An overview of Hybridoma technology

1. HYBRIDOMA TECHNOLOGY
Prashant Kumar
Animal Biochemistry
NDRI Karnal, Haryana
COURSE: ADVANCED TECHNIQUES IN BIOCHEMISTRY

2. Contents
History and introduction
Methods of hybridoma technology
Procedure and Steps involved in hybridoma generation
Production of monoclonal antibodies by hybridoma technology
Role of spleen cell and myeloma cell in hybridoma technology
Difference between polyclonal and monoclonal antibodies
Selection of hybrid cells in HAT medium
Cloning of hybridoma cell lines by limiting dilution
Conclusion and Future perspective
References

3. History
1975 : Georges Köhler and César Milstein
They invented hybridoma technology for production of
monoclonal antibodies. they were also awarded by
Nobel Prize along with Niels Kaj Jerne in Physiology and
Medicine field during 1984.
1976-77: Leonard Harzenberg
He coined the term Hybridoma during his sabbatical in César
Milstein's laboratory.
1890 : Emil von Behring
German physiologist who received the 1901 Nobel Prize in
Physiology and Medicine, for his discovery of antibodies
(diphtheria antitoxin).

4. Hybridoma technology is used to produce a hybrid cell. These hybrid cells are produced by
fusing B-lymphocyte with tumor cell and they are called as myeloma cells.
Hybridomas are cells formed via fusion between a short-lived antibody-producing B-cell and
an immortal myeloma cell. that have been engineered to produce a desired antibody in large
amounts.
Antibodies are produced in host in response to administration of molecule which are foreign
to blood circulation.
Different plasma cell make different antibody. Genetically identical plasma cells will make
identical antibodies.
Cloned cells are genetically identical and thus cloned plasma cells will make identical
(homogeneous) antibodies.
These identical antibodies produced from single clone of plasma cell are called monoclonal
antibodies.
MABs are antibodies that arise from a single clone of cells Antibodies produced by different
clones of plasma cell are called polyclonal antibodies.
Introduction

5. ƒ
The original hybridoma technology was aimed to generate hybridoma cells.
Although the technology is simple, large numbers of undesired fused cells are produced
because of nonspecific fusion.
There are two critical points for successful production of novel monoclonal antibodies based
on hybridoma technology.
i. The first point is immunization to facilitate differentiation of B lymphocytes into more
matured forms.
ii. The second point is selective fusion of targeted antigen-sensitized B lymphocytes with
myeloma cells.
Hybridoma Technology
1. Conventional hybridoma technology
Figure 1. Conventional hybridoma technology based on polyethyleneglycol and hemagglutinating virus of Japan.
Hemagglutinating virus
of Japan
lymphocyte Hybridoma cell

6. 2. Improved hybridoma technology
Hybridoma Technology
Improved hybridoma technology has the characteristic of eliciting relatively specific fusion
between a spleen cell and a myeloma cell.
Myeloma cells and spleen cells, including immunized B lymphocytes, form a monolayer on
electrode surfaces by the application of a nonuniform alternating current field and become fused
with application of electric field pulses to produce hybridoma cells. This method certainly increased
the fusion efficiency, but it still brought about Nonspecific spleen cell–spleen cell and also myeloma
cell–myeloma cell fusion.
Figure 2. Improved hybridoma technology based on pearl-chain formation.
(a). Pearl-chain formation

7. 2. Improved hybridoma technology
Hybridoma Technology
Figure 3. Improved hybridoma technology based on laser radiation.
Myeloma cell
Another improvement was also achieved with laser radiation. Performed under a
microscope, a lymphocyte is transferred by trapping laser so that it comes into contact
with a myeloma cell (SP2 cell). The contact surface of the lymphocyte–myeloma cell is
then irradiated with a pulse laser beam.
(b). Laser radiation
Consequently, while the pearl-chain formation and laser radiation methods have certain
advantages over conventional hybridoma technology regarding the frequency of fusion, they
still fail to control selective fusion of an aimed B-lymphocyte with a myeloma cell to solely
generate hybridoma cells secreting the desired monoclonal antibodies.

