www.tjprc.org editor@tjprc.

org

International Journal of Nanotechnology
and Application (IJNA)
ISSN(P): 2277-4777; ISSN(E): 2278-9391
Vol. 4, Issue 3, Jun 2014, 9-20
TJPRC Pvt. Ltd.


COLLOIDAL NANOGOLD IMMUNE ASSAY FOR RAPID DETECTION OF
EDWARDSIELLA TARDA IN LABEO ROHITA
CHANDRA BHUSHAN KUMAR
1
, KURCHETI PANI PRASAD
2
, K. H. D. T. KASAGALA
3
, AYATHULLAH
RABBANI SYED
4
, KUNDAN KUMAR
5
& GAYATRI TRIPATHI
6

1,2,3,4
Central Institute of Fisheries Education, Off Yari Road, Panch Marg, Versova,
Mumbai, India
5,6
Senior Scientist, Central Institute of Fisheries Education, Mumbai, Maharashtra, India

ABSTRACT
Edwardsiella tarda infections resulting in important economic losses have been reported from a variety of
cultured fish in Asia, especially India and Japan. Disease is enzootic in different parts of India also. To date, the disease
has been reported from various fish species, including channel catfish, Japanese eel, mullet, tilapia, Chinook Salmon,
Flounder, common carp, stripped bass, turbot, koi, European eel and Senegalese sole. The symptoms are lethargy, "hang"
at the surface, and swim in a spiraling or erratic pattern. The fish often develop small, cutaneous ulcerations; in advanced
cases, however, larger depigmented areas mark the sites of deep muscle abscesses. Detection of a disease causing organism
is the pre-requisite for the diagnosis of any disease. The diagnosis of E. tarda is based on isolation and identification of
etiological agent. Conventional plating and culturing methods commonly used to identify causative bacterial pathogens
have a low degree of accuracy and conclusive results typically require periods of 72 h or more. Although histopathology,
haematology, molecular diagnosis, Dot ELISA, indirect or competitive ELISA and latex agglutination test have been used
for the detection of E. tarda, these processes require time and sophisticated equipment for development of the tests. Sero-
diagnosis is a routine methodology in diagnosis. There is a constant need to improve the performance of current diagnostic
assays as well as develop innovative testing strategies to meet new testing challenges. The nanoparticles promise to help
promote in vitro diagnostics to the next level of performance. Quantum dots (QDs), gold nanoparticles (GNPs) and
superparamagnetic nanoparticles are the most promising nanostructures for in vitro diagnostic applications. Colloidal gold
conjugates have found widespread use due to their high stability and unique optical properties of GNP (Jain et al., 2006).
Nano size is within the size range of biomolecules and cellular organelles. This would allow one-on-one interaction
between the nanoparticles and the biomolecules of interest. Furthermore, nanoscale surfaces provided by colloidal gold
particles could accelerate antibody-antigen reaction sufficiently, which provide an amplified signal for immunoassay.
Especially, the results can be read directly without the use of instruments, which ensures the convenience of assay on-site.
It has been reported that although, the use of polyclonal antibody generated by immunization with whole bacteria as
antigen is associated with the problem of low specificity, cross reactivity hamper the application of polyclonal antibodies
but surprisingly, coupling of polyclonal antibodies with colloidal gold particles results in considerably high specificity of
detection. By considering these, an attempt was made to develop anti E. tarda rabbit IgG gold conjugate to detect E. tarda
in fishes. Herein we report the development of a specific immunoassay using gold-conjugated polyclonal antibodies for the
rapid detection of E. tarda. Positive reactions of development of purple red agglutination indicated presence of E. tarda.
10 Chandra Bhushan Kumar, Kurcheti Pani Prasad, K. H. D. T. Kasagala, Ayathullah Rabbani Syed, Kundan Kumar & Gayatri Tripathi

