- Email: [email protected]
Construction of bifunctional phage display for biological analysis and immunoassay
Analytical Biochemistry 396 (2010) 155–157
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Construction of bifunctional phage display for biological analysis and immunoassay Yongchao Guo a,1, Xiaosheng Liang a,b, Yafeng Zhou a, Zhiping Zhang a, Hongping Wei a, Dong Men a,b, Ming Luo a,b, Xian-En Zhang a,* a b
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Graduate School, Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e
i n f o
Article history: Received 26 June 2009 Available online 21 August 2009
a b s t r a c t A phage display-based bifunctional display system was developed for simple and sensitive immunoassay. The resulting bifunctional phage could simultaneously display a few single-chain variable fragment (ScFv) and many copies of the gold-binding peptide on its surface, thereby mediating antigen recognition and signal amplification. As a demonstration study, it was possible for bifunctional phage-based immunoassay to identify Bacillus anthracis spores from other Bacillus strains with detection sensitivity 10-fold higher than that of conventional phage enzyme-linked immunosorbent assay (ELISA). This protocol may be applied to build other bifunctional phage clones for broad applications (e.g., immunoassay kits, affinity biosensors, biorecognition assays). Ó 2009 Elsevier Inc. All rights reserved.
The phage display technique was first introduced in 1985 by Smith [1]. DNA sequences of interest are inserted at a location in the genome of the filamentous phage such that the encoded protein is expressed or ‘‘displayed” on the surface of the filamentous phage as a fusion product of one of the phage coat proteins [2]. Although phage display has traditionally been used more frequently in the development of medical preparations and for studying molecular recognition in biological systems [3,4], there have been some examples of its successful use for detection [5–7]. An interesting feature of the detection systems is that the selected phage itself is used as a probe, acting like a substitute for antibodies. There have been attempts to apply the phage display technique to immunological detection and in vivo imaging; for example, it has been applied to direct fluorochrome labeling or radiolabeling of phage display library clones and for incorporating alkaline phosphatase and antibodies or bifunctional ligand display systems [8–10]. Here we propose a new bifunctional phage display protocol for sensitive and convenient immunoassay. The coding sequences of a gold nanoparticle-binding peptide and a single-chain antibody were fused with pVIII and pIII coding genes, respectively. Thus, the resulting phage particle simultaneously displayed an antibody at the phage end and multiple copies of gold-binding peptide [11] on the surface. The resultant bifunctional gold phage, as a single phage, is like an antibody labeled with mark molecules that could mediate the antigen-binding and signal detection processes (Fig. 1A). Considering that there are approximately 2700 copies
* Corresponding author. Fax: +86 27 8719 9492. E-mail address: [email protected] (X.-E. Zhang). 1 Present address: Fok Ying Tung Graduate School, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.08.026
of pVIII proteins on a single phage virion, the gene fusion ensures that a large copy number of the gold-binding peptide can be codisplayed on one virion; this certainly increases the signal generation. This concept was demonstrated by the detection of Bacillus anthracis spores in this study [12]. M13K07 helper phage was chosen to construct a bifunctional display model system. In the most widely used phagemid antibody display system, the recombinant phage antibody system (RPAS),2 the phage rescued by the M13K07 helper phage displayed a singlechain antibody variable fragment (ScFv) on one end of the phage viron via the fusion of antibody and phage capsid protein pIII [13]. To construct the bifunctional display system, the gold-binding peptide sequence VSGSSPDS was inserted into the genome of the helper phage M13K07 through gene fusion with the major coat protein pVIII (see Supplementary material). This 8-mer gold-binding peptide motif had been previously screened through a type 8 library for specific binding to gold surface [11]. The resultant recombinant helper phage was named Bi-M13K07 phage. Thus, the rescued phage with Bi-M13K07 simultaneously displayed an antibody and a gold-binding peptide that could specifically bind antigen and gold nanoparticles, respectively. As compared with other recognizing molecules, the recombinant phage is a ready-to-use reagent that can be obtained by simply centrifuging an overnight Escherichia coli culture broth [14]. In this study, we selected a helper phage for the construction of the bifunctional phage. The helper phage can be used in the rescue of any antibody clone without being selective [15], meaning that
2 Abbreviations used: RPAS, recombinant phage antibody system; ScFv, single-chain antibody variable fragment; ELISA, enzyme-linked immunosorbent assay; TEM, transmission electron microscopy.
