hybridoma technology, an important topic of immunology

1. MONOCLONAL antibody (hybridoma technology)
Archana Soni
Assistant Professor
Shri Shankaracharya
Mahavidyalaya, Junwani,
Bhilai (C. G.) INDIA

2. Monoclonal Antibodies
Contents
1. Definition.
2. Introduction.
3. Pharmacology.
4. Effects of MAbs.
5. Principle for creation of MAbs.
6. Production of MAbs.
7. Human monoclonal antibodies.
8. Advantages of MAbs.
9. Limitations of MAbs.
10. Applications of MAbs.
11. Antibody fingerprinting.
12. Conclusion.
13. Reference.

3. Definition
MAbs are antibodies that are identical
because they were produced by one of
the immune cell (B cell), all clones of a
single parent cell.
Given any substance, it is possible to
create monoclonal antibodies that
specifically bind to that substance; they
can then serve to detect or purify that
substance. This has become an
important tool in biochemistry,

4. Introduction
• Antibodies or immunoglobulins are protein molecules produced by a specialized group of cells called
B-lymphocytes (plasma cells) in mammals.
• Monoclonal antibody (MAb) is single type of antibody that is directed against a specific
antigenic determinant (epitope). In the early years, animals were immunized against a specific antigen,
B-lymphocytes were isolated and cultured in vitro for producing MAbs. This approach was not
successful since culturing normal B-lymphocytes is difficult, and the synthesis of MAb was short-lived
and very limited.
• It is interesting that immortal monoclonal antibody producing cells do exists in
nature. They are found in the patients suffering from a disease called multiple myeloma (a cancer of B-
lymphocytes). It was in 1975. George Kohler and Cesar Milstein (Noble Prize, 1984) achieved large
scale production of MAbs. They could successfully hybridize antibody—production B-lymphocytes
with myeloma cells in vitro and create a hybridoma.
• The production of monoclonal antibodies by the
hybrid cells is referred to as hybridoma technology.

5. Pharmacology
• A) Mechanismof actionof MAbs:
• Blocking or steric hindrance of the function of target antigen i.e., T-lymphocytes, B-
lymphocytes, tumor necrosis factor-a (TNFa) and interleukin (IL) which are capable
of transducing intracellular signals.
• Cytotoxicity to the cell expressing target AG by ADCC or CDC.
• Inhibition of growth .
• B) Pharmacokinetics:
• MAbs are used by intravascular route and remain essentially intravascular.
Intravenous injection is not always be appropriate for long-term treatment for a
variety of reasons. Hour-long infusion require a hospital environment and are often
associated with mind to very severe side effects. Continous and sustained delivery
of antibodies can lead to induction of neutralizing anti-idiotypic immune
responses, which sometimes develop when massive doses of purified
immunoglobulins are repeatedly injected into patients. Additionally, the
bioavailability of therapeutic antibodies is often detrimental to the treatment
efficacy. They have small volume of distribution and limited tissue penetration.
They remain in circulation for 2 days to 2 weeks. Another limitation is the high cost
of recombinant proteins certified for human use.

6. Effects of MAbs
• 1.) Fever, chills
• 2.) Weakness
• 3.) Headache
• 4) Nausea
• 5) Diarrhea
• 6) Rashes
• 7) Changes in blood pressure
• 8) Flushes&faintness

7. PRINCIPLE FOR CREATION OF HYBRIDOMA
CELLS
• The myeloma cells used in hybridoma technology must not be capable of
synthesizing their own antibodies. The selection of hybridoma cells is based on
inhibiting the nucleotide synthesizing machinery. The mammalian cells can
synthesize nucleotides by two pathway—de novo synthesis and salvage pathway.
• The de novo synthesis of nucleotides
require tetrahydrofolate which is formed from dihydrofolate. The formation of
tetrahydrofolate can be blocked by the inhibitor aminopterin.
• The salvage pathway involves
the direct conversion of purines & pyrimidines into the corresponding nucleotides.
Hypoxanthine guanine phosphoribosyl transferase (HGPRT) is a key enzyme in the
salvage pathway of purines. Thymidine kinase (TK), involved in the salvage
pathway of pyrimidines converts thymidine monophosphate (TMP). Any mutation
in either one of the enzymes blocks the salvage pathway.
• When cells deficient in HGPRT are
grown in a medium containing hypoxanthine aminopterin & thymidine (HAT
medium), they cannot survive due to inhibition of de novo synthesis of purine
nucleotiodes. (salvage pathway is not operative due to lack of HGPRT). Thus, cells
lacking HGPRT, 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. Thus,
only the hybridoma cells can proliferate in HAT medium, & this procedure is
successfully used for their selection.