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0

The other bacteria gave a negative reaction indicating the specificity of the test developed. The antibody coated particles
were stable upto 21 weeks at 4
0
C and detected 10
5
CFU/ml.
KEYWORDS: Edwardsiella tarda, Cutaneous, Nanoscale, Nanoparticles
INTRODUCTION
Edwardsiella tarda infections resulting in important economic losses have been reported from a variety of
cultured fish in Asia, especially India and Japan (Herman and Bullock, 1986). Swain et al., (2003) collected and analysed
serum samples of L. rohita from Kolleru lake area of Andhra Pradesh, India and ponds of Central Institute of Freshwater
Aquaculture, India, which were showing typical signs of E. tarda infection. Disease is enzootic in different parts of India.
To date, the disease has been reported from various fish species, including channel catfish (Mayer and Bullock, 1973),
Japanese eel (Egusa, 1976), mullet (Kusuda et al., 1976), tilapia (Kubota et al., 1981), Chinook Salmon (Amandi et al.,
1982) flounder (Nakatsugawa, 1983), common carp (Sie-Qui et al., 1984), stripped bass (Herman and Bullock,1986),
turbot (Nougayrede et al., 1994), koi (Sahoo et al., 2000), European eel (Alcaide et al., 2006) and Senegalese sole (Castro
et al., 2012). The symptoms are lethargy, "hang" at the surface, and swim in a spiraling or erratic pattern. The fish often
develop small, cutaneous ulcerations; in advanced cases, however, larger depigmented areas mark the sites of deep muscle
abscesses (Meyer and Bullock 1973). Histologically, the lesions are characterized by focal necrosis often extending from
muscle, haemopoietic tissue and liver to parenchyma to perforate the abdominal wall (Miyazaki and Egusa, 1976). E. tarda
infection causes hypertrophy of the liver cells and enlargement of their nuclei (Miwa and Mano, 2000). Sahoo et al. (2000)
reported liquefaction and gaseous necrosis in the kidney.
Detection of a disease causing organism is the pre-requisite for the diagnosis of any disease. The diagnosis of E.
tarda is based on isolation and identification of etiological agent. Conventional plating and culturing methods commonly
used to identify causative bacterial pathogens have a low degree of accuracy and conclusive results typically require
periods of 72 h or more. Although histopathology, haematology, molecular diagnosis, Dot ELISA, indirect or competitive
ELISA and latex agglutination test have been used for the detection of E. tarda, these processes require time and
sophisticated equipment for development of the tests. Sero-diagnosis is a routine methodology in diagnosis. There is a
constant need to improve the performance of current diagnostic assays as well as develop innovative testing strategies to
meet new testing challenges. The nanoparticles promise to help promote in vitro diagnostics to the next level of
performance. Quantum dots (QDs), gold nanoparticles (GNPs) and superparamagnetic nanoparticles are the most
promising nanostructures for in vitro diagnostic applications.
Colloidal gold conjugates have found widespread use due to their high stability and unique optical properties of
GNP (Jain et al., 2006). Nano size is within the size range of biomolecules and cellular organelles. This would allow one-
on-one interaction between the nanoparticles and the biomolecules of interest (Azzay et al., 2006, 2007; Jain, 2005).
Furthermore, nanoscale surfaces provided by colloidal gold particles could accelerate antibody-antigen reaction
sufficiently, which provide an amplified signal for immunoassay (Faulk and Taylor, 1971; Richars, 1996). Especially, the
results can be read directly without the use of instruments, which ensures the convenience of assay on-site. It has been
reported that although, the use of polyclonal antibody generated by immunization with whole bacteria as antigen is
associated with the problem of low specificity, cross reactivity hamper the application of polyclonal antibodies but
surprisingly, coupling of polyclonal antibodies with colloidal gold particles results in considerably high specificity of
Colloidal Nanogold Immune Assay for Rapid Detection of Edwardsiella Tarda in Labeo Rohita 11

www.tjprc.org editor@tjprc.org

detection (Ying et al., 2010). By considering these, an attempt was made to develop anti E. tarda rabbit IgG gold conjugate
to detect E. tarda in fishes.
MATERIALS AND METHODS
Identification of E. tarda
E. tarda reference stain (ATCC-15947) and field strain from Aquatic Animal Health laboratory, CIFE, Mumbai,
India were used for the experiment. The stock of E. tarda was maintained at -20
o
C in 20% glycerol. Pure culture of
bacteria was cultured from stock by using BHI broth, BHI agar and Salmonella shigella agar. Pure culture stock was stored
at 4
o
C, until further use. All tests conducted were according to the procedure for identification methods of E. tarda by
Bergeys Manual of Systemic Bacteriology and VITEK32 system.
Production of Anti E. tarda Rabbit Sreum
Anti E. tarda rabbit hyperimmune serum was raised in rabbits using formalin killed whole cell of E. tarda
organisms as per the procedure of Dresser (1986). Clinically healthy New Zealand rabbit of 8-10 months old weighing
approximately 2.0 kg were used for the experiment. Prior to immunization, rabbits were bled through marginal ear vein
and blood was collected. The serum was separated from the blood and tested by agglutination test for the presence or
absence of anti E. tarda antibodies. This serum served as a control. The rabbits were acclimatized in the animal house of
CIFE. For the production of antibodies against whole cells of E. tarda, the PBS containing 1.0 X 10
9
colony-forming units
per mililiter (CFU/ml) of E. tarda was injected intravenously through the marginal ear vein as per the following schedule.
Day of Inoculation Dose
No. Of
CFU/ml
Route of
Inoculation
1 0.1 ml 1 x 10
9
Intravenous
3 0.2 ml 1 x 10
9
Intravenous
7 0.3 ml 1 x 10
9
Intravenous
11 0.4 ml 1 x 10
9
Intravenous
15 0.5 ml 1 x 10
9
Intravenous