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Notes & Tips / Anal. Biochem. 396 (2010) 155–157
Fig. 1. Bifunctional phage-based immunoassay. (A) Scheme for the construction of bifunctional phage. (B) Analysis of the gold nanoparticle size for the optimization of the binding capacity to bifunctional phage. (C) Detection sensitivity of bifunctional phage immunoassay with silver enhancement and phage ELISA for B. anthracis spores. The polystyrene microtiter plate was coated with 100 ll of 10-fold serial dilutions of B. anthracis spores. For the bifunctional phage immunoassay with silver enhancement, the development of silver was monitored spectrophotometrically at 490 nm. For the conventional phage ELISA, 3,30 ,5,50 -tetramethylbenzidine (TMB) was used for color development. The reaction was stopped with 2 M H2SO4, and absorbance was determined at 450 nm. (D) Specificity of bifunctional phage-based immunoassay. B. anthracis and Bacillus control strain spores B. cereus, B. mycoides, B. subtilis, and B. thuringiensis were examined by using the same method.
it can easily adapt to other ScFv phage clones to produce new bifunctional molecules without complicated genetic engineering. Therefore, the method could be applied for detection of many different targets because there are already a great number of antibodies and peptides available selected from phage libraries. In the previous study, we successfully selected a monoclonal antibody named 8G3 that specifically recognized B. anthracis spores [16]. The ScFv phage clone for B. anthracis was further constructed with the RPAS phagemid system (data not shown), and bifunctional phage was produced with the rescue of engineered helper phage Bi-M13K07. The antigen-binding capacity of bifunctional phage was proved to be similar to that of the parental phage with phage enzyme-linked immunosorbent assay (ELISA) (see Supplementary Fig. S1). The binding of the gold-binding peptide displayed on the phage surface to gold nanoparticles was characterized by three means: a precipitation test, a dot blot assay, and transmission electron microscopy (TEM) observation (see Supplementary material). All of the selected phage clones were amplified to a concentration of 108 pfu/ll. Approximately 10 ll of the phage solutions were mixed with 100 ll of the 10-nm gold colloidal suspension (Ted Pella). After 2–3 h of incubation, the mixtures of the control clones with the gold colloidal suspension remained clear, whereas a visible precipitate was observed in the mixtures with the bifunctional phage (Fig. 2A), indicating that gold colloids formed aggregates. In the dot blot assay, different phages were directly added onto the nitrocellulose membrane. After blocking, the membrane was soaked in gold nanoparticle solution followed by silver enhancement. As shown in Fig. 2B, significant staining was observed in Bi-M13K07 and Bi-8G3, indicating that the bifunctional phage could effectively bind gold nanoparticles. No staining was observed for the control phage clone. TEM studies of the bifunctional phage samples revealed that gold nanoparticles assembled into wire-like structures with lengths of approximately 1 lm; this morphology is consistent with that of the M13 virus (Fig. 2D). In contrast, the TEM image of the control phage clone samples stained with phosphotungstic acid clearly revealed that the gold nanoparticles were distributed randomly and were not templated by phages (Fig. 2C).
Fig. 2. Binding of bifunctional phage to gold nanoparticles. (A) Photograph of different phage clones mixed with 10-nm gold nanoparticles. Tubes 1, 2, 3, and 4 represent phage clones M13K07, Bi-M13K07, M13-8G3, and Bi-8G3, respectively. Tubes with Bi-M13K07 and Bi-8G3 showed visible precipitates, whereas tubes with M13K07 and M13-8G3 remained clear. (B) Dot blot of phage-binding gold nanoparticles. Dots 1, 2, 3, and 4 represent phage clones M13K07, M13-8G3, BiM13K07, and Bi-8G3, respectively. Significant staining was observed in Bi-M13K07 and 8G3 rescued from Bi-M13K07. (C) TEM image of phage M13-8G3 mixed with 10-nm gold nanoparticles stained with 2% phosphotungstic acid. (D) TEM image of bifunctional phage Bi-8G3 mixed with 10-nm gold nanoparticles. Distinguishable wire-like structures were observed.