9. Production of Mab’s
• The establishment of hybridomas
and production of MAbs involves
the following steps.
• 1.) Immunization.
• 2.) Cell fusion
• 3.) Selection of hybridomas.
• 4.) Screening the products.
• 5.) Cloning & propagation.
• 6.) Characterization & storage.

10. Human monoclonal
antibodies
• From SCID mouse by injecting human B and T
cells and grafting mouse by transplanting
spleens and lymph nodes.
• The transplanted mouse is immunized with
target antigens to produce human antibodies.
• Phage derived combinatorial antibody library,
phage display technique-mimics in vitro
affinity maturation.

11. ADVANTAGES OF MAB’S
• 1.) Homogeneity: Monoclonal antibody represents a single antibody molecule that
binds to antigen with the same affinity and promote the same effectors functions.
• 2.) Specificity: The product of a single hybridoma reacts with the same epitope on
antigens.
• 3.) Immunizing Antigen: Need not be characterized and is ultimately not needed in
large quantities to produce large quantities of antibody.
• 4.) Selection: It is possible to select for specific epitope specificities and generate
antibodies against a wider range of antigenic determinants.
• 5.) Antibody Production: Unlimited quantities of a single well-defined monospecific
reagent.

12. Limitations of MAb’s
• As they are specific to a particular antigen, they cannot distinguish
molecule as a whole.
• Some times they cannot distinguish groups of different molecules. Ex:
Presence of retro viruses as a part of mammalian chromosomes is not
distinguished.
• The presence of some of these viruses is detected in hybridomas. This poses
a great danger since there is no guarantee for MAb produced is totally
virus free.
• For this reason US food and drug administration insists that MAb for
human use should be totally free all pathogenic organism including
viruses.

13. APPLICATIONOF MAB’S
• 1.) Diagnostic applications.
• a) MAbs in biochemical analysis
• b) MAbs in diagnostic imaging
• 2.) Therapeutic uses
• a) MAbs as direct therapeutic agents
• b) MAbs as targeting agents in therapy
• 3.) Protien purification
• 4.) Miscellaneous applications
(ABZYMES).

14. ANTIBODYFINGERPRINTING
• The occurrence of antibodies and their
involvement in certain diseases is well known
(e.g., rheumatic arthritis). A new category
of individual specific (IS) autoantibodies
have been discovered in recent years.
These IS--autoantibodies are produced
after birth and reach maximum in number
by 2 years, and the remain constant for
the later part of life. Monoclonal
antibodies produced against IS--
autoantibodies can be used for their
detection, and identification of individuals.
This technique referred to as autoantibody

15. CONCLUSION
• Since for human health care, the subject of production and use
of antibodies has become a very important area of research, not
only for academic purposes but also for its relevance to
industrial growthfor diagnosis and therapeutics, monoclonal
antibodies can be utilized for it. In near future more such
monoclonal antibodies shouldbe produced for biochemical
research that will be of great commercial and medical value.

16. References
• http://www.searhpdfengine.com/HYBRIDOMA-
TECHNOLOGY-FOR-PRODUCTION-OF-
MONOCLONAL-ANTIBODIES.html
• grants.nih.gov/grants/policy/antibodies.pdf
• http://www.searchpdfengine.com/Therapeutic-
monoclonal-antibodies.html
• Biotechnology by U satyanaryana (pages:213-226)