Blood was collected as per method of Gupta and Arunan (1992). The rabbit was bled one week after the last
injection through the marginal ear vein. Aliquots of blood kept overnight at 4
0
C and

serum was separated by centrifugation
at 1,500 g for 10 min. The serum was stored in 1 ml aliquots at -20
0
C in Eppendorf tubes, until further use. Titration of
antisera was conducted in U shaped microtitre plates, as per procedure of Swain et al., 2003. The titre was calculated as the
reciprocal of the highest dilution of serum showing complete agglutination.
Separation of anti E. tarda immunoglobulin G (IgG)
The separation of anti E. tarda rabbit IgG was performed by using GeNei
TM
IgG Purification Kit. (Cat no. KT16A,
Bangalore Genie, India), as per manufactures instructions.
Preparation of Colloidal Gold Reagents
Colloidal gold reagents were prepared according to procedure followed by Upadhyay (1992) with slight
modifications. Colloidal gold solution of 10 nm size was procured from Sigma Aldrich, USA (Catalogue No. G.1527,
~0.01% HAuCl
4
, ~1 A
520
units/mL, 8.5-12.0 nm mean particle size (monodisperse),
12 Chandra Bhushan Kumar, Kurcheti Pani Prasad, K. H. D. T. Kasagala, Ayathullah Rabbani Syed, Kundan Kumar & Gayatri Tripathi

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0

SDS Polyacrylamide Gel Electrophoresis (SDS PAGE)
SDS-PAGE was carried out by using the method described by Laemmli (1970). The purified anti E. tarda
immunoglobulin G (IgG) was analyzed on a 12% gel for purity of IgG.
Determination of Optimal Condition of pH and Antibody Concentration for Coating Gold Nanoparticles
Stability of the colloidal gold was determined by placing 7 l of 100mM phosphate buffer (pH 6.5-8.5 in 0.5 unit
steps) to 93 l of gold solution in wells of a microtest plates and mixed by shaking the plate. The stability is assessed by
change in colour. The pH of the colloidal gold solution was adjusted by using 25mM potassium carbonate (K
2
CO
3
)

