Monoclonal antibodies (MAbs) are antibodies that are directed against a single antigen. They can be produced through hybridoma technology which involves fusing antibody-producing B cells with myeloma cells to form a hybrid cell line. This document outlines the process for producing MAbs including immunizing an animal, fusing B cells with myeloma cells, selecting antibody-producing hybridomas, cloning and mass producing the antibodies. MAbs have various diagnostic and therapeutic applications for diseases.

1. MONOCLONAL ANTIBODY
(MAb)
SUBJECT : IMMUNOLOGY
BY-
S. DASH

2. OVERVIEW :
INTRODUCTION
PRINCIPLE FOR
CREATION OF
HYBRIDOMA CELLS
STEPS INVOLVED IN
PRODUCTION OF
Mabs.
SECOND GENERATION
MAbs.
EXAMPLES
APPLICATIONS
DISADVANTAGES
ADVANTAGES
REFERENCES

3. INTRODUCTION :
• Monoclonal antibody is a single type of antibody that is directed against a specific
antigenic determinant(epitope).
• Natural source : Found in patients suffering from multiple myeloma.
• In 1975, George Kohler and Cesar Milstein achieved a large scale production of
MAbs. The pair won the 1984 Nobel Prize in Medicine and Physiology for this
discovery.
• The production of monoclonal antibodies by the hybrid cells is referred to as
Hybridoma Technology.

4. PRINCIPLE FOR CREATION OF HYBRIDOMA CELLS
• The mutated cells deficient in the enzyme HGPRT are grown in a medium
containing hypoxanthine, aminopterin and thymidine (HAT medium),
they cannot survive due to inhibition of de-novo synthesis of purine
nucleotides. Thus cells lacking the HGPRT enzyme, grown in HAT
medium die.
• The hybridoma cells possess the ability of myeloma cells to grow in vitro
with a functional HGPRT gene obtained from lymphocytes (with which
myeloma cells are fused). Thus, only the hybridoma cells can proliferate in
HAT medium and this procedure is used for selection.

6.  STEPS OF PRODUCTION OF
MONOCLONAL ANTIBODIES
1. Immunization
2. Cell fusion
3. Selection of hybridomas
4. Screening of products
5. Cloning and production
6. Characterization and storage.

7.  IMMUNIZATION
• The animal is immunized using an appropriate antigen. The antigen along with an
adjuvant (mainly Freund’s complete) is injected subcutaneously.
• The injections are administered at multiple sites to increase the production of B-
lymphocytes, which are responding to the antigen.
• Three days prior to the sacrifice of the animal, a final dose of antigen is given
intravenously. This allows the synthesis of antibodies to it’s maximum level.
• The concentration of the desired antibodies is assayed in the serum of the animal at
frequent intervals during the course of immunization.
• When the serum concentration of the antibodies is optimal, the animal is sacrificed.
The spleen is aseptically removed and disrupted by mechanical or enzymatic
methods to release the cells.
• The lymphocytes of the spleen are separated from the rest of the cells by density
gradient centrifugation.

8.  CELL FUSION
• The thoroughly washed lymphocytes are mixed with HGPRT defective
myeloma cells.
• The mixture of cells is exposed to polyethylene glycol (PEG) for a short
period of time.
• PEG is removed by washing and the cells are kept in a fresh medium.
• These cells are composed of a mixture of hybridomas (fused cells), free
myeloma cells and free lymphocytes.

9.  SELECTION OF HYBRIDOMAS
• Within the 7-10 days of culture, only the hybridoma cells are able to grow
whereas the rest starts disappearing.
• 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.

10.  SCREENING OF PRODUCTS
• The hybridomas must be screened for the secretion of the antibody of the
desired 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 and
the unbound antibody and other components can be washed off.
• Thus, the hybridoma cells producing the desired antibody can be identified
by screening.

11.  CLONING AND PROPAGATION
• The single hybrid cells producing the desired antibody are isolated and cloned.
• Two techniques are commonly employed for cloning hybrid cells-
i. Limiting dilution method
ii. Soft agar method
 LIMITING DILUTION METHOD
• The suspension of hybridoma cells are serially diluted and the aliquots of each dilution are
put into microculture wells.
• The dilutions are so made that each aliquot in a well contains only a single hybrid cell.
• This ensures that the antibody produced is monoclonal.
 SOFT AGAR METHOD
• The hybridoma cells are cultured in soft agar. It is possible to simultaneously grow many cells
in semisolid medium to form colonies. These colonies will be monoclonal in nature.

12.  CHARACTERIZATION AND STORAGE
• The monoclonal antibody has to be subjected to biochemical and biophysical
characterization for the desired specificity.
• It is also important to elucidate the Mab for the immunoglobulin class or sub-
class, the epitope for which it is specific and the number of binding sites it
possesses.

15. SECOND GENERATION MONOCLONALANTIBODIES
• By employing site-directed mutagenesis, cysteine residues are introduced at
the predetermined positions on the Mab.
• These cysteine residues which facilitate the isotope labelling. This would be
more useful in diagnostic imaging and radioimmunotherapy.

16.  ADVANTAGES
• Monoclonal antibodies truly represent a homogenous state of a single
molecular species.
• Each Mab is specific to a given antigenic determinant.
 DISADVANTAGES
• Laborious and time-consuming.
• Since they are produced against a single antigenic determinant, therefore
they cannot differentiate the molecules well.
• Mab for human use should be totally free from all pathogenic organisms,
including viruses, but since there is no guarantee it poses a great danger.

17. APPLICATIONS
 DIAGNOSTIC APPLICATIONS
 Biochemical analysis for the diagnosis of pregnancy, cancers, hormonal disorders, infectious
diseases.
 Diagnostic imaging for the detection of myocardial infarction, deep vein thrombosis, cancers,
atherosclerosis, bacterial infections.
 THERAPEUTIC APPLICATIONS
 Direct use as therapeutic agents to destroy disease causing organisms in the treatment of
cancers, AIDS, autoimmune diseases and organ transplantation.
 As targeting agents in therapy as immunotoxins (for treatment of cancers) in drug delivery, for
dissolving blood clots in radioimmunotherapy.
 PROTEIN PURIFICATION BY IMMUNOAFFINITY TECHNIQUES
 MISCELLANEOUS APPLICATIONS INCLUDES CATALYTIC AGENTS LIKE ABZYMES,
USED IN AUTOANTIBODY FINGERPRINTING.

19. REFERENCES :
• Textbook of Biotechnology by U. Satyanarayana. Edition-2013 ; Publisher-Books &
Allied Ltd.
• Textbook of Immunology (Kuby) by Judith.A. Owen, Jenni Punt, Sharon.A. Stanford
and Patricia.P.Jones. Edition-7th ; Publisher – W.H. Freeman and Company.
• www.Wikipedia.com
• www.genescript.com

20. THANK YOU