Notes & Tips / Anal. Biochem. 396 (2010) 155–157
To develop a sensitive bifunctional phage-based antigen detection technique, silver enhancement was applied in phage ELISA for signal amplification. First, the effect of gold nanoparticle size on the binding to bifunctional phage was analyzed using phage immunoassay to optimize the binding condition. Gold nanoparticles of different sizes (2, 5, 10, and 30 nm) were applied. As shown in Fig. 1B, the absorbance at 490 nm of the silver film depending on the well coated with antigen was significantly higher than that of the control antigen, and 10-nm gold nanoparticles exhibited better binding capacity to the bifunctional phage. The sensitivity of the bifunctional phage-mediated immunoassay using silver enhancement for detection of B. anthracis spores was determined in indirect immunoassay. The microtiter plate was coated with 10-fold serial dilutions of B. anthracis spores, and bifunctional phages bound with gold nanoparticles, premixed for 0.5–1 h, were then added. Because the bifunctional phages will aggregate after several hours of incubation with gold nanoparticles as described earlier, which is a problem for immunoassays, the ideal protocol for immunoassay would be binding phages to the antigens on the solid surface first, followed by the gold nanoparticle-binding process. Here the bifunctional phages and gold nanoparticles were premixed as bifunctional probes to develop a time-saving model. To reduce the interference of phage aggregation to immunoassay, the premixing time should be short, usually 0.5 h. After washing, silver enhancement was applied to detect the gold nanoparticles. The results indicated that the lowest level of B. anthracis spores detected by indirect phage immunoassay was 5 103 spores (Fig. 1C). This sensitivity is approximately 10-fold higher than that of the ELISA dose–response assay using conventional phages. To evaluate the species specificity of the method, other Bacillus strain spores, such as B. cereus, B. mycoides, B. subtilis, and B. thuringiensis spores, were also examined. The results revealed that the silver enhancement signal obtained by B. anthracis was significantly higher than that obtained by the control stains, indicating the specificity of the bifunctional immunoassay for identification of B. anthracis from other species (Fig. 1D). In conclusion, a bifunctional phage system for sensitive immunoassay has been proposed based on the phage display and gene fusion technology. This new strategy has obvious advantages over the conventional ELISA reagents. First, it produces intense signal because of multiple gold particle bindings. Furthermore, owing to the gold particle-promoted silver development, low amounts of antigen were detectable. Second, the bifunctional phage is easy to prepare and ready to use. With the antigen-binding and signal-generating capacity of bifunctional phage, it eliminates the need for secondary antibody. Besides, the principle may be used to exploit many other analytical systems, such as affinity biosensors and molecular recognition assays, and especially has potential applications in protein microarrays and surface plasmon resonance.
157
Acknowledgments We thank Ruifu Yang for providing B. anthracis samples. This work was supported by a grant from the National Natural Science Foundation of China (30700745). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.08.026. References [1] G.P. Smith, Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, Science 228 (1985) 1315–1317. [2] V.A. Petrenko, G.P. Smith, Phages from landscape libraries as substitute antibodies, Protein Eng. 13 (2000) 589–592. [3] W. Arap, M.G. Kolonin, M. Trepel, J. Lahdenranta, M. Cardo-Vila, R.J. Giordano, P.J. Mintz, P.U. Ardelt, V.J. Yao, C.I. Vidal, L. Chen, A. Flamm, H. Valtanen, L.M. Weavind, M.E. Hicks, R.E. Pollock, G.H. Botz, C.D. Bucana, E. Koivunen, D. Cahill, P. Troncoso, K.A. Baggerly, R.D. Pentz, K.A. Do, C.J. Logothetis, R. Pasqualini, Steps toward mapping the human vasculature by phage display, Nat. Med. 8 (2002) 121–127. [4] F. Curnis, A. Sacchi, L. Borgna, F. Magni, A. Gasparri, A. Corti, Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13), Nat. Biotechnol. 