51

CHAPTER 4

DEVOLEPMENT OF IMMUNO-DOT BLOT ASSAY USING

DUAL LABELED GOLD NANOPARTICLE PROBE TO

DETECT Cryptosporidum parvum

4.1 INDRODUCTION

Cryptosporidium parvum is a significant diarrhoea–causing

parasitic protozoan found both in humans and animals (Fayer 2004). About 30

% adults of eminent–rich countries and virtually of all adults in resource–poor

countries have serologic evidence of prior infection with this organism.

However, undersized minority of people subsist diagnosed with clinical

disease (David et al 2002). In general very less number of cysts found in

environmental water, but an infectious dose suggested to exist is as low as 30

cysts. Due to the efficient infectious behavior of C. parvum at lower

concentration, extremely sensitive detection methods are required to analyze

water and food samples. At present DNA based methods in use to detect cysts

in the environmental samples like water etc. Whereas PCR–based detection

methods demoed to be sensitive and specific for the detection of C. parvum in

clinical specimens and environmental samples (Xiao et al 2000, Amar et al

2001, Sturbaum et al 2001, Limor et al 2002). Quenching probe (QProbe)

PCR and nested PCR were used to quantitative detection of oocysts in water

and the detection limit ranging from 2.5 to 59 oocysts 100–1

(Morgan et al

2000, Masago et al 2006).

52

Though all the existing analytical methods could detect a small

number of biomolecules, it has various difficulties including time

consumption of the assay, high cost, and complicated equipment and lacking

expertise knowledge. To overcome such difficulties, enormous efforts have

been made in recent years to develop more sensitive and specific visual

detection methods for related issues. In the present study the development of

dual labeled gold nanoparticle (AuNP) coupled with both anti–cysts

monoclonal antibody (McAb) and enzyme alkaline phosphatase (ALP) and its

subsequent utilization in an immuno–dot blot assay to detect C. parvum was

reported. The results obtained confirm the eminence sensitivity of this new

approach as compared to that of customary method. This strategy offers a

simple instructive (visually monitored), utilization of low–cost reagents for

diagnostics of pathogen.

4.2 PRINCIPLE OF THE DUAL LABELED GOLD

NANOPARTICLE BASED IMMUNO-DOT BLOT ASSAY

The principle involved for enhanced immuno-dot blot assay using

dual labeled AuNP (ALP–AuNP–anti–cysts Mc Ab) is based on direct

sandwich immuno-dot blot assay and the occurance of an enzymatic catalytic

reaction. Initially, C. parvum was immobilized on the nitrocellulose

membrane, followed by the exposure of dual labeled (anti-cysts McAb and

ALP) AuNP. Finally the blot was developed with the buffer containing NBT

and BCIP. The colour intensity was amplified by manifolds due the presence

of many number of ALP molecules bounded gold nanoparticle in the assay.

The AuNP acted as a platform for the anchorage of both the antibody as well

enzyme ALP, which is clearly shown in Schematic diagram 4.1.

53

NC Membrane

Dual labeled (McAb and

ALP) gold nanoparticles

Anti-cysts Mc antibody

Cryptosporidium parvum

ALP Conjugated secondary

mouse antibody

Enhanced System Conventional System

Scheme 4.1 Schematic diagram of newly developed immuno-dot blot

assay for the detection of C. parvum using dual labeled gold

nanoparticle probe

4.3 CHARACTERIZATION OF DUAL LABELED GOLD

NANOPARTICLE PROBE

4.3.1 UV-vis Spectrophotometric Analysis of Gold nanoparticle and

Gold nanoparticle probe

Spectrophotometric analysis of gold nanoparticle before and after

coupling with antibody and ALP is shown in Figure 4.1. It was indicated

that the gold nanoparticle exhibited a sharp plasmon absorption band with

maximum absorbance at wavelength 518 nm due to the surface plasmon

resonance. After the conjugation of anti-cysts Mc antibody and ALP on gold

nanoparticle surface the surface plasmon band was red-shifted to 524 nm,

which indicated the formation of gold nanoparticle conjugate (kumar et al

2008).

54

450 500 550 600 650 700

0.1

0.2

0.3

0.4

0.5AuNP

AuNP/Ab/ALP

Wavelength (nm)

Ab

sorb

an

ce (

a.u

)

Figure 4.1 UV-Vis Spectral analysis for gold nanoparticle and dual

labeled (anti-cysts McAb and ALP) gold nanoparticle

conjugates

4.3.2 High–Resolution Transmission Electron Microscopy (HR-

TEM) Analysis of Gold nanoparticle and Gold nanoparticle

probe

HR-TEM images display (Figure 4.2 (A)) the gold nanoparticle and

gold nanoparticle conjugates. It was ascertained that the colloidal gold

nanoparticle has an average diameter of 16 ±0.2 nm and it was enhanced to an

average diameter 18 ±0.2 nm after the antibody and ALP coupled on

nanoparticles (Figure 4.2 (B)). The diameter of the nanoparticles was

increased by 2 nm after the interaction of the monoclonal antibodies and ALP

with gold nanoparticle and was coincides with that of UV-vis absorption

spectra (Zhang et al 2009).

