37

CHAPTER 3

DEVOLEPMENT OF AN ENHANCED IMMUNO-DOT

BLOT ASSAY TO DETECT WHITE SPOT SYNDROME

VIRUS IN SHRIMP USING ANTOBODY CONJUGATED

GOLD NANOPARTICLE PROBE

3.1 INTRODUCTION

White Spot Syndrome Virus (WSSV) is a double-stranded (305 kb)

DNA invertebrate virus (Wang et al 1998) that causes White Spot Syndrome

(WSS) in almost all commercially available crustaceans. Though 22 different

viruses are known to cause diseases in shrimp (Hsu et al 2000), WSSV is

particularly of great interest to cause mass mortality in 3-10 days of disease

blitz. The estimated annual global economic losses due to WSSV are close to

three billion dollars (Lundin et al 1996).

Early detection and routine monitoring of WSSV plays a key role in

sustaining shrimp hatcheries and farms. Presently, WSSV detection relies

mostly on different PCR-based protocols (Takahashi et al 1994, Peng et al

1998, Thakur et al 2002) for finding virus in various tissues of the infected

shrimp and detected up to 300 pg of viral DNA. Real-time PCR (Durand et al

2002) able to detect up to 5 to 10 pico mol of virus particles and in addition

used to screen the post larvae before introducing into the culture pond.

However, the requirement for sophisticated equipment and skilled

professionals make it impractical for farmers to employ such a method.

38

Protein- based techniques have also been employed to detect WSSV such as,

dot blot (Anil et al 2002), immunodot-blot (Zhana et al 2004, Rout et al 2005)

and Enzyme Linked Immuno Sarpant Assay (ELISA) could detect up to 300

pg of purified WSSV virus using monoclonal antibody (McAb)

(Liu et at 2002).

Gold nanoparticle based bioanalytical methods have beneficial

applications, which can influence the development of remarkable industrial

and engineering applications including biotechnological systems.

Nanoparticles facilitated the development of highly sensitive biomolecular

detection strategies for at least two major advantages, (i) compared to

conventional methods, nanoparticles provide a large surface area that

promotes the efficient macromolecular interactions; and (ii) the effective

signal amplification (Li et al 2006, Peng et al 2007, Feng Duan et al 2008,

Chia-Hsien et al 2009, Chirathaworm et al 2009, Fang et al 2009, Ambrosi et

al 2010). In recent years gold nanoparticle based immuno assays have been

reported for the detection of Hepatitis B and C viruses (Wang et al 2003,

Young Kim et al 2004 and Mikawa et at 2009), Human immunodeficiency

virus type 1(HIV-1) (Lianlian et al 2005), Respiratory Syncytial Virus (RSV)

in vitro and in vivo (Tripp et al 2007), Escherichia coli in water (Temur et al

2010), chronic gonadotropin hormone (hCG) in human serum (Ryo et al

2006) and staphylococcal enterotoxin B (Hwa et al 2010). Though all the

analytical method can be detected the biomolecules at nano gram level, it

entails various disadvantages including high cost, long time of assay and

involving highly toxic substances. In the present study, it is proposed to

address this hypothetical issue by developing a simple alkaline phosphatase

conjugated secondary antibody coupled gold nanoparticle based immuno-dot

blot assay to detect WSSV infection in shrimp.

39

3.2 PRINCIPLE OF THE GOLD NANOPARTICLE BASED

IMMUNO-DOT BLOT ASSAY FOR THE DETECTION OF

WSSV IN SHRIMP

In the present experiment, it is aimed to find out the easy and early

detection method of WSSV occurrences in the shrimp based on colour

appearance by the newly developed enhanced techniques. The appearance of

colour is directly proportional to the amount of virus immobilized onto the

membrane as well as the concentration of the antibody conjugate either plain

or nano–coupled. The comparative analysis has indicated that the colour

development was higher in gold nanoparticle coupled experiments than in

plain antibody. The lower colour intensity development in the plain antibody

experiments indicated that there was a limitation in signal amplification. It

might be due to equal number of antibody molecules bound to the antibody

raised against virus was not enough to develop the visible colour. The colour

intensity was amplified by manifold since each nanoparticle was bound by

many numbers of antibody conjugate in enhanced method. Theoretically, one

primary anti serum antibody molecule is bound to many molecules of

secondary antibody coupled gold nanoparticle. The chemical nature of the

gold nanoparticle acting as a platform for the anchorage of multiple numbers

of antibody conjugates. The principle of newly developed immuno–dot assay

and its features are explained in the flow chart (Scheme 3.1).

