The paper describes the superiority of monoclonal antibodies (MAb) over conventional polyclonal antisera. Studies undertaken indicate that Aeromonas hydrophila isolates are highly heterogenous and variation exists even between isolates from a farm, requiring a large number of MAbs for classification and use of information in vaccine development. However, some of the MAbs could be used for detection of homologous isolates in fish kidney by immunodot assay and evaluation and standardization of biofilm of A. hydrophila for oral vaccination in carps

Anil, T.M.

Mohan, C.V.

Shankar, K.M.

Vidhya, R.

English

Aquatic Commons

10 Naga, The ICLARM Quarterly (Vol. 23, No. 4) October-December 2000
Aquaculture Section
of the Network of Tropical Aquaculture
and Fisheries Professionals (NTAFP)Editor : M.V. Gupta
Abstract
The paper describes the superiority of monoclonal antibodies (MAb) over conventional polyclonal antisera.
Studies undertaken indicate that Aeromonas hydrophila isolates are highly heterogenous and variation exists even
between isolates from a farm, requiring a large number of MAbs for classification and use of information in vaccine
development. However, some of the MAbs could be used for detection of homologous isolates in fish kidney by
immunodot assay and evaluation and standardization of biofilm of A.hydrophila for oral vaccination in carps.
Monoclonal Antibodies in Fish and
Shellfish Health Management in India
K.M. Shankar, C.V. Mohan, T.M. Anil  and R. Vidhya
Introduction
Fish and shellfish aquaculture
has been established as an industry
in India in the last decade. Indian
major carps (Catla catla, Labeo
rohita and Cirhinus mrigala) are
cultured on a large scale particularly
in the states of Andhra Pradesh,
Uttar Pradesh, Punjab and Haryana.
In Andhra Pradesh alone, more than
40,000 tonnes of Indian major carp
are raised in farms annually, valued
at more than Rs. 600 million1 .
Shrimp are cultured in al l
maritime states of India, covering
about 100 700 ha. Due to intensive
culture of fish and shellfish, diseases
of microbial etiology of economical
significance have surfaced in rearing
and growout ponds. Annual losses
due to diseases in carp culture in the
state of Andhra Pradesh are
estimated at about Rs. 40 million.
Viral disease in shrimp culture has
become a stumbling block and the
economic loss was estimated at
about Rs. 9 450 million in 1995-96.
Hence, to sustain the aquaculture
industry, appropriate health
management measures involving
development of diagnostics and
vaccines are necessary. Deve-
lopment of specific, sensitive and
rapid diagnostic methods are
essential to detect the various stage
of disease such as peracute, acute to
sub acute and chronic infection. In
addition, there is a need to develop
simple and sensitive methods for
stereotyping pathogens for
epidemiological study. At present
diagnosis/stereotyping is performed
in many laboratories using the
conventional biochemical methods
which are tedious, expensive and not
sufficiently sensitive to differentiate
the large number of heterogeneous
isolates. Another area in health
management, which needs greater
attention, is the development of
effective vaccines for disease
prevention. Since a large number of
isolates of different virulence and
stereotype exists in a disease
situation, there is a need to detect
common immunogenic antigens
shared by several isolates to develop
an appropriate polyvalent vaccine.
Antibodies in
health management
The importance of antibodies in
health management measures such
as diagnosis, identification, stereo-
typing, antigen characterization,
epidemiology and vaccine
development is well known.
Conventionally, polyclonal anti-
bodies and antigen preparations
raised in rabbits are used in disease
diagnosis, stereotyping and other
biological applications. However,
due to the limitation of polyclonal
antisera such as cross reaction,
limited quantity, unwanted
background reactions and inability
_______________
1 US$ 1= Rs 47 (May 2001)
AQUABYTE
11Naga, The ICLARM Quarterly (Vol. 23, No. 4) October-December 2000
to discriminate antigen at epitope
level, monoclonal antibodies (MAb)
are becoming increasingly popular.
The monoclonal antibodies are
superior in a number of aspects to
conventional polyclonal antisera
(Table 1).
Since their initial description by
Kohler and Mielstein in 1975, MAbs
have made a profound impression in
all areas of biology and bio-
technology. MAbs production
involves obtaining  antibody
producing lymphoid cells from
immunized  mice. These cells are
then immortalized by hybridization
with an established myeloma cell
line followed by separation of
individual hybridoma clones. The
clones obtained are grown in tissue
culture, so that the MAbs produced
by them can be harvested. A
schematic illustration of the
production of monoclonal
antibodies is given in Fig. 1.
MAbs are widely used in the
diagnosis, stereotyping and analysis
of antigens in microbial pathogens