18 (2000) 1185–1190. [5] J. Dong, M. Ihara, H. Ueda, Antibody Fab display system that can perform opensandwich ELISA, Anal. Biochem. 386 (2009) 36–44. [6] S.S. Iqbal, M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt, J.P. Chambers, A review of molecular recognition technologies for detection of biological threat agents, Biosens. Bioelectron. 15 (2000) 549–578. [7] S.H. Wang, J.B. Zhang, Z.P. Zhang, Y.F. Zhou, R.F. Yang, J. Chen, Y.C. Guo, F. You, X.E. Zhang, Construction of single chain variable fragment (ScFv) and BiscFv– alkaline phosphatase fusion protein for detection of Bacillus anthracis, Anal. Chem. 78 (2006) 997–1004. [8] L. Chen, A.J. Zurita, P.U. Ardelt, R.J. Giordano, W. Arap, R. Pasqualini, Design and validation of a bifunctional ligand display system for receptor targeting, Chem. Biol. 11 (2004) 1081–1091. [9] C.M. Liu, Q. Jin, A. Sutton, L. Chen, A novel fluorescent probe: europium complex hybridized T7 phage, Bioconjug. Chem. 16 (2005) 1054–1057. [10] E.J. Slootweg, H.J. Keller, M.A. Hink, J.W. Borst, J. Bakker, A. Schots, Fluorescent T7 display phages obtained by translational frameshift, Nucleic Acids Res. 34 (2006) e137. [11] Y. Huang, C.Y. Chiang, S.K. Lee, Y. Gao, E.L. Hu, J. De Yoreo, A.M. Belcher, Programmable assembly of nanoarchitectures using genetically engineered viruses, Nano Lett. 5 (2005) 1429–1434. [12] K.A. Edwards, H.A. Clancy, A.J. Baeumner, Bacillus anthracis: toxicology, epidemiology, and current rapid-detection methods, Anal. Bioanal. Chem. 384 (2006) 73–84. [13] K. Chockalingam, H.D. Lu, S. Banta, Development of a bacteriophage-based system for the selection of structured peptides, Anal. Biochem. 388 (2009) 122–127. [14] Y.C. Guo, Y.F. Zhou, X.E. Zhang, Z.P. Zhang, Y.M. Qiao, L.J. Bi, J.K. Wen, M.F. Liang, J.B. Zhang, Phage display mediated immuno-PCR, Nucleic Acids Res. 34 (2006) e62. [15] R.A. Kramer, F. Cox, M. van der Horst, S. van der Oudenrijn, P.C. Res, J. Bia, T. Logtenberg, J. de Kruif, A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein, Nucleic Acids Res. 31 (2003) e59. [16] R. Hao, D. Wang, X. Zhang, G. Zuo, H. Wei, R. Yang, Z. Zhang, Z. Cheng, Y. Guo, Z. Cui, Y. Zhou, Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor, Biosens. Bioelectron. 24 (2009) 1330–1335.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Construction of bifunctional phage display for biological analysis and immunoassay Yongchao Guo a,1, Xiaosheng Liang a,b, Yafeng Zhou a, Zhiping Zhang a, Hongping Wei a, Dong Men a,b, Ming Luo a,b, Xian-En Zhang a,* a b
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Graduate School, Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e
i n f o
Article history: Received 26 June 2009 Available online 21 August 2009
a b s t r a c t A phage display-based bifunctional display system was developed for simple and sensitive immunoassay. The resulting bifunctional phage could simultaneously display a few single-chain variable fragment (ScFv) and many copies of the gold-binding peptide on its surface, thereby mediating antigen recognition and signal amplification. As a demonstration study, it was possible for bifunctional phage-based immunoassay to identify Bacillus anthracis spores from other Bacillus strains with detection sensitivity 10-fold higher than that of conventional phage enzyme-linked immunosorbent assay (ELISA). This protocol may be applied to build other bifunctional phage clones for broad applications (e.g., immunoassay kits, affinity biosensors, biorecognition assays). Ó 2009 Elsevier Inc. All rights reserved.
