- Email: [email protected]
Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e5, 2017 www.elsevier.com/locate/jbiosc
Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor Takenori Shimizu,1 Torahiko Tanaka,1 Shigeyuki Uno,1 Hiroki Ashiba,2 Makoto Fujimaki,2 Mutsuo Tanaka,3 Koichi Awazu,2 and Makoto Makishima1, * Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan,1 Electronics and Photonics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan,2 and Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan3 Received 31 October 2016; accepted 10 January 2017 Available online xxx
In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion. However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect antigeneantibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus (HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant changes in the reflectance spectra, which indicate specific antigeneantibody interaction for anti-HBs antibody and antiHCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for antiHBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be applied to on-site blood testing in emergency settings. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Waveguide-mode sensor; Hepatitis B virus; Hepatitis C virus; Antibody; Horseradish peroxidase; Aminoethyl carbazole]
During large-scale disasters, such as significant earthquakes, many victims need surgical treatment and/or blood transfusion. In this setting, clinical laboratories cannot work effectively for blood typing and screening of infectious diseases due to physical damage, disrupted electric supply and transport system, and damage or injury to laboratory workers (1). Although volunteer blood donors can contribute greatly to saving the lives of victims, blood typing in the ABO and Rh(D) systems for donors and recipients is necessary before transfusion, and viral examinations of donor blood are required to decrease the risk of transfusion-transmissible viral infections, such as hepatitis B virus (HBV) and hepatitis C virus (HCV) (2,3). We have developed waveguide-mode (WM) sensors for Influenza virus detection and blood typing (4,5). A WM sensor utilizes electric field enhancement in a sensor chip, similar to a surface plasmon resonance (SPR) sensor, and is more sensitive than a reflectance absorption spectrometer (6,7). A WM sensor detects target proteins on a monolithic sensing chip with a layer of silicon sandwiched with SiO2 layers (8). Excitation of the waveguide mode in a sensing chip decreases light reflectance at a specific wavelength, and a change in surface conditions, such as a change in the refractive index caused by binding of large molecules, results in
altered reflectance spectra (6). Biological interactions, including antigeneantibody reactions for the detection of Influenza virus, can be observed on a sensing chip as a change in reflectance spectra (4). Utilization of conjugates with gold particles or dyes efficiently increases detection sensitivity of biological interactions, because the presences of such molecules on a chip surface influence reflectance property, wavelength and depth of the change in reflectance (6,9). A SPR sensor has been successfully used for detection of specific antibodies in human serum samples (10e12). Although a WM sensor is based on a principle similar to that of a SPR sensor, there are several differences between WM and SPR sensing systems. A WM sensor uses waveguide modes, instead of SPR (6,8). While the material used to induce the SPR restricts the wavelength of incident light for a SPR sensor, there is no such restriction for a WM sensor. In addition, a stable sensing surface made of glass is available for a WM sensor. Thus, the wavelength of a WM sensor is widely controllable over the visible light region by adjusting the thicknesses of the top silicon oxide and embedded silicon layers. A WM sensor has sensitivity similar to enzyme-liked immunosorbent assay (ELISA) and consumes shorter experimental time than ELISA (13). Although a WM sensor can detect leptin diluted in human serum by immunoassay using anti-leptin antibody (14), it has been unclear whether a WM sensor can be applied for detection of endogenous antibodies in blood samples.
* Corresponding author. Tel./fax: þ81 3 3972 8199. E-mail address: [email protected] (M. Makishima).
