Abstract
Preparation of colloidal gold conjugated antibodies specific for influenza A/H5N1 and its use in developing a virus A/H5N1 rapid diagnostic kit is presented. Colloidal gold nanoparticles (AuNPs) were prepared through citrate reduction. Single chain antibodies specific to H5N1 (scFv7 and scFv24) were produced using pTI2 + vector and E. coli strain HB2151. These antibodies were purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The method for preparation of antibody–colloidal gold conjugate was based on electrostatic force binding antibody with colloidal gold. The effect of factors such as pH and concentration of antibody has been quantitatively analyzed using spectroscopic methods after adding 1 wt% NaCl which induced AuNP aggregation. The morphological study by scanning electron microscopy (SEM) showed that the average size of the spherical AuNPs was 23 nm with uniform sizes. The spectroscopic properties of colloidal AuNPs showed the typical surface plasmon resonance band at 523 nm in UV-visible spectrum. The optimal pH of conjugated colloidal gold was found between 8.0 and 10.0. The activity of synthesized antibody labeled AuNPs for detection of H5N1 flu virus was checked by dot blot immunological method. The results confirmed the ability in detection of the A/H5N1 virus of the prepared antibody labeled gold particles and opened up the possibility of using them in manufacturing rapid detection kit for this virus.
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1. Introduction
An influenza pandemic is known as a global outbreak of disease that occurs when a new strain of influenza A virus appears in the human population, causes serious illness, and then spreads easily from person to person worldwide [1]. Highly pathogenic avian influenza viruses of the H5N1 subtype are circulating in eastern Asia with unprecedented epizootic and epidemic effects [2]. The three viral envelope proteins of influenza A virus are most medically relevant. The hemagglutinin (HA), neuraminidase (NA) and M2 are essential viral proteins targeted by host antibodies or antiviral drugs such as oseltamivir and rimantadine [3–5]. Influenza A became a topic of much discussion since its appearance in humans, having caused deaths in the first 18 cases reported by Hong Kong Special Administration Region (SAR), China, in 1997 [6]. Because of certain characteristics, H5N1 is of particular concern because it mutates rapidly and has a propensity to acquire genes from viruses that infect other animal species. Its ability to cause severe disease and death in humans has been documented.
Single-chain fragment variable (scFv) antibody is a small antibody engineered by connecting the gene fragments for the variable regions of the heavy and light chains of an immunoglobin with a linker. The resulting scFv antibody usually retains the affinity and specificity of its parent antibody [7]. The scFv antibody has great advantages in diagnostic applications because of its small molecule and targeting drug-delivery agent potential [8].
The development of nanotechnology has resulted in new methods and approaches in designing rapid, accurate and sensitive diagnostic techniques. Gold nanoparticles (AuNPs) have attracted a wide range of biomedical applications because of their unique surface chemistry, electronic and optical properties [9–12]. Colloidal AuNPs exhibit a unique phenomenon, known as surface plasmon resonance (SPR) in the visible wavelength range which depends on their size, shape, particle–particle distances and surrounding medium [13]. AuNPs are nanomaterials that possess excellent biocompatibility and ease of conjugation to various biological molecules such as peptides, aptamers, antibodies etc. These properties allow colloidal AuNPs to be a prominent candidate for bioassay techniques; examples of such techniques are colorimetric sensor [14–16], lateral flow test trip [17–20], dot blot immunoassay [21–24], which have been used successfully in biological and medical aims as well. Thus, binding of AuNPs to biomolecules offers a promising approach for facile tracking of desired targets in aqueous samples.
In this study we developed, novel nanoprobes for H5N1 bird flu diagnosis by combining colloidal AuNPs with expressed antibody specific to HA surface antigen of H5N1. AuNPs were synthesized through the reduction of chloroauric acid (HAuCl4) by sodium citrate. Two kinds of ScFv (scFv7 and scFv24) antibodies specific to HA surface antigen of H5N1 virus were produced recombinantly in E. coli. The purified scFv7 was then labeled on AuNPs to produce antibody/AuNPs nanoprobes. The reactivity of these nanoprobes was qualitatively tested using dot blot in a sandwich mechanism in which scFv24 was used as secondary antibody for detection. As a result, these nanoprobes were highly evaluated and proved to be a good material for the further development of H5N1diagnotic kit.
2. Materials and methods
2.1. Materials
Chloroauric acid (HAuCl4) and sodium citrate (C6H5O7Na3) were obtained from Merck. Deionized water was used throughout the experiment. Nitrocellulose membrane (BIO-RAD) and all other chemicals in this study were of high quality.
