used in diagnostic tests to
identify specific antigens or markers associated with diseases. For instance,
pregnancy tests use mAbs to detect human chorionic gonadotropin (hCG) in urine.
Research Tools. mAbs are essential tools in biomedical research. They help
scientists study various proteins, receptors, and cells by specifically targeting them.
This aids in understanding their functions, interactions, and roles in diseases

Immunotherapy. Apart from cancer treatment, mAbs are used in immunotherapy to stimulate or enhance
the body's immune response against diseases. They can activate immune cells, like T cells, to target
infected or cancerous cells more effectively.
Drug Delivery. mAbs can serve as carriers for drugs or imaging agents, specifically delivering them to
targeted cells or tissues, reducing side effects, and improving treatment efficacy.
Imaging and Diagnostics: Monoclonal antibodies can be labeled with radioactive isotopes or other
imaging agents to detect specific cells or tissues in imaging techniques such as positron emission
tomography (PET) and single-photon emission computed tomography (SPECT).
Biotechnology and Industrial Applications. mAbs are utilized in various biotechnological processes,
including protein purification, as well as in the development of biosensors and diagnostic devices.
Passive Immunization: In certain situations, mAbs can be administered passively to provide immediate,
short-term protection against infections or toxins. This approach is used in cases of exposure to certain
diseases or as a preventive measure.

POLYCLONALANTIBODIES
Antibodies which are derived from a multiple clone of plasma cells, all
antibodies having the different antigen specificity, i.e. produced against a
various epitope of an antigen is called polyclonal antibody
Advantages of polyclonal antibodies.
Easy, cheap and quick preparation.
PAbs are heterogeneous, bind to a wide range of antigen epitopes.
PAbs can be made in large quantities.

THE GENERAL PROCEDURE TO PRODUCE POLYCLONALANTIBODIES
Selection of Purity form of immunogen
Animal selection
Immunization
Isolation of Polyclonal antibodies

Procedure To Produce Polyclonal Antibodies
A host Sp. is chosen for the production
of Polyclonal antibodies considering
three main aspects:-
1. Quantity of antibodies required.
2. Antigen source.
3. Final application of the
polyclonal antibodies.
1. Molecular Weight Of
Immunogen Should
Be 1000 kDa. Or
2. Have To Be Coupled
To Larger
Immunogenic
Molecules.
3. Adjuvants help to
achieve stronger
responses.
Antibodies purification
Affinity chromatography is used for
Polyclonal antibodies purification
There are two processes
 Antibody / Immunoglobulin
specific.
 Antigen specific purification.
Validation of
antibodies/ Quality
control.
1. concentration is
measured taking
absorbance at
280mm.
2. Purity is checked
by SDS-PAGE
3. Titer is
estimated by main
ly ELISA

PURIFICATION OF POLYCLONALANTIBODIES
Affinity chromatography

APPLICATION
Immunoassays. Polyclonal antibodies are widely used in various immunoassays, such as enzyme-
linked immunosorbent assays (ELISA), Western blotting, and immunohistochemistry. They can be
used to detect and quantify the presence of specific antigens in biological samples.
Diagnostic testing. Polyclonal antibodies are employed in clinical diagnostic tests for detecting
diseases and infections. For example, they are used in rapid diagnostic tests for detecting viral or
bacterial infections like HIV, hepatitis, and influenza.
Therapeutic Applications. Some polyclonal antibodies have therapeutic uses. They can be
administered to patients to boost the immune response against certain pathogens or toxins.
Additionally, polyclonal antibodies may be used to neutralize toxins or pathogens in cases where
specific monoclonal antibodies are not available.

In Vivo Research. Polyclonal antibodies are utilized in animal studies and
research to investigate specific biological processes, identify protein
expression patterns, and examine cellular localization of proteins.
Flow Cytometry. Polyclonal antibodies labeled with fluorescent tags are
employed in flow cytometry, allowing researchers to analyze and sort cells
based on specific surface markers and intracellular antigens.
Immunoprecipitation. Polyclonal antibodies can be used to isolate and pull
down specific proteins or protein complexes from a mixture of proteins using
immunoprecipitation techniques.

Chromatin Immunoprecipitation (ChIP). In ChIP assays, polyclonal
antibodies are used to investigate the interactions between proteins and DNA
by isolating specific DNA sequences bound to a particular protein of interest.
Neutralization Assays. Polyclonal antibodies can be employed in
neutralization assays to determine their ability to block the activity of
pathogens, toxins, or other antigens.
Purification of Proteins. Polyclonal antibodies can be used to purify proteins
of interest through affinity chromatography, where the target protein is
specifically captured by the antibody.

PRODUCTION OF MONOCLONAL AND POLYCLONAL ANTIBODIES PRESENTED BY SANJAY D.pptx

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