1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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In this study, we evaluated the potential of a WM sensor as a method for detection of antibodies against HBV and HCV in blood samples. Since a WM sensor-based method can utilize a portable, small-sized instrument and work with battery power source, it should be useful during large-scale disasters. We increased the detection sensitivity for anti-HBs antibody more than 300-fold by introducing a peroxidase reaction in the detection process and succeeded in detecting endogenous anti-hepatitis B surface (HBs) antibody in human plasma. MATERIALS AND METHODS WM sensors Optical arrangement of a WM sensor device was based on the Kretschmann configuration as reported previously (6). When light enters through a prism at a particular angle of incidence, light of a specific wavelength is propagated in a slab waveguide of a sensing chip, a phenomenon known as excitation of the WM, which causes decrease in the reflectance of light (Fig. 1A). Sensing chips were composed of a glass substrate, an embedded Si layer, and a top SiO2 layer. The Si and SiO2 layers function as the slab waveguide. We used sensing chips for colorless and colored targets as reported previously (8,9). When a spectrum of reflected light from a white LED is measured by the spectrometer, a specific wavelength appears as a dip in reflectance spectra. When target molecules (particles) are captured on a sensing chip, the interaction influences the reflective property of the waveguide surface and the complex refractive index near the waveguide surface can be evaluated as a change in the depth and/or wavelength of the dip in reflectance spectra (Fig. 1B). The interaction of colorless target molecules on a chip preferentially induces a shift of the dip peak position (Dl) (6). Dl levels are dependent on both the amount and size of bound molecules. For detection of colored target molecules, a change in the depth (DR) on a chip is also useful to obtain high sensitivity (6,15). The detection limit was defined as the lowest concentration of antibody that showed a statistically significant difference from control. All measurements were performed at room temperature with a WM sensor device EVA-001 (Optex, Otsu, Japan). Antigens and antibodies HBs adr antigen was purchased from ProsPec (East Brunswick, NJ, USA), and HCV core domain 1 protein was generated using an expression plasmid of core domain 1, which was made from the pON/C-5B/KE plasmid harboring HCV strain O genome (kindly provided by Prof. Nobuyuki Kato of Okayama University, Japan) (16). Mouse monoclonal antibodies, anti-HBs Hs33 and anti-HCV core protein H6-29, were purchased from HyTest Ltd. (Turku, Finland) and BioAcademia Inc. (Osaka, Japan), respectively. Immobilization of antigens on sensing chips surfaces To capture antibodies, HBs antigen and HCV core antigen were immobilized on chips. For antigen immobilization, a silica surface of a sensing chip was chemically modified with
J. BIOSCI. BIOENG., triethoxysilane derivatives, C12Es (silane bearing succinimide ester moiety) and M3EG (silane bearing methoxytriethylene glycol moiety), as described previously (14). Chips were immersed in a mixture of C12Es and M3EG toluene solutions for 72 h at 50 C, where the molar ratio of C12Es and M3EG was 1:10. M3EG and C12Es were used for blocking and immobilization of proteins, respectively. The modified chip was rinsed with acetone and dried. The succinimide ester group of C12Es was reacted to form a covalent bond with amine residues of antigens. Detection of mouse monoclonal anti-HBs antibody and anti HCV core antibody diluted in plasma with a WM sensor Before antigeneantibody reaction, phosphate buffered saline (PBS) was placed on a chip immobilized with HBs antigen or HCV core antigen at room temperature and a baseline spectrum was measured. Each chip has a material-dependent characteristic baseline spectrum. To develop a detection system for anti-HBs antibody on a WM sensor, human plasma containing the mouse anti-HBs antibody Hs33 was utilized. Antibody was diluted in plasma from HBs antibody-negative volunteers at final concentrations from 0.03 to 100 nM. Antibody containing plasma was added for antigeneantibody reaction on a chip for 30 min. The chip surface was washed with PBS five times and a spectrum was measured. To develop a detection system for anti-HCV core antibody, anti-HCV core antibody H6-29 was diluted in plasma from HCV antibody-negative persons, and was incubated on a chip immobilized with HCV core domain 1 antigen for 30 min. The chip surface was washed with PBS five times and a spectrum was measured. Measurement of anti-HBs antibody with a WM sensor and signal enhancement method For enhancement of detection sensitivity, a method utilizing a peroxidase reaction was evaluated. For this reaction, after incubation of a chip immobilized with HBs antigen with a test sample as described above for a method without enhancement, HRP-conjugated anti-mouse or anti-human IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in Can Get Signal Solution 2 (Toyobo Co., Osaka, Japan) was incubated on a chip for 30 min. The chip surface was washed with PBS-0.1% Tween 20 (PBST) instead of PBS, and then washed with PBS. Finally, aminoethyl carbazole (AEC) solution (Enzo Life Sciences, Farmingdale, NY, USA) was incubated as substrate of peroxidase reaction on a chip for 5 min. HRP catalyzes the conversion of AEC to red water-insoluble precipitates. The chip surface was washed with PBST and with PBS to remove unreacted AEC, and a spectrum was measured. Human plasma from healthy vaccinated and non-vaccinated volunteers were also examined with the peroxidase method for anti-HBs antibody detection. Anti-HBs antibody-positive blood was taken from a medical worker, who was inoculated with recombinant hepatitis B vaccine three times and was confirmed to be immunized by a hospital laboratory. We also utilized a polymerized secondary antibody, MAHG-SZ (Anti-human IgG [H, g-chain specific]-Strongzyme HRP Conjugate, Mouse-Mono; Stereospecific Detection Technologies, Baesweiler, Germany), which harbors multiple molecules of an anti-human IgG antibody and HRP on dextran polymers instead of the conventional HRP-conjugated antihuman IgG. This study was approved by Research Ethics Committee of Nihon University School of Medicine (reference number 25-6-0).