8. Hybridoma Technology
Next generation of hybridoma technology consist of three critical steps that feature selective fusion
of antigen-selected B lymphocytes with myeloma cells by electrical pulses.
ƒ
Antigen-based preselection of B lymphocytes preferentially selects those producing high-affinity
antibodies.
ƒ
Theoretically, all hybridoma cells could secrete monoclonal antibodies against antigens of interest.
3. Next generation of hybridoma technology :
(A). B-cell targeting
Three critical steps involved:
(i). antigen-based preselection of sensitized B lymphocytes based on immunoglobulin receptors.
(ii). the antigen-selected B lymphocytes are brought into contact with myeloma cells by strong and
specific binding between biotin and avidin.
(iii). B lymphocyte– myeloma cell complexes are selectively fused by electrical pulses.
These three critical steps realize selective production of hybridoma cells secreting novel monoclonal
antibodies against antigens of interest. This advanced technology is termed as pulsed electric field
method or B-cell targeting (BCT).
Formation of B lymphocyte & myeloma cell complexes: B lymphocytes, obtained from antigenimmunized
mouse, are preselected by either antigen–biotin or antigen–avidin (or antigen–streptavidin) conjugates and
combined together with myeloma cells by harnessing the power of biotin and avidin.

9. (a). Antigen–biotin selection
Two protocols of BCT have been developed, depending on the conjugates for selecting B-
lymphocytes.
(b). Antigen–avidin selection

10. Hybridoma Technology
3. Next generation of hybridoma technology : (B). Based on multi-targeting
Multitargeting new technology was developed that each B lymphocyte sensitized by multiple antigens is
selected in advance with the corresponding antigen based on immunoglobulin receptors on B-
lymphocytes. Since single B lymphocytes produce single specific antibodies against one epitope in an
antigen, selection of sensitized B-lymphocytes by each antigen is possible. This step is of critical
importance in the entire pathway of the multi-targeting technique.

11. Hybridoma Technology
3. Next generation of hybridoma technology : (C). Stereospecific targeting based
Recently, conformation-specific monoclonal antibodies have become an extremely
important focus, especially for the purpose of therapies for cancer and diseases caused by
self-immunodeficiency.
Demand for stereospecific monoclonal antibodies will surely increase in the future.
However, no practical protocols have been reported until now.

12. Hybridoma technology is a method for producing large numbers of identical monoclonal
antibodies. The production of Hybridoma cells involves a number of well defined steps given
below.
Steps involved in production of hybridoma
1. Immunization of mouse with antigen
2. Collection of myeloma cells and spleen
cells
3. Fusion of myeloma cells and spleen cells
4. Selection of fused cells in HAT medium
5. Culturing of fused cells
6. Screening of fused cells for production of
antibodies
7. Cloning of fused cells
8. Freezing of Cells
9. Production of monoclonal antibodies

13. Procedure of hybridoma generation
Only the hybridoma cells have got the ability to divide and proliferate on the HAT medium
The Specific antibody producing B-lymphocytes are unable to divide continuously in the culture
medium, therefore eventually they die
The HGPRT myeloma cells can not divide in the HAT medium due to the presence of aminopterin
This mixture of cell population is then cultured in selective media known as HAT medium along with the
drug aminopterin
HGPRT myeloma cells are fused with specific antibody producing B-lymphocytes, the resulting cells will
have the mixture of cell population such as hybrid cells, myeloma cells, B-lymphocytes
The myeloma cells are selected based on some criteria like these cells themselves should not produce
antibodies and also they should contain a genetic markers such as HGPRT
This specific antibody producing B-lymphocytes are then mixed with the selected myeloma cells and are
induced to fuse to form hybrid cells
This immunization with specific antigen increases the specific antibody producing B-lymphocytes
B-lymphocytes are extracted from the spleen of an animal (usually mouse), which has been immunized
with the required antigen against which monoclonal antibodies are produced

14. Procedure of hybridoma generation

15. Production of monoclonal antibody by hybridoma technology

16. Role of spleen cell and myeloma cell
The two cells (spleen cells and myeloma cells) are fused together to produce a
hybrid cell (hybridoma) that has the best properties of both cells – the hybridoma grows in
culture and makes antibody. Hybridoma cells are cloned to ensure that all cells in the culture
are making exactly the same mAbs
Figure: A general representation of the hybridoma method used to produce monoclonal antibodies.
Fusion of myeloma cells and spleen cells-
Myeloma and spleen cells are separately
washed with serum free medium.
Appropriate volume from myeloma and spleen
cells were transferred to 50 ml disposable plastic
centrifuge tube to obtain ratio of myeloma cells to
spleen cells 1:10.
Cell suspension is centrifuged.
Cells in cell pellet are fused with polyethylene
glycol (PEG) which has power to bind with water
and thus can remove water molecules trapped
between two adjacent membrane.
PEG is toxic to cells and thus its addition is
done in defined way to minimize toxicity and also
fusion results in reasonable number of fused
cells.

