Hybridoma technology

1. HYBRIDOMA TECHNOLOGY
By
Anita Devi P.hD
15099009

3. o Developed by Georges
J.F. Kohler and Cesar
Milstein in 1975
o They shared nobel
prize for this
discovery in 1984
o The term hybridoma
was coined by Leonard
Herzenberg in 1975

4. o A hybridoma is a hybrid cell obtained by
fusion of B lymphocyte with usually a
tumor cell of antibody forming system
or B lymphocyte (these are called
myelomas)

5. o The hybrid cell has the capacity of
antibody production derived from B
cells
o At the same time it can divide
continuously by the quality derived from
Myeloma cells
o By combining the desired qualities of
both the cells, the technology ensures
large scale Antibody production of
single specificity

6. o Specific hybridomas are either cultured
in vitro or passed through mouse
peritoneal cavity to obtain monoclonal
antibodies, this is called as hybridoma
technology

7. o Isolation of B cells
-Mice , 2-4 weeks old are immunized
with the antigen against which
monoclonal antibodies are to be raised
by subcutaneous injection
-Later B cells are isolated from the
spleen of an immunized mouse

8. from
o Isolation of myeloma cells
-Myeloma cells are isolated
bone marrow
-The myeloma cells used are
HGPRT(Hypoxanthine-guanine
phosphoribosyl transferase) mutant
cells ( raised by mutations using 8-
azaguanine)

9. o Somatic cell fusion
-Electrofusion : cells are allowed to
fuse with the application of an
electric field
-Done by using PEG medium
-PEG stands for Poly Ethylene Glycol

11. o Selection of hybrid cells
-HAT medium is used for the
selection of hybrid cells
-HAT stands for Hypoxanthine
Aminopterine Thymidine

12. • Nucleotide synthesis is essential for
cell survival
• In HAT medium, aminopterine blocks
the cellular synthesis of purines and
pyramidines from simple sugars (denovo
pathway)
• But cells can thrive by using
hypoxanthine and thymidine present in
the medium by salvage pathway using
the enzyme HGPRT

13. o How HAT medium works in the selection of hybrid
cells
 -B cells are HGPRT+ and can survive in the HAT
medium, but they undergo normal cell death after
some division
- In hybridoma technology, the myeloma cells
used are HGPRT deficient
-So these cells can’t survive in HAT medium
as Aminopterine blocks the Denovo pathway

14. o Hybrid cells has HGPRT enzyme from
the B cell as well as they have the
ability to multiply repeatedly as
myeloma cells
o So only hybrid cells can survive in HAT
medium

15. H
A
T
S
E
L
E
C
T
I
O
N

16. o Identification and isolation of the
hybridoma cells
• The first screening technique used is
ELISA
-Done by incubating the hybridoma
culture supernatant, secondary enzyme
labeled conjugate and chromogenic
substrate
-Formation of a coloured product
indicates a positive hybridoma

17. • Two methods have been used for
multiplying the hybridoma cells
1.In-vivo
2.In-vitro

18. o Iv-vivo procedure involves introduction
of hybridoma cells into the peritoneal
cavity of the animal , then ascetic fluid
is isolated and then antibodies are
isolated from it
o In-vitro method involves culturing of
hybridoma cells in suitable culture
media and then antibodies are isolated
and purified

19. • Once a hybridoma colony is established,
it will continually grow in culture medium
like RPMI-1640 and produce antibodies

20. • Multiwell plates are used initially to
grow the hybridomas
• After selection, they are changed to
tissue culture flasks
• This provides enough cells for
cryopreservation and supernatant for
subsequent investigations
• The supernatant can yield 1 to
60micrograms per ml which can be
maintained at lower temperatures for
future use

21. • availability of these antibodies with the
unmatched ability to identify highly
specific protein targets, has been
extensively exploited for both in vitro
diagnostics and in vivo therapeutics
• Monoclonal Antibody Development for
Quantitative Analysis of Pancreatitis
Associated Protein
• Immuno-PET: PET Imaging using
Radiolabeled Antibodies

22. oThe pancreatitis-associated protein (PAP) is a
pancreatic stress protein which is not produced in a
healthy pancreas but synthesized in high amounts in
pancreatic cells in response to acute and chronic
pancreatitis, hypoxia, toxins, diabetes and organ
transplant
oStrong induction of PAP observed during the early
phase of pancreatic diseases suggests that PAP
serum levels may be used as a valuable biological
marker. Pancreatic stress is among the symptoms of
Cystic Fibrosis (CF), so, PAP can also be used as a
marker of CF and numerous studies emphasize the
use of PAP in early diagnosis of CF in neonates

23. Positron Emission Tomography (PET) is a highly sensitive functional imaging
modality that provides 3-dimensional and quantifiable visualization of
biological processes.
The effectiveness of PET imaging, is dependent on the specificity of the
biomolecule portion of the injectable radiotracer, to the desired target.
PET imaging quality can be compromised by high background signal due to
unbound isotopes or nonspecifically targeted agents
Monoclonal Antibodies (mAbs) are high affinity molecules that can be used
for specific binding and delivery to cell surface molecules
Antibody targets can be identified and generated for a variety of
applications including cancer detection and staging, tumor and metastasis
phenotyping, stratification of patients into treatment groups and the
evaluation of tumor targeting and therapy response. Once the optimal
antibody has selected, it can be labelled with a radionuclide, a combination
used for Immuno-PET imaging

24. • B.D. Singh, Biotechnology Expanding Horizones
(3rd Revised edition) 2010, Kalyani
Publishers
• https://www.ncbi.nlm.nih.gov/pcm/articl
es/pm428445
• http://www.biologyexams4u.com
• http://www.biotecharticles.com/others-
article/hybridoma-technology