in medical and veterinary sciences.
Currently, they are also used in
disease research and health
management in aquaculture systems
of developed countries. However,
their application in commercial
aquaculture systems in developing
countries is yet to be implemented
Fig. 1. Schematic illustration of production of monoclonal antibodies.
at any significant level. The Fish
Pathology and Biotechnology
Laboratory of the Department of
Aquaculture, College of Fisheries,
Mangalore, established the first
hybridoma laboratory in fisheries in
India in 1993 with funding support
from the Department of
Biotechnology, Government of
India, and initiated a project on
monoclonal antibodies to
Aeromonas hydrophila. Among the
fish bacterial diseases in freshwater,
bacterial hemorrhagic septicemia
caused by A.hydrophila is a major
concern, causing a mortality rate of
30-70% in rearing ponds. Several
isolates of the pathogen are also
associated with human and other
aquatic vertebrate diseases. The
pathogen is highly heterogeneous
biochemically, phenotypically,
a
q
u
a
b
y
t
e
Table 1. Comparison between conventional antiserum and monoclonal antibody.
Conventional antiserum Monoclonal antibody
Determinant Several Single
Specificity Variable with animal and bleed Standard
Partial cross-reactions with common Unexpected cross reactions may occur;
determinants May be too specific for requirements
Seldom too specific
Affinity Variable with bleed May be specific during cloning
Yield of useful antibody Up to 1 mg/ml Up to 100 mg/ml in tissue culture; up to
20 mg/ml in acidic fluid
Contaminating immunoglobulin Up to 100% None in culture; 10% in acidic fluid
Purity of antigen Either pure antigen or serum absorption Some degree of antigen purification
desirable but not essential
Spleen Cells Myeloma Cells
Fusion employing
Polyethylene GlycolImmunisation of Balb / c Mice
with Antigen
Production of MAbs in vitro
Recloning
Screening of Hybridoma
Clones by ELISA
Hybridomas in
Tissue Culture Plates
Production of MAbs in vivo (Ascites)
MONOLCLONAL
ANTIBODIES
12 Naga, The ICLARM Quarterly (Vol. 23, No. 4) October-December 2000
serologically and in virulence.
Under this project, a panel of five
MAbs recognizing various epitomes
of A.hydrophila on lipopoly-
saccharides of the cell wall was
raised. These antibodies were
employed first for development of
an ELISA/immunodot for epitope
analysis of the isolates collected
from several hosts/environments of
the country. Epitopes shared or
otherwise by the different isolates
were then identified for serotyping
and determining the antigenic
relationship between isolates from
different regions and hosts. The
result indicated that the A.hydrophila
isolates are highly heterogeneous
and variation exists even between
isolates from a farm. Hence a battery
of a large number of MAbs is
required for classification. The
information should then be used in
vaccine development. Although a
MAb recognizing  a common
epitope of A.hydrophila could not be
obtained, some of the MAbs could
be used for detection of homologous
isolates in fish kidney by
immunodot assay (Fig. 2). Further,
MAbs could be conveniently used
for evaluation and standardization of
biofilm of A.hydrophila for oral
vaccination in carps. A biofilm
preparation of A.hydrophila was
found to be superior to a
conventional free cell preparation
(Azad et al. 1997, in press) which
was also confirmed by localization
of the vaccine antigen by MAb
based immunoflouresence.
Conclusion
Understanding a pathogen itself
is a main step in disease
Fig. 2. Detection of a A.hydrophila in kidney of Indian major carp by MAb based
immunodot assay.
management. Therefore, there is
scope for the application of MAbs
in fish and shellfish health
management in the Indian
subcontinent. At present, MAbs of
white spot syndrome virus (WSSV)
and Aphanomyces invadan
associated with EUS are being
produced mainly for diagnosis and
epidemiological analysis.
Acknowledgement
Authors acknowledge funding
support from the Department of
Biotechnology, New Delhi and
International Foundation for
Science-Sweden.
References
Azad, I.S., K.M. Shankar and C.V. Mohan.
1997. Evaluation of Aeromonas
hydrophila for oral vaccination of carp.
In T.W. Flegel and I.H. Macrae (eds.),
Diseases in Asian Aquaculture III. Fish
health section, Asian Fisheries Society,
Manila, p. 181-185
Azad, I.S., K.M. Shankar and C.V. Mohan
and B. Kalita. (In press). Uptake and
processing of biofilm and free cell
vaccine of Aeromonas hydrophila in
Indian major carps and common carp
following oral vaccination-antigen
localization by monoclonal antibodies.
Dis. Aquat. Organism
__________________
K.M. Shankar, C.V. Mohan, T.M.
Anil and R. Vidhya are from the
Fish Pathology and Biotechnology
Laboratory, Department of
Aquaculture, College of Fisheries,
Mangalore-575 002, India.


Monoclonal antibodies in fish and shellfish health management in India

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