The phage display technique was first introduced in 1985 by Smith [1]. DNA sequences of interest are inserted at a location in the genome of the filamentous phage such that the encoded protein is expressed or ‘‘displayed” on the surface of the filamentous phage as a fusion product of one of the phage coat proteins [2]. Although phage display has traditionally been used more frequently in the development of medical preparations and for studying molecular recognition in biological systems [3,4], there have been some examples of its successful use for detection [5–7]. An interesting feature of the detection systems is that the selected phage itself is used as a probe, acting like a substitute for antibodies. There have been attempts to apply the phage display technique to immunological detection and in vivo imaging; for example, it has been applied to direct fluorochrome labeling or radiolabeling of phage display library clones and for incorporating alkaline phosphatase and antibodies or bifunctional ligand display systems [8–10]. Here we propose a new bifunctional phage display protocol for sensitive and convenient immunoassay. The coding sequences of a gold nanoparticle-binding peptide and a single-chain antibody were fused with pVIII and pIII coding genes, respectively. Thus, the resulting phage particle simultaneously displayed an antibody at the phage end and multiple copies of gold-binding peptide [11] on the surface. The resultant bifunctional gold phage, as a single phage, is like an antibody labeled with mark molecules that could mediate the antigen-binding and signal detection processes (Fig. 1A). Considering that there are approximately 2700 copies
* Corresponding author. Fax: +86 27 8719 9492. E-mail address: [email protected] (X.-E. Zhang). 1 Present address: Fok Ying Tung Graduate School, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.08.026
of pVIII proteins on a single phage virion, the gene fusion ensures that a large copy number of the gold-binding peptide can be codisplayed on one virion; this certainly increases the signal generation. This concept was demonstrated by the detection of Bacillus anthracis spores in this study [12]. M13K07 helper phage was chosen to construct a bifunctional display model system. In the most widely used phagemid antibody display system, the recombinant phage antibody system (RPAS),2 the phage rescued by the M13K07 helper phage displayed a singlechain antibody variable fragment (ScFv) on one end of the phage viron via the fusion of antibody and phage capsid protein pIII [13]. To construct the bifunctional display system, the gold-binding peptide sequence VSGSSPDS was inserted into the genome of the helper phage M13K07 through gene fusion with the major coat protein pVIII (see Supplementary material). This 8-mer gold-binding peptide motif had been previously screened through a type 8 library for specific binding to gold surface [11]. The resultant recombinant helper phage was named Bi-M13K07 phage. Thus, the rescued phage with Bi-M13K07 simultaneously displayed an antibody and a gold-binding peptide that could specifically bind antigen and gold nanoparticles, respectively. As compared with other recognizing molecules, the recombinant phage is a ready-to-use reagent that can be obtained by simply centrifuging an overnight Escherichia coli culture broth [14]. In this study, we selected a helper phage for the construction of the bifunctional phage. The helper phage can be used in the rescue of any antibody clone without being selective [15], meaning that
2 Abbreviations used: RPAS, recombinant phage antibody system; ScFv, single-chain antibody variable fragment; ELISA, enzyme-linked immunosorbent assay; TEM, transmission electron microscopy.
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Notes & Tips / Anal. Biochem. 396 (2010) 155–157
Fig. 1. Bifunctional phage-based immunoassay. (A) Scheme for the construction of bifunctional phage. (B) Analysis of the gold nanoparticle size for the optimization of the binding capacity to bifunctional phage. (C) Detection sensitivity of bifunctional phage immunoassay with silver enhancement and phage ELISA for B. anthracis spores. The polystyrene microtiter plate was coated with 100 ll of 10-fold serial dilutions of B. anthracis spores. For the bifunctional phage immunoassay with silver enhancement, the development of silver was monitored spectrophotometrically at 490 nm. For the conventional phage ELISA, 3,30 ,5,50 -tetramethylbenzidine (TMB) was used for color development. The reaction was stopped with 2 M H2SO4, and absorbance was determined at 450 nm. (D) Specificity of bifunctional phage-based immunoassay. B. anthracis and Bacillus control strain spores B. cereus, B. mycoides, B. subtilis, and B. thuringiensis were examined by using the same method.
it can easily adapt to other ScFv phage clones to produce new bifunctional molecules without complicated genetic engineering. Therefore, the method could be applied for detection of many different targets because there are already a great number of antibodies and peptides available selected from phage libraries. In the previous study, we successfully selected a monoclonal antibody named 8G3 that specifically recognized B. anthracis spores [16]. The ScFv phage clone for B. anthracis was further constructed with the RPAS phagemid system (data not shown), and bifunctional phage was produced with the rescue of engineered helper phage Bi-M13K07. The antigen-binding capacity of bifunctional phage was proved to be similar to that of the parental phage with phage enzyme-linked immunosorbent assay (ELISA) (see Supplementary Fig. S1). The binding of the gold-binding peptide displayed on the phage surface to gold nanoparticles was characterized by three means: a precipitation test, a dot blot assay, and transmission electron microscopy (TEM) observation (see Supplementary material). All of the selected phage clones were amplified to a concentration of 108 pfu/ll. Approximately 10 ll of the phage solutions were mixed with 100 ll of the 10-nm gold colloidal suspension (Ted Pella). After 2–3 h of incubation, the mixtures of the control clones with the gold colloidal suspension remained clear, whereas a visible precipitate was observed in the mixtures with the bifunctional phage (Fig. 2A), indicating that gold colloids formed aggregates. In the dot blot assay, different phages were directly added onto the nitrocellulose membrane. After blocking, the membrane was soaked in gold nanoparticle solution followed by silver enhancement. As shown in Fig. 2B, significant staining was observed in Bi-M13K07 and Bi-8G3, indicating that the bifunctional phage could effectively bind gold nanoparticles. No staining was observed for the control phage clone. TEM studies of the bifunctional phage samples revealed that gold nanoparticles assembled into wire-like structures with lengths of approximately 1 lm; this morphology is consistent with that of the M13 virus (Fig. 2D). In contrast, the TEM image of the control phage clone samples stained with phosphotungstic acid clearly revealed that the gold nanoparticles were distributed randomly and were not templated by phages (Fig. 2C).