55

50 nm 50 nm

(A) (B)

Figure 4.2 HRTEM images of (A) gold nanoparticle and (B) dual

labeled (antibody and ALP) gold nanoparticle conjugate

4.4 OPTIMIZATION OF ASSAY CONDITIONS FOR THE

CONJUGATION OF ANTI–CYSTS McAb AND ALP ON

GOLD NANOPARTICLE

The conjugation process is influenced greatly by the pH, which

determines the charges and stability of the protein ensuring the best gold

surface coverage while preserving protein biofunctionality. In the present

work the optimum pH condition for the conjugation of antibody with gold

nanoparticle was examined at different pH values by salt induced aggregation

test. The AuNP solution adjusted to pH with 6, 7, 8, 9, 10 and 11 before the

antibody incubation. The optimal pH condition for conjugation was

determined by measuring the differential absorbance (A520–A670). The

maximum rate of A520–A670 was reached at pH 7, whereas at pH 6 the

absorbance rate was lower than that obtained at pH 7 and there was no

significant change occurred from pH 7 to 11 (Figure 4.3). Similarly, the

optimum quantity of anti cysts McAb to saturate the gold nanoparticle

solution was also quantified. Figure 4.4 (A) shows that the 20 of anti cysts

McAb was 100 % saturated the gold nanoparticle surface, simultaneously 10

µg/mL of McAb was used to prepare the AuNP conjugate. Followed by the

56

uncoated gold nanoparticle surface was conjugated with enzyme ALP and it

was also determined by salt induced aggregation test. It was inferred that the

200 µg/mL of ALP was suuficient to completely saturate the gold

nanoparticle present in the solution (Figure 4.4. B).

pH of the gold nanoparticles solution

Ab

sorb

an

ce

(A520-A

670)

0.366

0.37

0.374

0.378

5 6 7 8 9 10 11

Figure 4.3 Optimization of pH for the preparation of dual labeled

(antibody and ALP) gold nanoparticle (n=3)

0.2

0.24

0.28

0.32

0.36

0 20 40 60 80

(A)

[anti cysts Mc Ab] (µg/mL)

Ab

sorb

an

ce

(A5

20-A

670

)

(B)

[ALP ](µg/mL)

0.26

0.28

0.3

0.32

0.34

0 100 200 300 400 500

Figure 4.4 Quantification of the concentrations of (A) anti-cysts Mc

antibody and (B) ALP needed to saturate the gold

nanoparticle present in the solution (n=3)

57

4.5 EFFECT OF ANTIBODY CONCENTRATION FOR

ENHANCED IMMUNO-DOT BLOT ASSAY

The optimum concentration of anti-cysts McAb conjugated AuNP

required to carry out the immuno-dot blot assay was established as follows. A

wide range of concentrations of anti–cyst antibody coupled–nanoparticles was

applied to detect C. parvum. Each row of the strips indicated the choice of

amount of C. parvum and each column represented the particular

concentration of the antibody coupled gold nanoparticle. In the immuno–dot

blot assay, the colour appearance was noticed in all the concentration of

antibody coupled gold nanoparticle. As expected, the antibody concentration

was correlated with the colour intensity of the dot blot. The enhanced

immuno-dot blot was effectively detected the lowest number of 10 oocysts in

2 and 3 µg/mL of antibody; simultaneously 40 to 50 oocysts were detected

using very low concentration of 0.6 and 1 µg/mL respectively (Figure 4.5). It

was concluded that the optimum concentration of 2 µg/mL of anti–cysts Mc

antibody coupled AuNP was adequate to detect the lowest number of oocysts

spotted in the immuno assay (Figure 4.6).

Concentration of antibody

(µg/mL)

Oocysts/mL

3 2 1 0.6

40 cells

50 cells

30 cells

20 cells

10 cells

PBS

5 cells

100 cells

Figure 4.5 Effects of concentration of the dual labeled gold nanoparticle

(anti-cysts McAb and ALP) on immuno-dot blot assay

58

120

100

80

60

40

20

0

Inte

nsi

ty

Oocysts/mL

020406080100

3 ug anti cyst-Ab 2 ug anti cyst-Ab1 ug anti cyst-Ab 0.6 ug anti cyst-Ab

Figure 4.6 Intensity curve for enhanced immuno-dot blot assay (n=3)

4.6 EFFECT OF DURATION OF THE ENHANCED IMMUNO

ASSAY ON C.parvum DETECTION

The effect of duration of the enhanced immuno-dot blot assay was

predicted as explained below. Under optimized conditions the immuno assay

was placed at different time periods of 15, 30, 45 and 60 minutes and 50

oocysts/mL in each intervals of time was spotted. Figure 4.7 shows the colour

intensities of all the spots and were decreased with decreasing the duration of

the experiment. However, there was no significant difference observed at 60

and 45 minutes of duration and the intensity was slightly decreased at 30

minutes duration of assay. At 15 minutes duration of assay all the spots were

not significantly observed through visually. It was suggested that 30 minutes

could be the optimum reaction duration to perform the immuno dot blot assay

(Figure 4.8). Accordingly the entire enhanced immuno–dot blot procedure

was completed by 90 minutes including the wash procedure, which is

comparatively 240 minutes lower than that of used in the conventional

immuno–dot blot assay procedure.