17. Identification and Isolation of the Hybridoma Cells
1. The specific antibodies present in the each microwell are identified using one of the
methods such as precipitation method or agglutination method by most commonly used
and most sensitive and rapid method is ELISA (Enzyme Linked Immunosorbant Assay)
2. Wells which contain the antibodies specific to the antigens are identified and hybridoma
cells are isolated from these wells and cultured (cloned). This ensures that these hybridoma
cells have the capacity to produce same single type of antibodies specific to the antigen used.
3. After these hybridoma cells are multiplied using in vitro or in vivo method.
Mass Production of Antibodies:
4. The in vivo production involves injecting hybridoma cells into the peritoneal cavity of the
animal (mouse), then ascetic fluid is isolated and then antibodies are isolated from it.
5. In vitro method hybridoma cells are cultured in suitable culture media and then antibodies
are isolated and purified.
Uses:
This hybridoma technology is used to produce monoclonal antibodies.
Hybridoma cells producing specific antibodies for the antigen used to immunize the animal
(mouse) are identified and isolated by following methods :

18. Polyclonal and monoclonal antibodies
An antigen (immunogen) injected into animals induces them to produce and secrete high
levels of antibodies into the blood.
Several months after repeated immunization, the blood (plasma, serum) is collected, and
antibodies are purified.
The antibodies generated by this method are called polyclonal antibodies because they are
derived from different B cell clones and the resulting antiserum contains numerous different
antibodies that react to the injected immunogen.
1. Polyclonal antibodies

19. 2. monoclonal antibodies
Polyclonal and monoclonal antibodies
Monoclonal antibody can be stably produced if a single B cell producing the antibody is isolated
and cultured indefinitely. This is achieved by artificially fusing the antibody-producing B cells with
immortalized cancer cells (myeloma) to generate hybridomas that live indefinitely and contain
genes encoding specific antibodies, and by selecting the hybridoma clones that produce the
desired monoclonal antibodies with high affinity and specificity.
It usually takes 4-6 months from immunization of animals to production of monoclonal antibodies.
Direct biochemical purification of a monoclonal antibody from a polyclonal antibody preparation
is not feasible.

20. Difference between polyclonal and monoclonal antibodies
Polyclonal Monoclonal
Animal species Rabbit, guinea pig, goat, sheep,
rat, mouse, etc.
Rat mouse, chicken, rabbit, human,
etc.
Form Antiserum Hybridoma
Class, subclass Mixed classes Single class
Epitope React to multiple epitope React to a single epitope
Specificity Lower than the monoclonal
antibodies because multiple types
of antibodies are present
High if good quality antibodies are
selected
Reproducibility Variable among lots Same antibodies are produced
indefinately
Stability Binding ability tends to be
unaffected by
fixation/denaturation of the
antigen, because multiple
different antibody molecules are
present. Tolerate modification
such as labeling and removal of
Fc region.
Binding ability may be loss if the
epitope is lost by fixation/
denaturation of the antigen, because
the monoclonal antibodies are
homogeneous. Tend to be sensitive to
modification, such as labeling and
removal of the Fc region.

21. Fusion of cells results in mixture of parent cells, hybrid of each parent cells to itself and hybrids
between on parent to other parent.
Selection is made by culturing of cells after fusion in selection medium containing hypoxanthine,
aminopterin and thymidine and medium is referred as HAT medium.
Aminopterin is folic acid antagonist, inhibits dihydrofolate reductase and blocks main synthetic
pathway for purine and pyrimidine synthesis. However, cell can still synthesize DNA via salvage
pathway provided cell is provided hypoxanthine and thymidine and cell has HGPRT and TK
enzyme.
Myeloma cell lacks either HGPRT or TK and therefore it can not make use of salvage pathway
and therefore it will die in culture.
Spleen cells lack capability to continuously divide in culture and therefore unfused spleen cells
will die in culture at its own. Although myeloma lacks HGPRT or TK, fused cell derived these
enzymes from parental spleen cell and therefore fused cells have both of these enzymes.
The substrate of HGPRT is hypoxanthine and that of TK is thymidine.
These substrates are present in HAT medium. Thus, fused cell can survive in HAT medium.
Other cells in HAT medium die out and therefore selection in Hat is called positive selection.
Selection of HAT medium