2.2. Synthesis of AUNPs
Citrate-stabilized AuNPs were prepared according to Turkevich et al [25]. Briefly, 100 μl of an aqueous HAuCl4 (5%) was added into a flask containing 80 ml deionized water and then the solution was brought to boil, with constant stirring. As quickly as possible, 3.5 ml of an aqueous sodium citrate (1%) solution was added. The solution was heated for another 20 min until a deep-red solution was observed. The particles solution was centrifuged for 10 min at 3500 rpm and supernatant was collected.
2.3. Characterization of the colloidal gold particles
The morphology and elemental information of AuNPs were characterized by scanning electron microscopy (SEM) (S-4800 FESEM equipped with energy dispersive x-ray (EDX) spectroscopy) in which SEM images were used to physically measure the size of the AuNPs. UV-Vis spectroscopy (Shimadzu, UV-1650PC) determined the optical characteristics of colloidal AuNPs by SPR measurement.
2.4. Expression and purification of single chain antibodies (scFv) specific to H5N1
Human Single Framework scFv Libraries A + B (I. Tomlinson, MRC, University of Cambridge, UK) [26] were used to screen specific recombinant antibodies against E. coli-expressed HA1 protein. The screening procedure was described by Gahrtz and Conrad [27]. DNA fragments encoding scFv7 and scFv24 were cloned into the pTI2 + plasmid. The recombinant plasmid was then introduced into E. coli strain HB2151. The expression of scFv7 and scFv24 was induced using 1 mM of isopropyl-D-1-thiolgalactopyranoside (IPTG) for 5 h at 30 °C. The bacteria were then collected and lyzed by lysozyme and sonication. The inclusion bodies were collected by centrifuging, followed by washing with buffer (50 mM Tris, 1% triton X-100 and 100 mM NaCl, pH 7.5). The scFv7 and scFv24 proteins were refolded and further purified by affinity chromatography using HiTrap Chelating HP columns pre-charged with Ni2 + . The expression and purity of the scFv were further characterized by sodium dodecyl sulfate polyacrylamide gel electrophotoresis (SDS-PAGE).
2.5. Preparation of binding antibody/gold probes
2.5.1. Determining the optimal pH.
The colloidal AuNPs formed by citrate reduction were stable in a colloidal state by a repulsive force which exists along particles and maintained by a net negative charge on their surface. Typically, these charged particles were very sensitive to changes in solution dielectric. The presence of cations in salt solutions negated this charge repulsion and caused these particles to agglomerate and eventually precipitate [28]. Thus, for typical citrate stabilized particles, the addition of aqueous NaCl covered the surface charge, decreased the interparticle distance and eventually induced particles' aggregation. Therefore, binding of proteins to AuNPs or other stabilizing agents to surface of particles would keep a suspended state by blocking the salt-induced precipitation of colloidal AuNPs.
The binding of scFv7 antibody to colloidal AuNPs depends on the pH of the colloidal gold and the antibody solutions. To determine the optimal pH for binding, the pH of 2 ml colloidal gold aliquots was adjusted from pH 5 to 11 by 1 N NaOH. Purified scFv7 was diluted to a concentration of 100 μg ml−1 in 3 mM of TRIS-HCl pH 8.0. Then, 100 μl of this stock solution was added to the 7 aliquots of adjusted gold solution and incubated for 15 min followed by addition of 100 μl of a 10% NaCl solution to each of the aliquots to induce particle precipitation. The pH binding optimum was defined at the pH that allowed antibody to bind to AuNPs to prevent their agglomeration by NaCl.
2.5.2. Binding procedure.
Binding protocol of scFv7 to AuNPs was carried out according to the method described by Horisberger and Leuvering et al [29]. The optimal concentration of antibody scFv7 for conjugation was determined by titrating aliquots of diluted antibody with colloidal AuNPs. The purified scFv7 was diluted to a concentration of 0.1 mg ml−1 in phosphate buffered saline (PBS) buffer (0.001 M, pH 7.4). The pH of colloidal gold solution and the diluted scFv7 was adjusted to pH 8.0 with 0.1 N NaOH. Ten aliquots of variable concentrations (0.01–0.1 mg ml−1) of the diluted antibodies were prepared in 0.1 ml PBS buffer, and added separately to 1 ml of colloidal gold solution. The tubes were incubated for 15 min and 0.1 ml of 10% NaCl was added. The UV-visible absorbance was recorded and estimated at the wavelength of around 523 nm. The least amount of protein required to sufficiently bind to the colloidal gold was determined from the curves of the absorbance.
After determining the optimal concentration of binding scFv7, the antibody/gold nanoprobes were prepared. An aliquot (50 μl) of scFv7 prepared in PBS (0.01 M, pH 7.4) was added slowly to 2 ml colloidal gold solution pH8 (adjusted by 1 N NaOH) followed by the addition of bovine serum albumin (BSA) (100 μl, 10%) under gentle stirring after 45 min. The mixture was incubated for another 1 h at 4 °C and then centrifuged (10 000 rpm for 15 min at 4 °C) to remove supernatant unconjugated antibody. The pellet obtained was washed with PBS once again. The pellet was finally redispersed in 0.5 ml PBS (pH7.4) containing 2% BSA and stored at 4 °C.