for
effective conjugation with immunoglobulins. 100 l of gold solution was dispensed into wells of microtitre plate. The final
concentration of the IgG was adjusted to 1g/l. 1, 2, 5, 10, 15, 20, 25 and 30 l of IgG solution was added serially to each
well containing gold solution. They were mixed thoroughly and allowed to stand for 15 min. Then 10 l of 10% sodium
chloride (NaCl) solution was added to all the wells. They were mixed and checked for the change in colour. Optimum
conditions of pH and antibody concentration for the coating was determined by comparing the absorption between 520 and
580nm as per the procedure of (Xiulan et al., 2005), where blue colour indicated flocculation and no change in colour
denotes stability (red colour).
Preparation of ant E. tarda IgG-coated Gold Nanoparticles
Anti E. tarda IgG coated gold nanoparticles were prepared as per the procedure of Zhao et al. (2008) with slight
modifications. Briefly, the pH of 1ml colloidal gold was adjusted to pH 8.0, using 25mM potassium carbonate (K
2
CO
3
) to
which 20 g/ l anti E. tarda IgG added. They were stirred vigorously and incubated for 30 min at room temperature. Then
100 l of 10% bovine serum albumin (BSA) was added and mixture was further incubated for 10 min. The suspension was
washed with washing solution [PBS pH 8.0, containing 1% (v/v) BSA] by centrifugation at 20,000 g for one hour at 4
0
C.
The supernatant was discarded and the pellet resuspended in 1ml washing buffer. The gold conjugate was stored at 4
0
C,
until further use.
Detection of E. tarda from Infected Fish by using Antibody-coated Gold Nanoparticles Immunoassay
E. tarda infected L. rohita fish weighing 90-100 g were dissected aseptically and inoculums from kidney, liver
and ascitic fluid were inoculated on BHI petri plates and incubated at 28
0
C for 24 h. The bacterial colonies were harvested
from petri plates and suspended in PBS. Forty microlitres from each bacterial suspension were mixed with 100 l of
antibody-coated GNPs in a microcentrifuge tube. Negative controls were taken for the study, in one control 40 l of sterile
PBS was mixed with 100 l antibody-coated GNPs and another one by adding 40 l of bacterial suspension to 100 l of
uncoated GNPs. Colour change and agglutination of GNPs indicates a positive result. Presence of E. tarda in bacterial
suspension was verified by using biochemical tests also.
Specificity of E. tarda Antibody Coated Colloidal Gold Immunoassay
The specificity of antibody-coated GNPs solution was tested with other common fish bacteria viz Aeromonas
hydrophila, Pseudomonas aeuroginosa and Vibrio alginolyticus and tissue extracts of healthy fish. The 24 h cultures of
bacterial suspension from respective media were serially diluted in PBS, pH 7.0. The PBS containing 1 x 10
6
CFU mL
-1

was added to E. tarda coated and uncoated GNPs.
Colloidal Nanogold Immune Assay for Rapid Detection of Edwardsiella Tarda in Labeo Rohita 13

www.tjprc.org editor@tjprc.org

Sensitivity of E. tarda Antibody Coated Colloidal Gold Immunoassay
Bacterial culture of E. tarda was serially diluted 10 folds in PBS, pH 7. 0.1 ml from each dilution was inoculated
in brain heart infusion and incubated at 28
0
C. Plates containing 30-300 colonies were selected to estimate colony-forming
unit per millilitre (CFU mL
-1
) of the initial concentration. Each bacterial dilution was tested with E. tarda antibody-coated
GNPs to estimate the sensitivity of assay. The highest bacterial dilution which demonstrated agglutination of GNPs was
considered the lowest detection limit of the E. tarda immunoassay.
Shelf life of anti E. tarda Rabbit IgG Gold Conjugate
The stability of the reagent developed was assessed by storing the reagent at different temperatures viz 37
0
C,
24
0
C and 4
0
C.
RESULTS
The two strains, field strain and ATCC 15947 were confirmed biochemically and by VITEK 32 system confirmed
to E. tarda. The results of various biochemical tests are shown in Table 1. Bacteria were enumerated by TPC before
immunization of rabbit. The number of bacteria was found to be 1.09 X 10
9
CFU/ml. The titre of anti E. tarda rabbit serum
was found to be 1:2048. Anti E. tarda rabbit IgG separated by GeNei
TM
IgG Purification Kit. The OD at 280 nm was
plotted against the eluted fraction of antibody in their respective tubes (Fig 1). The tubes containing highest absorbance
(A
280nm
0.4) were only taken for IgG elute. The fraction representing the first peak was tested for the purity of IgG by
SDS PAGE (Fig 5). Fractions containing IgG were measured at 280 nm. IgG concentration was determined as 1.12 using
1.45 as extinction coefficient (A
280
/1.45=concentration in mg/ml) (Fremy and Usleber, 2003; Gasparyan, 2005). The
stability of colloidal gold was found to be optimum at pH 8.0 (Fig 2.). The optimum amount of IgG required to stabilize
the colloidal gold was 20 g/ml of gold solution (Fig 3). All the tissue homogenates and inoculum from different organs of
infected fish were tested positive for the presence of E. tarda. The detection of E. tarda was confirmed by antibody-coated
GNPs immunoassay. The positive results produced reddish purple agglutination, while no agglutination was observed in
the negative control sample. No reddish purple agglutination was observed using uncoated GNPs with E. tarda (Fig 6).
Agglutination in tube no. 2, also confirmed by change in absorption (Fig 4). Colour change from reddish to bluish was
observed 10-12 minutes, while agglutination formation taken after 90 minutes of incubation in a microcentrifuge tube.
The absence of reddish purple agglutination with other fish bacteria and tissue homogenates of healthy fish was
also confirmed by this immunoassay. The assay was able to detect lower detection limit upto 1X 10
5
cfu/ml E. tarda.
The result of stability of kit at different temperatures viz 37
0
C, 24
0
C and 4
0
C showed that kit is stable both at 24
0
C and
4
0
C, but at 4
0
C for longer period of time ie for 21 weeks (Table 2)
DISCUSSIONS
The present study successfully developed immunoassay of 10 nm colloidal gold that can detect E. tarda.
The positive reaction was visible with the naked eye in the form of colour change in 10-12 minutes and agglutination
within 90 min of incubation at room temperature (24-28
0
C). For production of polyclonal antibodies, identification of
E. tarda reference stain (ATCC-15947) and field strain was confirmed by biochemical tests and VITEK 32 system.
The results obtained by biochemical tests and VITEK32 system were similar to the results of Mohanty et al. (2007) and
14 Chandra Bhushan Kumar, Kurcheti Pani Prasad, K. H. D. T. Kasagala, Ayathullah Rabbani Syed, Kundan Kumar & Gayatri Tripathi

