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HYBRIDOMA TECHNOLOGY

Hybridoma technology for the production of


monoclonal antibodies (MABs) has contributed
significantly to aquaculture.
Monoclonal antibodies are being employed in disease,
pathogen classification, epidemiological analysis and
development of vaccines.
Hybridoma technology is a technology of forming
hybrid cell lines (called hybridomas) by fusing a
specific antibody-producing B cell with a myeloma (B
cell cancer) cell that is selected for its ability to grow in
tissue culture.
The antibodies produced by the hybridoma are all of a
single specificity and are therefore monoclonal
antibodies (in contrast to polyclonal antibodies ).
The idea of a magic bullet was first
proposed by Paul Ehrlich.
In the 1970s the B-cell cancer multiple
myeloma was known, and it was understood
that these cancerous B-cells all produce a
single type of antibody.
This was used to study the structure of
antibodies, but it was not yet possible to
produce identical antibodies specific to a
given antigen.
Production of monoclonal antibodies involving
humanmouse hybrid cells was described by
Jerrold Schwaber in 1973.
The invention was conceived by Prof. Pieczenik,
with Prof. John Sedat, and produced by Kohler and
Milstein.
Kohler, Milstein and Jerne in 1975; who shared
the Nobel Prize in Physilogy or Medicine in 1984
for the discovery.
The key idea was to use a line of myeloma cells
that had lost their ability to secrete antibodies,
come up with a technique to fuse these cells with
healthy antibody-producing B-cells, and be able to
select for the successfully fused cells.
Production of monoclonal antibodies
MAbs production is initiated by the immunisation of
BALB/c mice with immunogens.
Antibodies are produced by differentiated B-cells
(plasma cells) of a particular specificity, the antibodies
are identical and therefore, is a source of homologous
antibodies.
Plasma cells are, however, short-lived and cannot be
grown in culture.
Therefore, fusion of these cells with immortal myeloma
cells produces hybridoma cells with the ability to grow
in culture and to secrete antibody with a defined
specificity.
Chemical selection, screening of the antibodies produced
and cloning of the hybridoma cells lead to the ultimate
production MAbs.
Monoclonal antibodies can be prepared against a
wide variety of immunogens, e.g., protein,
carbohydrate, nucleic acid or combinations of
these.
They can also be produced from impure antigen
by selecting single cell clone after the fusion.
The resulting MAbs are extremely specific and
are therefore, very useful diagnostic tools.
In addition, hybridoma cell lines have the
advantage of providing an unlimited supply of
the antibody in the cell supernatant, which
allows standardisation of the MAb reagents.
Myeloma cell lines used in fusions have been selected
because they do not produce antibody molecules,
although some of the commercially available cell lines
do produce immunoglobulin heavy or light chain
molecules.
For this reason P3x63. Ag8-653 (653) and Sp2/0-Ag14
(Sp2/0) are the most frequently used cell lines in
hybridoma technology.
Hybridoma cells can be prepared by fusing myeloma
cells and antibody producing cells which have been
isolated from different mouse species.
But the success rate of fusion is greatly increased if
both cell types come from the same strain of mouse
(e.g., BALB/c).
Kohler and Milstein used Sendai virus as the fusion
agent.
Polyethylene glycol (PEG) is now routinely used to fuse
the cells.
Even in efficient fusions, only approximately 1% of the
initial cell numbers result in fusion.
This leaves a large number of unfused cells, both
spleen and myeloma cells still present in the culture.
The spleen cells from the mouse die within 3 days of
culture and therefore, do not pose a problem.
However, the myeloma cells quickly adapt to the
culture conditions and will outgrow the hybridoma
cells resulting from the fusion.
Removal of the myeloma cells is therefore, essential
and is achieved by chemical selection.
Commercially available myeloma cells are defective in
one of the enzymes of the salvage pathway of purine
nucleotide biosynthesis.
Cell lines 653 and SP2 have mutations of
hypoxanthine-guanine phosphoribosyl transferase
(HGPRT) gene.
Addition of aminopterin to the culture medium blocks
the de novo nucleotide synthesis pathway and forces
the cell to use the salvage pathway in which HGPRT
uses exogenous hypoxanthine and thymidine.
Myeloma cells defective in HGPRT are unable to use
this pathway and therefore, die in culture.
The only cells able to grow in HAT (hypoxanthine,
aminopterin, thymidine) culture medium are the
hybridoma cells, which are unable to synthesize DNA
via de novo nucleotide synthetase pathway and rely
on the salvage pathway for DNA synthesis (a
characteristic provided by the spleen cell part of the
hybridoma).

Positive clones producing specific antibodies are


usually identified by ELISA and are selected, expanded
and cloned using a limiting dilution technique.
Positive hybridomas are normally cloned three times
before they are considered MAb producing cells.
Application of Monoclonal Antibodies in Fish Farming

Our knowledge of the immune system of fish and fish


diseases is extremely limited when compared to our
knowledge of large animals.
At present, fish farming is becoming an increasingly
important food production industry, and may play a
significant role as a food source in the future.
For this reason, application of the latest
biotechnological advances, including MAbs, to the
aquaculture industry, is extremely important.
MAbs are being adopted for purposes of
immunoassay and immunotherapy.
Though the technology for MAb production has been in
existence for more than 25 years, yet this application to
fish farming is still in its infancy.
Today, monoclonal antibodies to several viral and bacterial
pathogens of fish and shellfish are available in the market.
It has been possible to develop rapid, simple, cheap,
specific and sensitive MAb based immunodiagnostic kits
for several microbial pathogens.
MAb based diagnostic kits such as ELISA and immunodot
have even been simplified to the field level for use by
farmers.
Furthermore, detection of minute serological difference
among bacterial and viral variants of fish and shellfish is
possible by MAb based epitope analysis.
This has helped immensely in serological and
epidemiological studies.
Monoclonal antibodies were produced against
enterotoxin of Vibrio cholerae, a brackishwater
and estuarine bacterium which causes cholera.
MAbs based ELISAs have been used for studies of
Vibrio anguillarum strains and for rapid diagnosis
of clinical cases of Enteric Red mouth (Yersinia
ruckeri) and furunculosis (Aeromonas
salmonicidae) in fish farms.
MAbs are also used to study piscine parasities.
MAbs have been developed against Bonamia
ostreae, Ceratomyxa shastia, Cryptobia
salmonsitica, Perkinsus maximus are pathogenic
protozoan of shell fish.
MAbs have also been employed for analysis of
lymphocyte receptors and characterization of
lymphocyte population in carp,
for immunopurification of salmon prolactin and for
development of sandwich ELISA system for both salmon
prolactin and somatotropin.
MAbs to A. hydrophila, EUS fungus Aphanomyces
invadans and white spot virus of shrimp have been
produced and being used in diagnosis in India.
The use of monoclonal antibody techniques would
prove extremely useful in the development of
diagnostic tests for certain important diseases of fish,
whose incidence is likely to increase with the higher
stocking densities necessary for successful fish farming.
1. E.g. Detection of Infectious Pancreatic
Necrosis virus ( IPNV ) by ELISA. ELISA could
be used for the identification of different
serotype of IPNV.
2. Application of a MAb against virus:-
Infectious hematopoietic necrosis (IHN),
caused by IHN virus (IHNV), is a severe and
acute epizootic among salmonid fish. This
disease is now widespread. MAbs against
IHNV HV - 7601, were produced.
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