40

NC Membrane

Enhanced MethodConventional

Method

ALP Conjugated 20Ab+AuNP

ALP Conjugated 20Ab

WSSV

10 anti-serum

Scheme 3.1 Schematic diagram of newly developed enhanced immuno–

dot blot assay for the detection of WSSV.

3.3 CHARACTERIZATION OF SECONDARY ANTIBODY

COUPLED GOLD NANOPARTICLE

3.3.1 UV-vis Spectroscopic Analysis

Spectrophotometric analysis of gold nanoparticle before and after

coupling with secondary antibody indicated that the absorption maximum

shifted from 518 nm, the typical plasmon resonance band of the nanoscale

gold to 524 nm upon coupling is shown in Figure 3.1. Earlier (Kumar et al

2008) similar criterion (Plasmon resonance peak shift) was used as evidence

for antibody coupled on gold nanoparticle surface. Thus, UV-spectral analysis

41

used as initial stage of confirmation of antibody coupling with nanoparticles.

The interaction between gold nanoparticle and protein occurred by means of

three separate but dependent phenomena, (a) ionic attraction between the

negatively charged gold and the positively charged protein; (b) hydrophobic

attraction between the antibody and the gold surface and (c) dative bonding

between the gold conducting electrons and sulfur atoms which may occur

with amino acids of the protein (Ambrosi et al 2010).

0

0.1

0.2

0.3

0.4

0.5

0.6

400 450 550500 700650600

Ab

sorb

an

ce (a

.u)

Wavelength (nm)

a b

Figure 3.1 UV-Vis Spectral analysis of (a) gold nanoparticle and (b)

secondary mouse antibody coupled gold nanoparticle

3.3.2 High–Resolution Transmission Electron Microscopy (HR-

TEM) Analysis

HR-TEM images of gold nanoparticle and nanoparticles conjugates

are presented in Figure 3.2. From the figure it was estimated that the colloidal

gold nanoparticle has an average diameter of 16 ±0.2 nm (Figure 3.2 (A)),

after the antibody coupled with the nanoparticles surface has an average

diameter increased to 18 ±0.2nm (Figure 3.2 (B)) and were dispersed

uniformly. The antibody coupled nanoparticles are stable and do not

aggregate. Under higher magnification grayish halos around the modified

42

nanoparticles surface was observed, which indicates the biomolecules were

coupled on the nanoparticles surface (Zhang et al 2010).

50nm

50 nm

20 nm

50 nm

(b)(a)(A) (B)

Figure 3.2 HR-TEM images of (A) gold nanoparticle and (B) secondary

mouse antibody coupled gold nanoparticle

3.4 EFFECT OF pH OF THE GOLD NANOPARTICLE

SOLUTION FOR THE PREPARATION OF SECONDARY

ANTIBODY COUPLED GOLD NANOPARTICLE

CONJUGATE

The conjugation of biomolecules with gold nanoparticle is highly

influenced by the pH, which determines the charges and stability of the

protein ensuring the feasible nanoparticles surface coverage while preserving

protein bio-functionally. Different pH values were tested to determine the

optimal pH condition for the conjugattion of secondary antibody with gold

nanoparticle by salt induced aggregation method. The optimum pH condition

for the conjugation was determined by measuring the differential absorbance

(A520–A620) that the maximum value of A520–A620 was attained at pH 7.

Whereas at pH 6 the absorbance rate was lower than that of obtained at pH 7

and there was no significant change observed from pH 7 to 11 and is shown in

Figure 3.3.