59

60 45 30 15

Figure 4.7 Time kinetics study to evaluate the optimum duration

needed to perform the enhanced immuno-dot blot assay

Inte

nsi

ty

Duration of the assay (min)

70

60

50

40

30

20

10

0

102030405060

Figure 4.8 Intensity curve for time kinetics study to evaluate the

optimum duration needed to perform the enhanced immuno

dot blot assay (n=3)

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4.7 SPECIFICITY AND DETECTION LIMIT OF GOLD

NANOPARTICLE BASED IMMUNO ASSAY

The efficiency, specificity and least detection limit were analyzed

using freshly developed enhanced immuno–dot blot assay under the optimum

reaction conditions of pH 7.0, 2 µg/mL anti-cysts McAb coupled

nanoparticles at 90 minutes of duration. A series of oocysts were quantified

from 200, 150, 100, 50, 40, 30, 20, 10, 5 and 3 oocysts/mL of cells spotted in

the immuno blot. A non specific antigen E. coli and PBS employed as

negative control. Results noticeably found that under optimized conditions the

colour intensity of the enhanced immuno–dot blot assay decreased linearly

with the decreasing number of antigen and could be detected visually up to 10

oocysts and the colour intensity of the dot was saturated from 150 oocysts/mL

(Figures 4.9 and 4.10). In contrast, the negative control E. coli and PBS were

not found any changes while using the enhanced immuno–dot blot assay.

Oocysts /mL

Figure 4.9 Efficiency of enhanced immuno–dot blot assay for the

detection of C.parvum

Inte

nsi

ty

Oocysts /mL

120

100

80

60

40

20

0

050100150200

Figure 4.10 Colour intensity curve of the enhanced Immuno–dot blot

assay (n=3)

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4.8 EVALUATION OF SPECIFICITY OF THE GOLD

NANOPARTICLE BASED IMMUNO ASSAY

In order to validate the specificity and cross reactivity of the

enhanced immuno-dot blot assay, the experiment was performed with other

water borne pathogens such as C.parvum, E.coli, Shigella,Vibriocholerae and

Salmonella. It was indicated that the enhanced immuno-dot blot was more

specific to the C.parvum and there was no false positive or non specific

detection found in the assay (Figure 4.11). Moreover, the result obtained was

in good agreement with PCR analysis.

1 2 3 4 50

20

40

60

80

Inte

nsi

ty

M 1 2 3 4 5

556 bp

(A)

(B)

M- marker; 1- C.parvum; 2- E.coli; 3- C.parvum+Shigella; 4- Vibriocholerae; 5-

Salmonella. The concentrations of used microorganisms were 1x102/mL. The relative

standard deviation (RSD) of the assay was less than 5 %.

Figure 4.11 Evaluatio of specificity of the enhanced immuno-dot blot

assay (A) and PCR (B)

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4.9 FIELD LEVEL EVALUATION OF ENHANCED IMMUNO–

DOT BLOT ASSAY

Enhanced immuno–dot blot assay was used to analyze C. parvum

present in the environmental samples. The field evaluation study was carried

out with the random samples using PCR based technique, enhanced and

conventional immuno dot–blot assay. In effects of PCR analysis the presence

of C. parvum was found in the zonal samples 1, 2 and 4, whereas C.parvum

was not found in the zonal samples of 3 and 5 (Figure 4.12). Figure 4.13 (A),

the enhanced immuno–dot blot assay was also detected the PCR confirmed

water samples of zonal 1, 2 and 4. However, the similar behaviour was not

observed in conventional method that the C. parvum found in the zonal

sample 2 alone at very low colour intensity (Figure 4.13 (B)).

M 1 2 3 4 5 6 7

556 bp

M– Marker; –ve – Negative control; +ve – Positive control (1000 cysts/mL); from lane 3 to

7 – Zonal 1 to 5

Figure 4.12 Field level analysis of C.parvum present in drinking water

samples

63

1 2 3 4 5 6 7

(A)

(B)

1 – Negative control E.coli; 2- +ve – Positive control (1000 cysts/mL); from 3 to 7– Zonal

1 to 5

Figure 4.13 Field level analysis of C.parvum present in drinking water

samples, (A) Enhanced immuno–dot blot assay and

(B) Conventional method