CHAPTER 4. DEVOLEPMENT OF IMMUNO-DOT BLOT ASSAY USING DUAL LABELED GOLD NANOPARTICLE PROBE TO DETECT Cryptosporidum parvum

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1 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 (Morgan et al 2000, Masago et al 2006).

2 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.

3 53 Enhanced System Conventional System Cryptosporidium parvum Anti-cysts Mc antibody Dual labeled (McAb and ALP) gold nanoparticles ALP Conjugated secondary mouse antibody NC Membrane 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 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).

4 54 Absorbance (a.u) AuNP AuNP/Ab/ALP Wavelength (nm) Figure 4.1 UV-Vis Spectral analysis for gold nanoparticle and dual labeled (anti-cysts McAb and ALP) gold nanoparticle conjugates 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).

5 55 (A) (B) 50 nm 50 nm 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

6 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) Absorbance (A 520-A 670) ph of the gold nanoparticles solution Figure 4.3 Optimization of ph for the preparation of dual labeled (antibody and ALP) gold nanoparticle (n=3) (A) 0.36 (B) 0.34 Absorbance (A 520-A 670) [anti cysts Mc Ab] (µg/ml) [ALP ](µg/ml) 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)

7 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). Oocysts/mL Concentration of antibody (µg/ml) cells 50 cells 40 cells 30 cells 20 cells 10 cells 5 cells PBS Figure 4.5 Effects of concentration of the dual labeled gold nanoparticle (anti-cysts McAb and ALP) on immuno-dot blot assay

8 ug anti cyst-ab 2 ug anti cyst-ab 1 ug anti cyst-ab 0.6 ug anti cyst-ab Intensity Oocysts/mL 20 0 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.

9 Figure 4.7 Time kinetics study to evaluate the optimum duration needed to perform the enhanced immuno-dot blot assay Intensity Duration of the assay (min) 10 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)

10 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 Intensity Oocysts /ml Figure 4.10 Colour intensity curve of the enhanced Immuno dot blot assay (n=3)

11 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. (A) 80 Intensity (B) M bp M- marker; 1- C.parvum; 2- E.coli; 3- C.parvum+Shigella; 4- Vibriocholerae; 5- Salmonella. The concentrations of used microorganisms were 1x10 2 /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)

12 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 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

13 (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