2.6. Dot blot immunoassay for rapid detection of H5N1 surface antigen
The dot blot immunoassay was carried out by using sandwich reaction mechanism in which scFv24 was used as a secondary antibody (figure 4(a)). A nitrocellulose membrane used for dot blotting test was drawn in a grid by pencil to indicate the region of blot before spotting 2 μl of different concentrations of scFv24 antibodies onto the membrane at the center of the grid. Six areas on the membrane were spotted and allowed to dry in air. The membrane was soaked in a petri dish and immersed in PBS buffer containing 5% BSA for another 45 min to block all non-specific sites. Then, the membrane was washed three times with PBS buffer to remove all the remaining BSA and dried by air. For the dot blot test, the membrane was soaked into the diluted solution containing H5N1 viruses and antibody/gold nanoprobes for rapid specific interaction.
Surface plasmon properties of red AuNPs were enhanced as the AuNPs moved to scFv24-spotted circular template due to the sandwich specific interaction. As a result, the circular templates were directly observed on the surface of nitrocellulose membrane.
3. Results and discussion
3.1. Characterization of AuNPs
Colloidal AuNPs were synthesized according to standard wet chemical methods using sodium citrate as a reducing agent. Colloidal gold solution appears intensely red in color. A characteristic SPR band of AuNPs was shown in the UV-Vis spectrum (the typical peak at 523 nm) which confirms the presence of spherical AuNPs (figure 1(c)). An SEM image (figure 1(a)) shows mostly monodispersed AuNPs with an average particle diameter of 23 nm (figure 1(b)). In addition, EDX spectrum (figure 1(d)) clearly confirms the presence of pure gold in the sample with the prominent peaks corresponding to Au element. The prepared colloidal gold solution was stable for months and this stability of colloids allowed AuNPs to be used for further conjugation with antibody without other stabilizing agent.
Figure 1. (a) SEM micrograph of AuNPs, (b) size distribution histogram of AuNPs, (c) UV-Vis spectrum of colloidal gold solution and (d) EDX spectrum of the AuNPs.
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Standard image3.2. Expression and purification of scFv7 and scFv24
The plasmid of pTI2 + was designed to carry DNA fragments encoded to generate protein scFv7 and scFv24. These plasmids were then transformed into E. coli strain HB2151, and the expression of recombinant scFv proteins was induced by IPTG (figure 2(a)).
Figure 2. Expression and purification of recombinant scFv7 and scFv24. The pTI2 + plasmids were transformed into E. coli HB2151 and the expression of recombinant scFv was induced by IPTG. The expressed recombinant proteins were separated on 12% SDS–PAGE, stained with Coomassie blue R-250. (a) SDS-PAGE analysis of induced total bacteria proteins. M: protein marker; lane 1 and 2: the induced total bacteria proteins; lane 2: the uninduced total bacteria proteins. (b) Characterization of the purified scFv7 (lane 1), scFv24 (lane 2).
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Standard imageThe recombinants scFv7 and scFv24, as expected, have the same molecular weight of approximate 25 kDa. Following sonication and washing with appropriate buffers, inclusion bodies were subjected to purification by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + , refolded and finally characterized by SDS-PAGE. The generated scFv have a purity of 90%. Approximately, 5 and 12.5 mg of refolded scFv7 and scFv24 were yielded from 1 l of bacterial suspension, respectively. The purified scFv7 and scFv24 proteins provide useful reagents for further testing their bioactivity.
3.3. Preparation of antibody labeled AuNPs
3.3.1. Optimal pH for binding.
It is evident that optically transparent red-colored gold solution was obtained when appropriate pH was applied. The color change from red to blue occurred immediately after adding NaCl 1 N at low pH 4 and pH 5, causing the aggregation of the nanoparticles in solution. The optimal pH for binding antibody to colloidal gold was determined between 8.0 and 10.0. At lower pH values, the addition of the antibody caused the particles to agglomerate, while adjusting the pH of the colloidal gold above 10 resulted in the generation of unstable nanoprobes preparation, since the addition of large quantities of NaCl caused agglomeration of the gold particles before the further addition of protein [30].
3.3.2. Determining optimal concentration of binding antibody to AuNPs.
Figure 3(a) illustrates the color change of gold suspension containing scFv7 coupled AuNPs with different ratios compared with the dispersed particles. The color of the suspension of dispersed AuNPs changed into blue after addition of a low amount of antibodies in the presence of NaCl. In contrast, at enough or excess amount of antibodies, AuNPs remained in a stable state even after addition of NaCl to induce precipitation.
