1. The production of monoclonal antibodies involves immunizing an animal like mice with an antigen, fusing their spleen cells that produce antibodies with myeloma tumor cells, and selecting the resulting hybridoma cells that continuously produce the desired antibodies. 2. The fused hybridoma cells are selected using HAT medium, which allows their growth while killing off unfused spleen and myeloma cells. 3. Individual hybridoma cells are further isolated by limiting dilution to produce monoclonal antibody-secreting clones from which a specific monoclonal antibody can be produced at large scale for commercial purposes.

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MONOCLONAL ANTIBODY PRODUCTION STRATEGY
Following main steps are involved in making a hybridoma and enabling it to successfully grow
in culture medium to produce monoclonal antibodies:
Purity and form of immunogen
The purity of the antigen to be used as immunogen is crucial for generation of monoclonal
antibodies. Molecules of low molecular weight (1000 daltons) are poor immunogens and have to
be coupled to larger immunogenic molecules. Aggregated and particulate antigens elicit stronger
responses. Adjuvants help to achieve stronger responses.
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.
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 1-50 mg per
injection per mouse repeated two or three times is sufficient to evoke a strong antibody response.
For soluble antigens, the first injection is given in the presence of the adjuvant and the final
booster is given in aqueous solutions. Ten 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, they are injected i/p with 20-100g of soluble antigen
on the first day and on the following day, they are injected with the same dose intravenously.
Killing 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 the spleen fluid (and red blood cells) by density gradient centrifugation, and
washed.
Immortalization
The immortalization of antibody producing lymphocytes is the core of hybridoma technology.
The purpose of immortalization is to conserve the capability of individual lymphocytes to secrete
one single type of antibodies by unlimited in vitro growth. This can be done by:
(i) Immortalization by fusion with tumor cells
(ii) Immortalization by infection with a transforming virus
(iii) Immortalization by transfection with tumor or virus derived DNA
Fusosen Used
The cell fusion process between amyeloma cella and spleen lymphocytes is usually induced in
the presence of polyethylene glycol (PEG-1500). The first fusion experiments were performed
with sendai virus as a fusogenic agent. Both regents aggregate the cells which eventually lead to
fusion of cells. As compared with UV inactivated sendai virus, PEG increases the number of

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hybird cells in myeloma fusions because sendai virus has fewer agglutination points on the
membrane of B cells. It has recently been shown that hybridization frequencies could be
increased by virus by using short electric field pulses of high intensity (electric fusion).
A very recent development, called receptor-directed cell fusion, might be a significant
improvement of the fusion process. Lymphocytes carrying antigen-specific antibodies on their
surface, are coated with antigen to which avidin is linked. Bioten is bound to the myeloma cell
surface. The affinity of avadin for biotin makes the fusion of lymphocytes and mylelomas from a
poor efficiency and random process to a controlled and very specific process with high yields.
This method is combined with a newly developed hybridization system based on short electric
pulses of high intensity.
Condition for Fusion Procedure
Before carrying out the fusion, the following conditions should be met for the mouse system.
 Myeloma cells in the logarithmic growth phase
 Ratio of 2-5 lymphocytes per myeloma cell
 40% PEG (1000 dal); PEG should be protested for cell toxicity.
 Fusion at 37°C, pH 7.5-8.0, 3 min
Fusion
Membrane fusion consists of 2 distinct stages, cell agglutination, during which the plasma
membranes of adjacent cells are brought into close proximity, and the formation of cytoplasmic
bridges between cells. These stages are followed by osmotic cell swelling and heterokaryon
formation. Polyethylene glycol is used for induction of cells fusion. The mixture of cells is
exposed to this fusion promoting agent, but only for few minutes since it is cyotoxic. One minute
after exposure of the cells to PEG lipid probes spread from one plasma membrane into the other.
Cytoplasmic mixing is optimal at 37°C and is complete at 4h. Cytoplasmic mixing is optimal at
37 degree centigrade and requires 4h for completion.
Selection of myeloma cells
The myeloma cell line used must itself not be capable of synthesizing antibody otherwise
hybridoma cell line will produce a mixture of antibodies. It is worth noting that in most cases of
multiple myeloma there is an overproduction of a particular type of antibody. Such myeloma cell
lines that do not produce antibodies are readily available because there are instances where a
particular myeloma cell line multiples uncontrollably without antibody production. HPRT-
negative myeloma cell line is such an example. Mouse, rat and human myeloma cell lines are
available now.
Fusion of separated lymphocytes with myeloma cells
After washing, the lymphocytes are mixed with myeloma cell. The mixture of the two cell lines
is now exposed to polyethylene glycol, a fusion-promoting agent. The latter is cytotoxic and
therefore the exposure is limited only to a few minutes. The cells are then washed to make them
polyethylene glycol free. The washed cells comprise a mixture of hybridoma cells, unfused
lymphocytes and unfused myeloma cells.

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Selection of hybridoma cells
Since the mixture contains hybridoma cells, unfused myeloma cells and unfused normal
lymphocytes, the hybridoma cells have to be selected from the mixture for further requirements.
This is done by using HAT-medium for growth. HAT-medium represents a mixture of
hypoxanthine, aminopterin and thymidine. When the said mixture of cells is grown in the HAT-
medium, the unfused normal lymphocytes and the unfused myeloma cells fail to grow.
However, the hybridoma cells grow successfully in the HAT-medium because they possess the
ability of myeloma cells to grow in vitro and the normal HRPT gene inherited from normal
lymphocytes.
Isolation of a monoclonal antibody producing hybridoma cell
If all the hybridoma cells that have been selected using HAT-medium are grown together, a
polyclonal antibody mixture would be obtained. Consequently, a single antibody (monoclonal
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. Such cells are transferred to separate fresh media for growth. Each mass
of hybridoma cells (clone) produced from a single parent hybridoma cell is now examined to
determine whether it produces the desired monoclonal antibody.
Commercial Production of Monoclonal Antibodies (MAbs)
Once the correct hybridoma has been isolated, it can be stored, frozen (cryopreserved in liquid
nitrogen containing FCS and Dimethyl sulphoxide) and cultured whenever required. Since the
hybridoma is a transferred cell line, it grows readily in culture but the antibody titre is low, say
generally 5-10 mg/l. This rate of monoclonal antibody production, which is very low seeing its
requirements, has been considerably enhanced using large-scale production methods.