43

pH of the gold nanoparticles solution

Ab

sorb

an

ce

(A5

20-A

67

0)

0.366

0.37

0.374

0.378

5 6 7 8 9 10 11

Figure 3.3 Optimization of pH for the preparation of secondary

antibody coupled gold nanoparticle

3.5 COUPLING EFFICIENCY OF GOLD NANOPARTICLE

VERSUS SECONDARY ANTIBODY CONJUGATE

The optimum volume of gold nanoparticle required to bind

completely to the antibody present in the solution was analysed. As shown in

Figure 3.4 there is a positive correlation between the volume of gold

nanoparticle and the intensity of the dots, whereas it was not appeared in the

respective supernatant. The colour devolepment was niticed in the supernatant

of 0.5 mL and 1 mL of AuNP reaction, which may due to the presence of

unreacted ALP conjugated antibody in the supernatant. It was clearly

indicated that 0.5 mL and 1.0 mL gold nanoparticle solutions were

insufficient to bind the total antibody molecules present in the solution.

Whereas, 1.5 mL and 2.0 mL of gold nanoparticle solution were high enough

to completely saturate the antibody molecules. Since, the corresponding

supernatants from 1.5 mL and 2.0 mL reaction did not show any colour

development that confirming the absence of unreacted antibody molecules.

44

Thus, the results confirmed that 1.5 mL of gold nanoparticle is sufficient to

bind all the antibody molecules present in the solution.

2nd Ab coupled

AuNP (µL)

Supernatant

(µL)

2 4 6 50 25

0.5

1.5

1.0

2.0

PBS

Au

NP

(mL

)

Figure 3.4 Immuno-dot blot analysis for the identification of optimum

quantity of gold nanoparticle needed to bind antibody

present in the solution

3.6 COMPARISON OF DETECTION EFFICIENCY OF WSSV

BETWEEN ENHANCED METHOD (NANOPARTICLE-

COUPLED) AND CONVENTIONAL METHOD (ANTIBODY

ALONE)

The efficiency of enhanced method and conventional method for

the detection of WSSV was compared and the experiments were conducted as

follows. All rows of the strip indicated the various concentrations such as 100,

80, 40, 20, 10, 5 and 1 ng/mL of WSSV and each column stands for exact

dilution of 1:10,000, 1:50,000, 1:100,000 and 1:200,000 antibody. In

enhanced method, least amount of 1 ng/mL WSSV was able to detect at

1:10,000 dilution and lower concentration of 1:50,000, 1:100,000 and

45

1:200,000 dilutions were able to detect 10, 40 and 80 ng/mL of WSSV

respectively. Whereas, in the conventional method antibody concentration of

1:10,000 dilution was able to detect upto 80 ng/mL of virus (Figure 3.5).

However, in lower concentrations of 1:50,000, 1:100,000 and 1:200,000

dilution was not able to detect even 100 ng/mL of virus by conventional

method. Figure 3.6 shows the colour intensity of immuno-dot blot assays of

both the methods and clearly indicates that the efficiency of AuNP based

immuno-dot blot assay was enhanced when compared to that of conventional

method.

1 2 3 4 1 2 3 4

WS

SV

(n

g/ m

L)

Enhanced Method Conventional Method

01

80

20

40

05

10

100

PBS

1) 1 : 10,000 dilution, 2) 1 : 50,000 dilution, 3) 1 : 100, 000 dilution, 4) 1 : 200, 000 dilution

Figure 3.5 Comparison of immuno-dot blot analysis between enhanced

(Antibody coupled AuNP) and conventional method

(antibody alone) at various antibody dilutions

46

Concentration of WSSV ng/mL

Inte

nsi

ty

100 80 60 40 20 00

20

40

60

801: 10,000 Enhanced Method 1: 10,000 Conventional Method

1: 50,000 Enhanced Method 1: 50,000 Conventional Method

1: 100,000 Enhanced Method 1: 200,000 Enhanced Method

Figure 3.6 The graph generated from the colour intensity of the dots

verses the amount of virus in the dots (n=3)

3.7 EFFICIENCY AND LIMIT OF DETECTION OF GOLD

NANOPARTICLE BASED IMMUNO-DOT BLOT ASSAY

Efficiency and visually least detection limit of the enhanced

immuno-dot blot assay was investigated using various concentrations such as

200, 150, 100, 80, 40, 20, 10, 5, 1, 0.5 and 0.25 ng/mL of WSSV and the

assay was performed under optimized conditions. As shown in Figure 3.7, the

colour intensity of the dot blot was decreased linearly with decreasing the

concentration of WSSV and was visually able to detect up to 1 ng/mL of

purified WSSV. The colour intensity of dot blot was saturated from 100

ng/mL of WSSV (Figure 3.8).