22. Figure: HAT selection depicted by a plasmacytoma thymidine kinase mutant fused with a mortal splenic B-cell.
HAT Medium (hypoxanthine-aminopterin-thymidine medium) is a selection medium for selection of hybrid
cell, nucleotide synthesis is essential for cell survival. aminopterin, a drug that block the cellular synthesis
of purine and pyrimidines from simple sugar (de novo pathway) by inhibiting dihydrofolate reductase
with hypoxanthine (a purine derivative) and thymidine (a deoxynucleoside) which are intermediates in DNA
synthesis. But cells can thrive by utilizing hypoxanthine and thymidine present in the medium by salvage
pathways by using the enzyme HGPRT (hypoxanthine guanine phasphorybosyl transferase).
Selection of HAT medium

23. How HAT medium works in the selection of hybrid cells ??
In this process myeloma cells are HGPRT deficient. So these cells can not survive in HAT
medium as aminopterin block denovo synthesis.
This selection process involve the two pathways:
1. De novo pathway
2. Salvage pathway

24. Cloning of hybridoma cell lines by “limiting dilution” or expansion & stabilisation of
clones by ascites production
 Dilution cloning or cloning by limiting dilution describes a procedure to obtain a
monoclonal cell population starting from a polyclonal mass of cells.
This is achieved by setting up a series of increasing dilutions of the parent (polyclonal) cell
culture. A suspension of the parent cells is made. After the final dilutions are produced
aliquots of the suspension are plated.
A mouse is inoculated with cell and thereby becomes a factory for producing the mAb.
Small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture
followed by selection for antigen binding or grown by the mouse ascites method with cloning
at a later time.
The hybridoma now are ready to be diluted and grown, thus obtaining a number of different
colonies, each producing only one type of antibody.
Single hybridoma clones by limiting dilution (getting one clone or less per well)
Applications for the procedure including cloning of parasites, T cells, transgenic cells, and
macrophages.

27. ƒ
Utilizing innate functions of immune cells and cancerous cells is of critical
importance for hybridoma technology.
ƒ
Selective fusion of sensitized B lymphocytes with myeloma cells by electrical
pulses elicits efficient production of hybridoma cells secreting target novel
monoclonal antibodies.
B-cell targeting, multitargeting and stereospecific targeting may contribute to the
next generation of hybridoma technology.
Conclusion

28. Future perspective
Requirements of human monoclonal antibodies for medical purposes have been
dramatically increasing, these are very specific to target antigens, have a relatively
long half-life and also exert limited side effects on the body. They can be expected to
be an important component of the Next generation of promising medicines.
Stereospecific targeting technology will contribute greatly to this scenario
because of the advantages of generation of specific monoclonal antibodies against
tertiary structures of desired antigens.
Monoclonal antibodies as a molecule for understanding and monitoring the
biology of disease and its role in research, clinical, diagnostic, analytical, and
pharmaceutical applications.

29. References
 Tomita, M. and Tsumoto, K., 2011. Hybridoma technologies for antibody
production. Immunotherapy, 3(3), pp.371-380.
 Smith, S.A. and Crowe Jr, J.E., 2015. Use of human hybridoma technology to isolate human
monoclonal antibodies. Antibodies for Infectious Diseases, pp.141-156.
 Pandey, S., 2010. Hybridoma technology for production of monoclonal
antibodies. Hybridoma, 1(2), p.017.
 https://en.wikipedia.org/wiki/Hybridoma_technology
 https://en.wikipedia.org/wiki/HAT_medium
 https://www.biotecharticles.com/Others-Article/Hybridoma-Technology-A-Biotechnology-
Technique-378.html
 https://www.biologyexams4u.com/2013/02/what-is-monoclonal-antibody-how-it
is.html#.Xr9U6f8zbIU