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0

Panangala et al. (2006) respectively. The results of titration of antisera confirmed the presence of E. tarda antibodies in the
sera. The agglutination procedures in U shaped microtitre plate were in conformity to the Swain et al., 2003.
The concentration of anti E. tarda rabbit IgG found was close to the concentration found by Rudolf et al., 2009.
The purity of eluted IgG by SDS PAGE, heavy chain and light chain was similar to the well-known structure given by
Carayannopoulos and Capra, 1993 (Fig 5). After adding different concentrations of antibody/ml gold solution, sodium
chloride was added to identify the antibody coverage rate of the colloidal gold particles. If not, the whole gold nanoparticle
surface is covered with antibody and therefore free negatively charged binding sites remain, then the addition of positively
charged sodium ions leads to agglomeration of gold particles and due to this reaction a change in absorption maximum
occurs. The evaluation of the photometrical measurement allows an estimation of the optimal coupling ratio
(Rudolf et al., 2009). The optimum pH for GNPs and optimum amount of E. tarda specific polyclonal antibodies to
stabilize the GNPs was estimated by the minimum concentration of antibody that did not change the colour of solution
from red to blue after addition of 10% NaCl. The methods used for this were as per Upadhayay (1992) and stability
checked by comparing the absorbance between 520 and 580 nm (A520-A580) (Sun Xiulan et al., 2005). Anti E. tarda
rabbit IgG at the rate of 20 g/ml for colloidal gold was found to be optimum during the study but De Mey (1981a,b)
reported that 450 g/ml to 30 nm gold particles. De (1983) used 1mg/ml and Lucocq and Roth (1984) used 609 g/10ml of
14 nm colloidal gold and Smita et al., 2010 estimated 10 g/ml antibody for 40 nm colloidal gold were optimum
concentration for conjugation and stabilization. Thobhani et al. (2010) found that the conjugation, stability and
performance of gold bio-conjugates were dependent on the chemical nature, pH and concentration of buffering system
employed. If either the nanoparticles or antibody is changed, then the optimal conjugation condition will also differ. An
optimised protocol for one system will not necessarily be the correct one for another one. The specificity of immunoassay
was confirmed by the observation of reddish purple agglutination with the E. tarda type strain (ATCC 15947) and with the
field strain. There was no agglutination observed with other common fish bacterial pathogens. When E. tarda bacterial
suspensions and fish tissue extracts containing E. tarda were tested with GNPs without coated polyclonal antibody also
showed no agglutination. There by, developed kit confirmed the specificity of the assay. The E. tarda immunoassay
developed in this study is easy to perform and has the ability to detect as little as 10
5
CFU/ml in fish tissues. Moreover,
agglutination could be visually inspected with the naked eye without the aid of instruments. The kits developed is also
stable upto 21 weeks at 4
0
C. Further studies are required to increase the stability.
CONCLUSION
A kit to detect using anti E. tarda rabbit IgG colloidal gold conjugate to detect E. tarda in fishes was developed.
The developed kit confirmed the specificity and rapidity of the assay. The E. tarda immunoassay developed in this study is
easy to perform and has the ability to detect as little as 105 CFU/ml in fish tissues. Moreover, agglutination could be
visually observed with the naked eye without the aid of instruments. The kits are stable upto 21 weeks when stored at 40C.
ACKNOWLEDGEMENTS
The authors acknowledge with thanks Dr. W. S. Lakra, Director/Vice Chancellor for the encouragement and help
extended in completing the research work.