FIG. 1. A schematic diagram of a WM sensor for detection of antigeneantibody complexes. (A) A frame format of a WM sensor. A sensing chip is composed of 3 layers of glass (SiO2 and Si). (B) Illustration of dips detected in reflectance spectra. Interaction of target molecules on a chip changes a dip shape as DR in depth and/or Dl in wavelength.
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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Measurement of anti-HBs antibody with a WM sensor To evaluate a detection system for anti-HBs antibody on a WM sensor, we chose anti-HBs monoclonal antibody Hs33 after comparing several commercial anti HBs antibodies for the sensitivity of antigeneantibody interaction. First, we examined whether we can detect interaction of anti-HBs antibody suspended in human plasma with HBs antigen immobilized on a chip using a WM sensor without a enhancing method. Although plasma used for dilution of antibody contain immunoglobulin, we used control mouse monoclonal antibody at the same concentrations to verify specificity of anti-HBs antibody. While addition of control antibody did not, incubation with anti-HBs antibody resulted in a dip shape spectra (Fig. 2A). An anti-HBs antibody-dependent Dl change was observed at 100 nM, although it was not significant at 10 nM (Fig. 2B). A Dl value obtained from anti-HBs antibody at 100 nM was larger than that at 10 nM (Fig. 2B). The difference between Dl values from control antibody at 10 nM and 100 nM was not significant. Measurement of anti-HCV antibody with a WM sensor Similarly to detection of anti-HBs antibody, anti-HCV core antibody H6-29 was dissolved in human plasma and was detected with a WM sensor. We observed a dip for 10 nM antiHCV antibody, which was not statistically significant (Fig. 3A). This method significantly detected anti-HCV antibody at 100 nM in a WM sensor (Fig. 3B). Enhanced sensitivity in detection of anti-HBs antibody We used chips modified with M3EG-C12Es, which were reported to show the highest nonspecific adsorption-resistant effect in human serum (14). Although we detected antigeneantibody interactions for anti-HBs antibody and anti-HCV antibody with a WM sensor (Figs. 2 and 3), we investigated how to enhance detection sensitivity and/or to reduce nonspecific signals. We
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could decrease background signals by washing a tip surface with PBST instead of PBS. Furthermore, in order to increase detection sensitivity, we examined a method using peroxidase-conjugated secondary antibody with a peroxidase reaction on AEC. This signal enhancement method successfully detected anti-HBs antibody at 0.03 and 0.1 nM (Fig. 4A). The color change was not recognized with naked eye. We observed a concentrationdependent change from 0.01 nM to 10 nM, and the detection limit was 0.03 nM (Fig. S1). Compared to the detection sensitivity without enhancement (Fig. 2), the method using peroxidaseconjugated secondary antibody and AEC staining increases detection sensitivity of anti-HBs antibody in a WM sensor more than 300-fold. Finally, we applied a WM sensor with peroxidase enhancement to detect anti-HBs antibody in human blood samples. Using a chip immobilized with HBs antigen and HRP-conjugated anti-human IgG antibody with peroxidase reaction, we observed a significant difference in DR between plasma from non-vaccinated and vaccinated persons (Fig. 4B). Interestingly, when we used the polymerized secondary antibody MAHG-SZ and the peroxidase reaction, the difference in DR became larger (Fig. 4C). Therefore, a WM with enhanced by conjugated secondary antibody and dye reaction can be applied to detection of anti-HBs antibody in human blood samples. DISCUSSION We report here a novel method to detect anti-viral antibodies in human blood samples using a WM sensor. This is the first report describing the use of a WM sensor for detection of endogenous antibody in human blood. First, we evaluated a detection system by using mouse monoclonal antibody (anti-HBs antibody or anti-HCV core antibody) diluted in human plasma, and found that anti-HBs antibody (100 nM) and anti-HCV core antibody (100 nM) can be detected in a WM sensor. Next, we developed a signal enhancement
FIG. 2. Detection of anti-HBs antibody with a WM sensor. (A) Spectral changes by antigeneantibody reaction on a sensor chip. Left, control antibody. Right, anti-HBs antibody (Hs33). (B) Anti-HBs antibody is detected with a WM sensor. *p < 0.05; **p < 0.01; n.s., not significant (one-way ANOVA followed by Tukey’s multiple comparisons). Data are presented as means S.D. (n ¼ 3).