Fig. 2. Binding of bifunctional phage to gold nanoparticles. (A) Photograph of different phage clones mixed with 10-nm gold nanoparticles. Tubes 1, 2, 3, and 4 represent phage clones M13K07, Bi-M13K07, M13-8G3, and Bi-8G3, respectively. Tubes with Bi-M13K07 and Bi-8G3 showed visible precipitates, whereas tubes with M13K07 and M13-8G3 remained clear. (B) Dot blot of phage-binding gold nanoparticles. Dots 1, 2, 3, and 4 represent phage clones M13K07, M13-8G3, BiM13K07, and Bi-8G3, respectively. Significant staining was observed in Bi-M13K07 and 8G3 rescued from Bi-M13K07. (C) TEM image of phage M13-8G3 mixed with 10-nm gold nanoparticles stained with 2% phosphotungstic acid. (D) TEM image of bifunctional phage Bi-8G3 mixed with 10-nm gold nanoparticles. Distinguishable wire-like structures were observed.
Notes & Tips / Anal. Biochem. 396 (2010) 155–157
To develop a sensitive bifunctional phage-based antigen detection technique, silver enhancement was applied in phage ELISA for signal amplification. First, the effect of gold nanoparticle size on the binding to bifunctional phage was analyzed using phage immunoassay to optimize the binding condition. Gold nanoparticles of different sizes (2, 5, 10, and 30 nm) were applied. As shown in Fig. 1B, the absorbance at 490 nm of the silver film depending on the well coated with antigen was significantly higher than that of the control antigen, and 10-nm gold nanoparticles exhibited better binding capacity to the bifunctional phage. The sensitivity of the bifunctional phage-mediated immunoassay using silver enhancement for detection of B. anthracis spores was determined in indirect immunoassay. The microtiter plate was coated with 10-fold serial dilutions of B. anthracis spores, and bifunctional phages bound with gold nanoparticles, premixed for 0.5–1 h, were then added. Because the bifunctional phages will aggregate after several hours of incubation with gold nanoparticles as described earlier, which is a problem for immunoassays, the ideal protocol for immunoassay would be binding phages to the antigens on the solid surface first, followed by the gold nanoparticle-binding process. Here the bifunctional phages and gold nanoparticles were premixed as bifunctional probes to develop a time-saving model. To reduce the interference of phage aggregation to immunoassay, the premixing time should be short, usually 0.5 h. After washing, silver enhancement was applied to detect the gold nanoparticles. The results indicated that the lowest level of B. anthracis spores detected by indirect phage immunoassay was 5 103 spores (Fig. 1C). This sensitivity is approximately 10-fold higher than that of the ELISA dose–response assay using conventional phages. To evaluate the species specificity of the method, other Bacillus strain spores, such as B. cereus, B. mycoides, B. subtilis, and B. thuringiensis spores, were also examined. The results revealed that the silver enhancement signal obtained by B. anthracis was significantly higher than that obtained by the control stains, indicating the specificity of the bifunctional immunoassay for identification of B. anthracis from other species (Fig. 1D). In conclusion, a bifunctional phage system for sensitive immunoassay has been proposed based on the phage display and gene fusion technology. This new strategy has obvious advantages over the conventional ELISA reagents. First, it produces intense signal because of multiple gold particle bindings. Furthermore, owing to the gold particle-promoted silver development, low amounts of antigen were detectable. Second, the bifunctional phage is easy to prepare and ready to use. With the antigen-binding and signal-generating capacity of bifunctional phage, it eliminates the need for secondary antibody. Besides, the principle may be used to exploit many other analytical systems, such as affinity biosensors and molecular recognition assays, and especially has potential applications in protein microarrays and surface plasmon resonance.
