Hybridoma technology revolutionized the field of immunology by enabling the production of monoclonal antibodies with high specificity and affinity. This presentation delves into the principles of DNA hybridoma technology, highlighting its significance in antibody production, therapeutic applications, and biomedical research. Learn about the key steps involved in generating hybridomas, from immunization to antibody screening, and discover the potential of recombinant DNA techniques in enhancing antibody engineering. Whether you're a student, researcher, or industry professional, this overview will provide valuable insights into the innovative world of hybridoma technology."
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Hybridoma Technology ( Production , Purification , and Application )
1. NAGPUR COLLGE OF PHARMACY
Present By :-
Ku . Sakshi R. Ghasle
TOPIC NAME :- HYBRIDOMA TECHNOL
( PRODUCTION , PURIFICATION ,
APPLICATION )
2. 1) Hybridoma technology was discovered in 1975 by two scientists,
Georges Kohler and Cesar Milstein.
2) The conventional or polyclonal antibodies which are isolated from
serum are represents a collection of antibodies from different B cells
that recognize multiple epitope on the same antigen .
3) Each of these individual antibodies recognizes a unique epitope
that is located on that antigen.
4) To overcome the limitations of the polyclonal antibodies ,
researchers invented new methodology to produce monoclonal
antibodies .
5) Monoclonal antibodies can be produced in specialized cells
through a technique now popularly known as HYBRIDOMA
TECHNOLOGY .
6) Monoclonal antibodies are single antibodies made by fusing
antibody producing cells with an immortalized cell line , resulting in
cell call as HYBRIDOMA .
Hybridoma Technology
.
3. MONOCLONAL
ANTIBODIES
POLYCLONAL
ANTIBODIES
1. Expensive production 1. Inexpensive production
2. Long production time 2. Rapid production
3. Large quantities of
specific antibodies
3. Large quantities of
nonspecific antibodies
4. Recognize a single
epitope on an antigen
4. Recognize multiple epitope
on an antigen
5. Production is continuous
and uniform .
5. Different batches vary in
composition .
4. It is used for producing hybrid cells by fusing B lymphocyte
with tumor or myeloma cells.
The hybrid cells produced using hybridoma technology , are
either cultured in laboratory or sub – cultured using mouse
peritoneal cavity .
Thus , due to the presence of B lymphocyte genetic
material the hybrid cells can produce antibodies.
The tumour cell used to produce hybrid cells make them
undergo indefinite division in the culture.
The B lymphocytes involved are pre-programmed to
respond to a single type of antigen or antigenic determinant
, thus they produce a single type of antibody that shows
specificity for a specific antigen.
The reaction of antigen with B – lymphocytes receptors
triggers the rapid division of the lymphocytes .
As a result , a clone of B cells is produce that generate
antibodies against that specific antigen.
This entire process is called as the CLONAL SELECTION.
5. • The myeloma cells used in
hybridoma technology
should not synthesise their
own antibodies .
• The hybridoma cells are
selected based on inhibiting
the nucleotide (
subsequently , the DNA )
synthesising machinery .
• The mammalian cells can
synthesise nucleotides
either by De novo synthesis
or salvage pathway .
6. In the de novosynthesis of nucleotides : -
Tetrahydrofolate formed from dihydrofolate is required . Aminopterin ( an inhibitor ) is
used to block the formation of tetrahydrofolate (and therefore nucleotides )
In the Salvagepathway :-
• The purines and pyrimidines are converted into the corresponding
nucliotides .
• Key enzymes is Hypoxanthine Guanine Phosphoribosyl Transferase (
HGPRT), Which convert hypoxanthine to guanine to inosine
monophosphate and guanosine monophosphate , respectively .
• Thymidine kinase ( TK ) is involved in the salvage pathway of pyrimidine
and converts thymine to thymidine monophosphate (TMP )
• When mutated cells ( deficient in HGPRT ) are grown in a medium
containing Hypoxanthine , Aminopterin , Thymine (HAT medium ) , they
fail to survive as the de novo synthesis of purine nucleotides is inhibited .
Thus , cells deficient in HGPRT and grown in HAT medium die .
7. PRODUCTIONOF MAbs
Involves following steps :-
• Immunisation
• Cell fusion
• Selection of hybridomas
• Screening of products
• Cloning and propagation and
• Characterisation and storage
8.
9. Immunisation :
1. Immunise a mouse with a suitable antigen
2. Antigen and Freund’s adjuvant are injection vis subcutaneous
route
3. Injection are repeated multiple times at many sites .
4. Increases the stimulation of B lymphocytes which are responding
to the antigen .
5. Give rises to large number of immune stimulated cells for the
synthesis of antibodies .
6. The animal is sacrifices when the conc . Of the antibodies in
serum becomes optimal .