Colloidal Nanogold Immune Assay for Rapid Detection of Edwardsiella Tarda in Labeo Rohita 15

www.tjprc.org editor@tjprc.org

REFERENCES
1 Alcaide, E., Herraiz, S. and Esteve, C., 2006. Occurrence of Edwardsiella tarda in wild European eels Anguilla
anguilla from Mediterranean Spain. Dis. Aquat. Org., 73:77-81.
2 Amandi, A., Hiu, S. F., Rohovec, J. S. and Fryer, J. L., 1982. Isolation and characterization of Edwardsiella tarda
from fall Chinook salmon (Oncorhynchus tshawytscha). Appl. Environ. Microbiol., 43:1380-1384.
3 Azzazy, H. M., Masour M. M. and Kazmierczak, S. C., 2006. Nanodiagnostics: a new frontier for clinical
laboratory medicine. Clin. Chem., 52:1238-46.
4 Azzazy, H. M., Masour M. M. and Kazmierczak, S. C., 2007. From diagnostics to theory: prospects of quantom
dots. Clin. Biochem., 40:917-27.
5 Carayannopoulos, L. and Capra, J. D., 1993. Immunoglobulins: structure and function. Fundament. Immunol.
Paul, W. E., Editor. Raven press, New York. 283-314.
6 Castro, N., Toranzo, A. E., Devesa, S., Gonzalez, A., Nunez, S. and Magarinos, B., 2012. First description of
Edwardsiella tarda in Senegalese sole, Solea senegalensis (Kaup). J. Fish Dis., 35: 79-82.
7 De mey, J., Moeremans, M., De Waele, M., Geuens, G. and De Brabander, M., 1981b.

In (Eds.), Peeters. Protides
of the Biological Fluids. J Cell Biol Inter Rep., 29: 943-947.
8 De Mey, J., Moeremans, M., Geuens, G., Nuydens, R. and De Bradander, M., 1981a. Protides of the Biological
Fluids. Cell Biol Inter Rep., 5: 889-899.
9 De Waele, M., De May, J., Moeremans, M., Smet, L., Broodtaerts, L. and Van Camp, B., 1983. Cytochemical
profiles of Immunoregulatory T- Lymphocyte Subsets defined by Monoclonal Antibodies. J.
Histochem. Cytochem., 31(4): 471-478.
10 Dresser, D. W., 1986. Immunization of experimental animals. In Weir, D.M. (ed.), Handbook of Experimental
Immunology. (IV edn.), vol I, Blackwell scientific publication, Oxford, 8.9: 16-17.
11 Egusa, S., 1976. Some bacterial diseases of freshwater fishes in Japan. Fish pathol., 10: 103.
12 Faulk, W. P. and Taylor, G. M., 1971. An immunocolloid method for the electron microscope. Immunochem.,
8:1081-1083.
13 Fermy, J. M., Usleber, E., 2003. Policy on characterization of antibodies used in immunochemical methods of
analysis for mycotoxins and phytotoxins. J. AOAC Int. 86: 4-868.
14 Gasparyan, V. K., 2005. Hen egg immunoglobin Y in colloidal gold agglutination assay: comparison with rabbit
immunoglobin G. J. Clin. Lab. Anl. 19:124
15 Gupta, S. K. and Arunan, K., 1992. Generation of Antisera - Techniques of immunization, use of Adjuvants. In
Talwar, G. P and Gupta, S. K., (Eds), A Handbook of Practical and Clinical Immunology. (II Edn.), Vikas
publishing House, New Delhi. Pp. 194-208.
16 Chandra Bhushan Kumar, Kurcheti Pani Prasad, K. H. D. T. Kasagala, Ayathullah Rabbani Syed, Kundan Kumar & Gayatri Tripathi