Figure 3. (a) Photograph of different aliquots of colloidal gold supplemented with different concentrations of antibodies. (b) Spectral analysis of AuNPs incubated with various concentrations of antibodies. Ten aliquots of variable concentrations of 0.01–0.1 mg ml−1 (curves from (a) to (j), respectively) of the diluted antibodies were prepared in 0.1 ml PBS buffer, and added separately to 1 ml of colloidal gold solution. SEM micrographs of (c) antibody stabilized AuNPs, compared to (d) citrate AuNPs without antibody.
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Standard imageThe binding of antibody to the colloidal AuNPs exhibited saturation kinetics. As shown in figures 3(a) and (b), at 5 μg of antibody per 1 ml of gold solution, the antibody was adequately bound to AuNPs. As the mass of antibody added to the gold solution increased, more protein bound to the AuNPs until all of the available binding sites were occupied.
The plasmon peak depends on the extent of colloid aggregation [31]. To monitor stability of the silver colloid, we measured the absorption of the colloid after addition of different amounts of antibodies. The evolution of UV-Vis spectra is shown in figure 3(b). The wavelength of absorption peak of citrate-stabilized AuNPs is 523 nm and the adsorption peak of stabilized AuNPs with scFv7 is red-shifted by several nanometers. This is due to the fact that the scFv7 antibody bound to the surface of AuNPs has slightly increased the size of particles. As the particles increase in size, the absorption peak usually shifts toward the red wavelengths [32]. The absorptions curves of aggregated AuNPs are much broader than those of stabilized AuNPs and shifted to the larger wavelengths, while their absorption intensity drastically decreased. Spectra change in maximum adsorption and the intensity of surface plasmon band determines the optimal amount of antibodies binding to AuNPs. The high stability of antibody stabilized AuNPs was further confirmed by SEM characterization. As a control, the SEM micrograph of citrate AuNPs without antibody was also taken (figures 3(c) and 3(d)). It is clearly seen that the antibody stabilized AuNPs remain as clusters of particles without aggregation. The addition of antibody into gold sol allows antibody to bind to the surface of particles and cause the change in surface energy, as a consequence, this results in a tendency of nanoparticles to clump together on the substrate in SEM.
3.4. Immunoblotting analysis
Figure 4(a) depicts the sandwich mechanism for detecting HA surface antigen on the H5N1 virus. The scFv24-immobilized membrane was soaked into nanoprobes solution and specific interaction between antibody stabilized AuNPs and HA surface antigen was allowed; then this complex further interacted with scFv24 on nitrocellulose membrane. Colloidal AuNPs solution shows a particular color due to collective oscillations of the surface electrons induced by visible light of suitable wavelength. The intensely red colored dots appeared within 5–10 min as many AuNPs moved together on the surface of membrane. The description of the test can be described as follows. First, scFv7 labeled AuNPs specifically interacted with HA surface antigen of H5N1 virus to form a complex between nanoprobes and antigen. Second, the complex moved forward along the scFv24-spotted membrane, the scFv24 captured other HA surface antigens by specific interaction and then formed the red-circular templates which could be directly observed by the naked eye. The intensely red color, which was produced by the accumulation of AuNPs in the dots, as a result, demonstrates the evidence for specifying nanoprobes in detecting antigen (figure 4(b)). As the concentration of scF24 increases, the color of the AuNPs-blotted dot changes from pale to strong. Therefore, we can speculate that this method is very sensitive at very low concentration of H5N1.
Figure 4. (a) Scheme of dot blot hybridization between scFv7 labeled AuNPs and H5N1 showing on nitrocellulose membrane. (b) Six dots were experimentally assayed in serial dilution of antigen of 0.1–1 μg ml−1 (a) to (f), respectively).
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Standard image4. Conclusion
In this work we have demonstrated a simple method to produce gold nanoprobes by labeling scFv7 antibody to AuNPs. Spherical monodispersed AuNPs were synthesized by citrate reduction method with an average size of 23 nm. The scFv antibodies specific to HA surface antigen of H5N1 virus were successfully expressed by IPTG induction and purified by affinity chromatography technique employing HiTrap Chelating HP columns pre-charged with Ni2 + . The optimal binding concentration of scFv7 antibody to the synthesized AuNPs occurring at 5 μg of scFv7 antibody per 1 ml colloidal gold was determined by using UV-visible spectra. Results showed that gold nanoprobes were tested with high activity by dot blot giving a rapid test result within 5 min. Gold nanoprobes demonstrated a well applicable material for further development of H5N1 diagnostic kit.
Acknowledgments
This work was supported by VAST's project 'Research on production of rapid detection kit of influenza A virus by application of recombinant single chain antibody scFv'. Several experiments were done using equipment of National Key Laboratory for Gene technology of Institute of Biotechnology, VAST.