CHAPTER INTRODUCTION

Transcription

1 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.

2 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.

3 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).

4 40 Conventional Method Enhanced Method NC Membrane ALP Conjugated 2 0 Ab+AuNP WSSV ALP Conjugated 2 0 Ab 1 0 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 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

5 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). 0.6 Absorbance (a.u) a b Wavelength (nm) Figure 3.1 UV-Vis Spectral analysis of (a) gold nanoparticle and (b) secondary mouse antibody coupled gold nanoparticle 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

6 42 nanoparticles surface was observed, which indicates the biomolecules were coupled on the nanoparticles surface (Zhang et al 2010). (A) (a) (b) (B) 20 nm 50 nm 50 nm 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.

7 Absorbance (A 520-A 670) ph of the gold nanoparticles solution 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.

8 44 Thus, the results confirmed that 1.5 ml of gold nanoparticle is sufficient to bind all the antibody molecules present in the solution. 2 nd Ab coupled AuNP (µl) Supernatant (µl) AuNP (ml) PBS 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

9 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. 100 Enhanced Method Conventional Method WSSV (ng/ ml) 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

10 : 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 Intensity Concentration of WSSV ng/ml 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).

11 47 (ng/ ml) Figure 3.7 Efficiency of enhanced immuno-dot blot analysis for the detection of WSSV 80 Intensity Concentration of WSSV ng/ml Figure 3.8 Colour intensity curve for the respective enhanced immunodot 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

12 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. (A) Intensity (B) M bp 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.

13 49 Table 3.1 Comparison of field level evaluation of enhanced immuno-dot blot on hemolymph from various shrimp culture farms with that of conventional PCR analysis Enhanced Immuno assay PCR analysis Performance Positive Negative Total Positive Efficiency (%) Sensitivity (%) Specificity (%) FP rate (%) FN rate (%) Negative Total Efficiency- (TP + TN)/100/Total; Sensitivity-TP/100/(TP + FN); Specificity-TN/100/(TN + FP); False-positive rate-fp/100/(fp + TN); False-negative rate- FN/ 100/(TP + FN); TP-True Positive; TF-True Negative.

14 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.