This PPT expalins the production of monoclonal antibody

1. Monoclonal Antibody Production
-
Hybridoma Technology
By
Rajamehala M

2. Monoclonal antibodies can be produced from
hybridoma cells by a techniques known as Hybridoma
Technology
The production of monoclonal antibodies was
invented by César Milstein and Georges J. F. Köhler
in 1975.

3. Kohler Experiment
Kohler(1974) successfully produced a hybridoma by
fusing a P3 myeloma cell (resistant to azaguanine)
and a lymphocyte(from the spleen of a mouse)
immunized against sheep red blood cells

4. These hybridomas retain the property of
immortality of the myeloma cells as well as that
of secreting an antibody specific for an unknown
antigen

5. WHAT IS HYBRIDOMA TECHNOLOGY???
Hybridoma techniques is a method of creating pure and
uniform antibodies against a specific target antigen.
Hybridoma are the hybrid cells of myeloma (Cancer) cells
with antibody producing cells (lymphocytes)from an
immunized donor(animal)

6. MONOCLONALANTIBODY
Monoclonal antibodies (mAbs) are antibodies that are
identical because they are produced by one type of
immune cell, all clones of a single parent cell.
Basically produced by white blood cell which is
called as plasma cell.

8. Monoclonal Antibody Production Process
Purity and form of immunogen
Selection of myeloma cell line
Choice of animals
Immunization procedure
Sacrifice of animal and separation of lymphocytes
Immortalization by fusion with tumor cells
Selection of hybridoma cells
Isolation of hybridoma cells
Commercial production of monoclonal Antibodies

10. Purity and Form of Immunogen
Purity of the antigen to be used as immunogen is crucial for
generation of monoclonal antibodies
Molecules of low molecular weight are poor immunogens and
have to be coupled to larger immunogenic molecules.
Aggregated and Particulate antigens elicit stronger responses.

11. Selection of Myeloma Cell line
No myeloma lymphocyte should be allowed to fuse with
normal lymphocyte
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.

12. Choice of Animals
The choice of the species and strain of the animal used as a source of
spleen(donor) for fusion is largely dependent on the myeloma cells
available and the origin of the immunogen.
Mice are the most common species used for immunization primarily
because there are more murine myeloma cell lines available.
The BALB/c strain of mice is preferred since the myeloma cell lines
available for fusion are derived from this strain.

13. 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.
For most proteins, a dose of 1-50mg per injection per mouse repeated
two or three times is sufficient to evoke a strong antibody response.
For soluble antigens, the first injection is give in the presence of the
adjuvant and the final booster is given in aqueous solutions.

14. They are injected i/p with 20-100 g of soluble antigen on the
first day and on the following day, they are injected with the
same dose intravenously.
10 to 14 days afterwards, individual animals are bled and the
titre of the antibodies is determined.
Animals that show the highest antibody titre are selected and
at least 3-4 weeks after the last booster

15. 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 spleen fluid by density gradient
centrifugation and washed.

16. Immunization by fusion with tumor cells
It requires PEG (poly ethylene glycol) as medium
for fusion
First fusion experiments were performed with
sendai virus as a fusogenic agent

17. Condition for Fusion process
Before carrying out the fusion, the following conditions should be met
for the mouse system.
1. Myeloma cells in the logarithmic growth phase.
2. Ratio of 2-5 lymphocytes per myeloma cell
3. 40% PEG
4. Fusion at 37o
C, pH 7.5-8.0, 3 min

18. Fusion Process
Membrane fusion consists of 2 distinct stages
Cell agglutination
Osmotic Cell swelling and heterokaryon formation

19. Cell Agglutination
Formation of cytoplasmic bridges between
cells
Osmotic Cell swelling and heterokaryon
formation
Polyethylene glycol is used for induction of
cell fusion.
The mixture of cells is exposed to this fusion
promoting agent, but only for few minutes
since it is cytotoxic

20. Inter-cytoplasmic communication begin in 1 to 3
min after dilution of PEG
Cell clumping is maximal within 30 min and
cytoplasmic mixing is almost complete after 40
min

21. Selection of Hybridoma Cells
The basis of hybridoma technonolgy was the development of suitable
myeloma mutant cell lines that are non-antibody secreting and
deficient in the enzyme Hypoxanthine Guainine 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.

22. The HAT
(Hypoxanthine-Aminopterin-Thymid
ine) medium is used for metabolic
selection of fused cells.
The medium containing HAT will
eliminate the parent myeloma cells
and allow the growth of hybridomas
which harbor the genes for HGPRT
from the parent lymphocytes

23. 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.
Lymphocyte parent cells are eliminated due to poor growth in vitro.

24. Isolation of Monoclonal Antibody producing
hybridoma cell
If all the hybridoma cells that have been selected using HAT medium,
a polyclonal antibody mixture would be obtained.
A single 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.

25. Such cells are transferred to
separate fresh media for growth.
Each mass of hybridoma cells
produced from a single parent
hybridoma cell is now examined to
determine whether it produces the
desired monoclonal antibody.

26. Commercial production of monoclonal Antibodies
Once the correct hybridoma has been isolated, it can be stored,
frozen(Cryo-preserved) and cultured whenever required.

28. Applications of Monoclonal Antibodies
Diagnostic Applications
Therapeutic Applications
Transplant rejection
Cardiovascular disease
Cancer
Infectious Diseases
Inflammatory disease
Clinical Applications
Purification of drugs, Imaging the target
Future Applications
Fight against Bioterrorism