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0

16 Herman, R. L. and Bullock, G. L., 1986. Pathology caused by the bacterium Edwardsiella tarda in striped bass.
Trans. Amer. Fish. Soc., 115:232-235.
17 Herman, R. L. and Bullock, G. L., 1986. Pathology caused by the bacterium Edwardsiella tarda in striped bass.
Trans. Amer. Fish. Soc., 115:232-235.
18 Jain, K. K., 2005. Nanotechnology in clinical laboratory diagnostics. Clin. Chim. Acta., pp. 358:37-54
19 Jain, P. K., Lee, K. S., El-Sayed, I. H. and El-Sayed, M. A., 2006. Calculated absorption and scattering properties
of gold nanoparticles of different size, shape, and composition: applications in biological imaging and
biomedicine. J. Phys. Chem. B., 110:7238.
20 Kubota,S.S., Kaige, N., Miyazaki, T. and Miyashita, T., 1981. Histopathological studies on Edwardsiellosis of
Tilapia-1 natural infection. Bull. Fac. Fish., Mie University, 9: 155-156.
21 Kusuda, R., Toyoshima, T., Iwamura, Y. and Sano, H., 1976. Edwardsiella tarda from an epizootic of mullets
(Mugil cephalus) in Okistu Bay. Bull. Japan. Soc. Scient. Fish., 42: 271-275.
22 Lucocq, M. and Roth, J., 1984. Application of immunocolloids in light microscopy III Demonstration of
Antigenic and Lection binding sites in semithin resin sections. J. Histochem. Cytochem., 32(10): 1075-83.
23 Meyer, F. P. and Bullock, G. L., 1973. Edwardsiella tarda, a new pathogen of channel catfish (Ictalurus
punctatus). Appl. Microbiol., 25: 155-156.
24 Miwa, S. and Mano, N., 2000. Infection with Edwardsiella tarda causes hypertrophy of liver cells in Japanese
flounder Paralichthys olivaceus. Dis. Aquat. Org., 42: 227-231.
25 Miyazaki, T. and Egusa, S., 1976. Histopathological studies on Edwardsiella tarda infection of Japanese eel-1.
Natural infection-supernative intestinal nephritis. Fish pathology, 13: 325-335.
26 Mohanty, B. R. And Sahoo, P. K., 2007. Edwardsiellosis in fish: a Brief review. J. Biosci., 32(2): 1331-1344.
27 Nakatsugawa, T., 1983. Edwardsiella tarda isolated from cultured young flounders. Fish Pathol., 18: 99-101.
28 Nougayrede, P. H., Vuillaume, A., Vigneulle, M., Faivre, B., Luengo, S. and Delprat, J., 1994. First isolation of
Edwardsiella tarda from diseased turbot (Scophthalmus maximus) reared in a sea farm in the Bay of Biscay. Bull.
Eur. Assoc. Fish Pathol., 14: 128-129.
29 Panangala, S. V., Shoemaker, A. C., McNulty, T. S., Arias, R. C. and Klesius, H. P., 2006. Intra- and interspecific
phenotypic characteristics of fish-pathogenic Edwardsiella ictaluri and E. tarda. Aquacul Res., 37: 49-60.
30 Richars, G. D., 1996. Preparation of colloidal gold particles of various sizes using sodium borohudride and
sodium cyanoborohydride. Anal. Biochem., 236: 168-170.
31 Rudolf, J., Fiihrer, M., Galler, B., Ansari, P., Hasenhindl, C. and Baumgartner, S., 2009. Differences in usability
of rabbit IgG and chiken IgY after clean-up and impact on gold labelling properties. J. Immuno. Methods, 350:
79-88.
Colloidal Nanogold Immune Assay for Rapid Detection of Edwardsiella Tarda in Labeo Rohita 17