157
Acknowledgments We thank Ruifu Yang for providing B. anthracis samples. This work was supported by a grant from the National Natural Science Foundation of China (30700745). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.08.026. References [1] G.P. Smith, Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, Science 228 (1985) 1315–1317. [2] V.A. Petrenko, G.P. Smith, Phages from landscape libraries as substitute antibodies, Protein Eng. 13 (2000) 589–592. [3] W. Arap, M.G. Kolonin, M. Trepel, J. Lahdenranta, M. Cardo-Vila, R.J. Giordano, P.J. Mintz, P.U. Ardelt, V.J. Yao, C.I. Vidal, L. Chen, A. Flamm, H. Valtanen, L.M. Weavind, M.E. Hicks, R.E. Pollock, G.H. Botz, C.D. Bucana, E. Koivunen, D. Cahill, P. Troncoso, K.A. Baggerly, R.D. Pentz, K.A. Do, C.J. Logothetis, R. Pasqualini, Steps toward mapping the human vasculature by phage display, Nat. Med. 8 (2002) 121–127. [4] F. Curnis, A. Sacchi, L. Borgna, F. Magni, A. Gasparri, A. Corti, Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13), Nat. Biotechnol. 18 (2000) 1185–1190. [5] J. Dong, M. Ihara, H. Ueda, Antibody Fab display system that can perform opensandwich ELISA, Anal. Biochem. 386 (2009) 36–44. [6] S.S. Iqbal, M.W. Mayo, J.G. Bruno, B.V. Bronk, C.A. Batt, J.P. Chambers, A review of molecular recognition technologies for detection of biological threat agents, Biosens. Bioelectron. 15 (2000) 549–578. [7] S.H. Wang, J.B. Zhang, Z.P. Zhang, Y.F. Zhou, R.F. Yang, J. Chen, Y.C. Guo, F. You, X.E. Zhang, Construction of single chain variable fragment (ScFv) and BiscFv– alkaline phosphatase fusion protein for detection of Bacillus anthracis, Anal. Chem. 78 (2006) 997–1004. [8] L. Chen, A.J. Zurita, P.U. Ardelt, R.J. Giordano, W. Arap, R. Pasqualini, Design and validation of a bifunctional ligand display system for receptor targeting, Chem. Biol. 11 (2004) 1081–1091. [9] C.M. Liu, Q. Jin, A. Sutton, L. Chen, A novel fluorescent probe: europium complex hybridized T7 phage, Bioconjug. Chem. 16 (2005) 1054–1057. [10] E.J. Slootweg, H.J. Keller, M.A. Hink, J.W. Borst, J. Bakker, A. Schots, Fluorescent T7 display phages obtained by translational frameshift, Nucleic Acids Res. 34 (2006) e137. [11] Y. Huang, C.Y. Chiang, S.K. Lee, Y. Gao, E.L. Hu, J. De Yoreo, A.M. Belcher, Programmable assembly of nanoarchitectures using genetically engineered viruses, Nano Lett. 5 (2005) 1429–1434. [12] K.A. Edwards, H.A. Clancy, A.J. Baeumner, Bacillus anthracis: toxicology, epidemiology, and current rapid-detection methods, Anal. Bioanal. Chem. 384 (2006) 73–84. [13] K. Chockalingam, H.D. Lu, S. Banta, Development of a bacteriophage-based system for the selection of structured peptides, Anal. Biochem. 388 (2009) 122–127. [14] Y.C. Guo, Y.F. Zhou, X.E. Zhang, Z.P. Zhang, Y.M. Qiao, L.J. Bi, J.K. Wen, M.F. Liang, J.B. Zhang, Phage display mediated immuno-PCR, Nucleic Acids Res. 34 (2006) e62. [15] R.A. Kramer, F. Cox, M. van der Horst, S. van der Oudenrijn, P.C. Res, J. Bia, T. Logtenberg, J. de Kruif, A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein, Nucleic Acids Res. 31 (2003) e59. [16] R. Hao, D. Wang, X. Zhang, G. Zuo, H. Wei, R. Yang, Z. Zhang, Z. Cheng, Y. Guo, Z. Cui, Y. Zhou, Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor, Biosens. Bioelectron. 24 (2009) 1330–1335.