47

(ng/ mL)

Figure 3.7 Efficiency of enhanced immuno-dot blot analysis for the

detection of WSSV

Concentration of WSSV ng/mL

Inte

nsi

ty

200 180 160 140 120 100 80 60 40 20 00

20

40

60

80

Figure 3.8 Colour intensity curve for the respective enhanced immuno-

dot blot assay

3.8 EVALUATION OF SPECIFICITY OF THE ENHANCED

IMMUNO-DOT BLOT ASSAY FOR THE DETECTION OF

WSSV

The specificity and cross reactivity of the enhanced immuno-dot

blot assay was also validated with other pathogens such as Yellow Head Virus

(YHV), Monodon Baculo Virus (MBV) and Taura Syndrome Virus (TSV).

Figure 3.9 shows that the enhanced immuno-dot blot was more specific to the

WSSV and there was no false positive or non specific detection found in the

48

assay. Moreover, the result obtained was coincides with the of PCR analysis.

It is inferred that the colour development was not due to non-specific

adherence of the gold nanoparticle to the modified membrane surface.

Inte

nsi

ty

1 2 3 4 50

10

20

30

40

50

60

70

624 bp

M 1 2 3 4 5

(A)

(B)

M- marker (100bp); lane 1- WSSV; lane 2- WSSV+ Yellow head virus (YHV); lane 3-

Yellow head virus (YHV); lane 4- Monodon baculo virus (MBV); lane 5- Taura syndrome

virus (TSV). The concentrations of used virus were 100 ng/mL. The presented values have

an average of triplicates (n=3)

Figure 3.9 Evaluation of specificity of the enhanced immuno-dot blot

assay (A) and conventional PCR (B)

3.9 FIELD EVALUATION OF WSSV BY ENHANCED

IMMUNO-DOT BLOT ASSAY

The major aim of the present study is to develop the enhanced

immuno-dot blot assay to monitor the WSSV present in the shrimp culture

farms at the earlier stage. The field evaluation study was carried out with the

randomly collected 36 hemolymph samples from different shrimp culture

farms using PCR based technique and enhanced method under optimized

conditions and are given in Table 3.1.

49

Tab

le 3

.1 C

om

pa

riso

n o

f fi

eld

lev

el e

valu

ati

on

of

en

ha

nce

d i

mm

un

o-d

ot

blo

t on

hem

oly

mp

h f

rom

vari

ou

s sh

rim

p c

ult

ure

farm

s w

ith

th

at

of

con

ven

tion

al

PC

R a

naly

sis

PC

R a

naly

sis

Per

form

an

ce

Posi

tiv

e N

ega

tiv

e T

ota

lE

ffic

ien

cy

(%)

Sen

siti

vit

y

(%)

Sp

ecif

icit

y

(%)

FP

ra

te

(%)

FN

ra

te

(%)

Posi

tive

28

129

Neg

ativ

e2

57

91.6

93.3

83.3

16.6

6.6

En

han

ced

Imm

un

o

assa

yT

ota

l30

636

Eff

icie

ncy-

(TP

+ T

N)/

10

0/T

ota

l; S

ensi

tiv

ity-T

P/1

00

/(T

P +

FN

); S

pec

ific

ity-T

N/1

00

/(T

N +

FP

); F

als

e-p

osi

tiv

e ra

te-F

P/1

00

/(F

P +

TN

);

Fal

se-n

egat

ive

rate

- F

N/

100

/(T

P +

FN

); T

P-T

rue

Po

siti

ve;

TF

-Tru

e N

egat

ive.

50

The enhanced immuno-dot blot assay was compared with that of

conventional PCR to monitor the presence of WSSV in the shrimp in the

culture farm. It was ascertined that the enhanced immuno-dot blot assay

shows the higher efficiency (91.6%), higher sensitivity (93.3%) and higher

specificity (83.3%) than those of conventional methods. It was also confirmed

and demonstrated that the enhanced immuno-dot blot can be used to detect the

WSSV in the hemolymph of the shrimp, which was comparable to

polymerase chain reaction (PCR) detected level. Further, the gold

nanoparticle based immuno-dot blot assay devoleped in the present study

provides a promising alternative rout to detect WSSV in shrimp in the culture

farms for early diagnostics.