www.tjprc.org editor@tjprc.org

32 Sahoo, P. K., Swain, P., Sahoo, S. K., Mukherjee, S. C. and Sahu, A. K., 2000. Pathology caused by the bacterium
Edwarsiella tarda in Anabas testudineus (Bloch). Asian Fish. Sci., 13: 357-362.
33 Sahoo, P. K., Swain, P., Sahoo, S. K., Mukherjee, S. C. and Sahu, A. K., 2000. Pathology caused by the bacterium
Edwarsiella tarda in Anabas testudineus (Bloch). Asian Fish. Sci., 13: 357-362.
34 Sie-Qui, D., Muroga, K. and Nakai, T., 1984. A case of Edwardsiella tarda infection in cultured coloured carp,
Cyprinus carpio. J Fish Pathol., 19: 197-199.
35 Swain, P. and Nayak, S. K., 2003. Comparative sensitivity of different serological tests for seromonitoring and
surveillance of Edwardsiella tarda infection of Indian major carps. Fish and shellfish immunol, 15: 333-340.
36 Thobhani, S., Attree, S., Boyd, R., Kumarswami, N., Nobel, N., Szymanski, M. and Porter, A. R., 2010.
Bioconjugation and characterization of gold colloid-labelled proteins. J. Immunolog.l Meth., 356: 60-69.
37 Upadhyay, S.N., 1992. Immunogold Technique. In Talwar, G.P. (ed.), A Handbook of Practical Immunology,
Vikas publishing House, New Delhi. 139-149.
38 Xiulan, S., Xiaolian, Z., Jian, T., Zhou, J. and Chu, F. S., 2005. Preparation of gold-labeled antibody probe and its
use in immunochromatography assay for the detection of aflatoxin B1. Inter. J. F. Microbio., 99: 185-194.
39 Ying Ju, Hao, H-J., Xiong, G-H., Geng, H-R., Zheng, Y-L., Wang, J., Cao, Y., Yang, Y-H., Cai, X-H. and Jiang,
Y-Q., 2010. Development of colloidal gold-based immunochromatographic assay for rapid detection of
Streptococcus suis serotype 2. Vet. Immunol. Immunopatho., 133: 207-2011.
40 Zhao Y., Zhang G., Lio Q., Teng M., Jang J. & Wang J. (2008) Development of a lateral flow colloidal gold
immunoassay strip for the rapid detection of enrofloxacin residues. J. F. Agri. A. F. Chem. 56, 12138-12142.
Table 1: Biochemical Characteristics of E. tarda
S.No. Characteristics ATCC Field strain
1 Grams stain - -
2 Motility
25
0
C + +
35
0
C + +
3 Methyl red + +
4 Voges-proskauer - -
5 Catalase + +
6 Oxidase (Kovacs) - -
7 Indole production + +
8 H
2
S production
Triple sugar iron agar + +
9 Ornithine decarboxylase + +
10 Simmons citrate agar - -
11 Arginine dihydrolase - -
12 Gelatin hydrolysis - -
13 Malonate utilization - -
14 Triple sugar iron agar K/AG* K/AG*
18 Chandra Bhushan Kumar, Kurcheti Pani Prasad, K. H. D. T. Kasagala, Ayathullah Rabbani Syed, Kundan Kumar & Gayatri Tripathi

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0

Table 1-Cond.,
15 Acid from
Glucose / Trehalose + +
D-Mannose / Sucrose - -
L-Arabinose / Dulcitol - -
Lactose / Raffinose - -
Xylose / Inositol - -
Salicin - -
16 Tolerance to NaCl
1.5% + +
3.0% + +
K = Alkaline/no reaction, AG
*
= Acid Gas



Figure 1: Separation of anti E. tarda rabbit IgG by IgG Purification Kit

Figure 2: Optimization of pH of Colloidal Gold

Figure 3: Optimization of Amount of Protein to be Adsorbed to Colloidal Gold
Colloidal Nanogold Immune Assay for Rapid Detection of Edwardsiella Tarda in Labeo Rohita 19

www.tjprc.org editor@tjprc.org


Figure 4: GNPs Aggregation in tube 2 Accompanying Change in Absorbance
















Figure 5: SDS PAGE of Purified Rabbit IgG
Lane 1
st
: Purified Rabbit IgG (Heavy Chain 52 kD; Light chain 24kD)
Lane 2
nd
: Protein Marker (250-10 kD)


20 Chandra Bhushan Kumar, Kurcheti Pani Prasad, K. H. D. T. Kasagala, Ayathullah Rabbani Syed, Kundan Kumar & Gayatri Tripathi

Impact Factor (JCC): 1.8003 Index Copernicus Value (ICV): 3.0









Figure 6: Detection of E. tarda antigen by the Immunoassay
Tube Nos.
GNPs-coated antibody with PBS
GNPs-coated antibody with bacterial suspension
GNPs without coated antibody with bacterial suspension
Table 2: Stability of kit at Different Temperatures
Period
in
Weeks
Storage Temperature
37
o
C 24
o
C) 4
o
C
1
+ + +
2 + + +
3
+ + +
4 + + +
5 + + +
6 + + +
7 + + +
8 + + +
9 + + +
10 + + +
11 + + +
12 + + +
13 + + +
14 + + +
15 - + +
16 - + +
17 - + +
18 - - +
19 - - +
20 - - +
21 - - +
22 - - -
+ Positive - Negative