7. The spleen is removed aseptically and distrupted mechanically or
enzymatically to release the cells
8. The spleen lymphocytes are separated from the remaining cells
by density gradient centrifugation
10. Cell fusion
• The lymphocytes are thoroughly
washed and mixed with HGPRT
defective myeloma cells
• The mixture of cells is treated with
polyethylene Glycol ( PEG ) but for few
minutes due to its toxicity .
• The cells are then washed to remove
PEG and kept it in fresh medium .
• These cells contain a mixture of
hybridomas ( fused cells ) , free
myeloma cells and free lymphocytes .
11. SELECTION OF HYBRIDOMA
• On culturing the cells in HAT medium , the hybridoma cells
grow and the remaining cells disapper slowly within 7-10
days .
• Selecting a single antibody producing hybrid cells is very
essential , and is possible if the hybridomas are isolated
and grown individually.
• The suspension of hybridoma cells is diluted to such
intensity that the individual aliquots contain one cell each .
• These cells are grown in a regular culture medium to
produce the desired antibody .
12. Screening of products :
• The hybridomas should be screened for the secretion of the
antibodies of desired specificity , and the culture medium
from each hybridoma culture should be tested occasionally (
by ELISA and RIA )for desired antibody specificity .
• In this , the antibody binds to the specific antigen (coated to
plastic plates ) and the unbound antibody and other
components of the medium are washed off .
• Thus , the hybridoma cells producing the desired antibody
are identified by screening .
• The antibody secreted by the hybrid cells is the Monoclonal
antibody .
13. Cloning and propagation
The single cells producing the desired
antibody are isolated and cloned by using 2
techniques :-
LIMITING DILUTION TEST :
• In this medthod , the suspension of
hybridoma cells is serially diluted and
aliquots of each dilation are transferred
into micro culture wells .
• The dilutions are made such that each
aliquot in a sell contains a single hybrid
cell , thus ensuring that the antibody
produced is monoclonal .
SOFT AGAR TEST :
• In this method , the hybridoma cells
are cultured in soft agar . Many cells
can be grown simultaneously in a
semisolid medium to form
monoclonal colonies .
14. Characterisation and storage
0
• The obtained monoclonal antibodies are subjected to
biochemical and biophysical characterization for the desired
specificity.
• The MAbs should also be elucidated for the immunoglobulin
class or sub class , its specific epitope , and its number of
binding sites .
• The stability of the cell lines and the MAbs is also important .
• Both are characterized to check their ability to withstand
freezing and thawing by freezing the desired cell lines in
liquid nitrogen at several stages of cloning and culture .
15. Production of MAbs :
• In cultured bottle is low ( 5-10 ug / ml ) , thus to
increase the yield the hybrid cells are grown as
ascites in the peritonial cavity of mouse .
• The ascites fluid contain 5-20 mg of Mabs /ml , which
is much superior to the in vitro cultivation technique .
• Collection of Mabs is heavy risk of contamination by
pathogenic organism of the animal .
• Worker prefer in vitro technique over using
animals . )(
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16. PURIFICATION OF MA bs
• For , purification the sample is conditioned by removing
cells , cell debris , lipids and clotted materials through
CENTRIFUGATION AND THEN FILTERATION through
a 0.45 um filter.
• Also , Ultra filtration or dialysis
• By ion exchange chromatography :
1.) At sufficiently low PH = Cation exchange
chromatography
2.) AT sufficiently high Ph = Anion exchange
chromatography
• Size exclusion chromatography :
• Chromatogram (analysis of final purity )
• Gel electrophoresis and capillary electrophoresis
18. DIAGNOSTIC APPLICATION :
MAbs are used in Biochemical analysis , as diagnostic reagents and in
imaging of diseases as diagnostic tools .
MAbs in Biological analysis :
Pregnancy ( by ,measuring HGC )
Cancer (estimation of plasma carcinoembryonic antigens in colorectal
cancer ,etc )
Hormonal Disorders (for detecting thyroid disorders )
MAbs in Diagnostic Imaging : ( Immunoscintigrapgy )
Myocardial infraction
Deep vein Thrombosis
Atherosclerosis
Cancer
Bacterial Infection
19. THERAPEUTICAPPLICATION:
MAbs as Direct Therapeutic Agents : Used for direct enhancement
of the immune function of the host .Minimises the toxicity to the
target tissues or the host .
In Destroying Disease –Causing organisms ( inc. phagocytosis )
In Cancer Treatment
In Immunosuppression of organ transplantation
In AIDS Treatment
In treatment of Auto immune Diseases
MAbs as Targeting Agents in therapy :
As immunotoxin
In drug delivery
In the dissolution of blood clots
In radio immunotheraphy