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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FIG. 3. Detection of anti-HCV core antibody with a WM sensor. (A) Spectral changes by antigeneantibody reaction on a sensor chip. Left, control plasma. Middle and right, anti-HCV core antibody (H6-29). (B) Anti-HCV antibody induces a concentration-dependent dip shift. **p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons). Data are presented as means S.D. (n ¼ 3).
method and obtained improved sensitivity for detection of antiHBs antibody by more than 300-fold (0.03 nM) using a HRP reaction on AEC dye. The peroxidase reaction generates red-colored water-insoluble precipitates from AEC, which exhibit optical absorption and strongly influence light waves in a WM sensor. Thus, the WM spectra reveal a change in the depth of a dip. Comparing between before and after the HRP reaction, the following two factors are considered to contribute to the sensitivity enhancement: (i) The changes in the extinction coefficient, k, caused by a colored substance induce a larger change in the spectra than that in the refractive index, n, caused by a colorless substance (9), and (ii) The HRP reaction continuously generates the colored product until the reaction is stopped by washing, and the change in k is enhanced by the progress of the reaction. Finally, our enhanced detection method could be applied for detection of human anti-HBs antibody in blood samples from a vaccinated individual. These results have encouraged us to develop a WM sensor as a portable blood test device. The limitation of the current enhancement method is a
reaction time of about 90 min, because it needs repeated washing to decrease nonspecific effects of plasma proteins. We are further developing an improved detection method using conjugated secondary molecules to increase sensitivity and to shorten reaction time. Usual methods to detect antibodies, including anti-HBs antibody and anti-HCV core antibody, are hemagglutination assay, chemiluminescent immunoassay and chemiluminescent enzyme immunoassay. These methods are sensitive and quantitative methods, but usually require a clinical laboratory and become impractical during large-scale disasters. Several portable devices for chemiluminescent reactions have been developed and may be useful for on-site testing (17). Immunochromatography is a handy method and can be used in cases of emergency. A method using SPR is similar to a WM sensor in detection of molecules on a chip and is useful in biochemical analysis (18e20). A portable SPR sensor has been developed for immunoassay (21). We have successfully developed microfluidic chips on a WM sensor for blood typing
FIG. 4. HRP-conjugated secondary antibody and HRP reaction increase detection sensitivity. (A) Increased sensitivity in detection of anti-HBs antibody (Hs33). HRP-conjugated antimouse IgG was added after antigeneantibody interaction and HRP reacted on AEC dye. (B, C) Anti-HBs antibody in human blood samples is detected with a WM sensor in combination with enhancing methods. While HRP-conjugated anti-human IgG was used in panels B and C, HRP reactions were performed on AEC in panel B like in panel A and on the combination of MAHG-SZ and AEC reactions in panel C. Plasma A and B were derived from a non-vaccinated individual and a vaccinated individual, respectively. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons for panel A; unpaired two-group Student’s t test for panels B and C). Data are presented as means S.D. [(A, C) n ¼ 3; (B) n ¼ 6].
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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(5,22,23). A method using a multichannel WM will be useful for both blood typing and screening of virus antibodies. In this study, we used a single-channel WM sensor, which is 30 cm wide, 15 cm tall and 20 cm deep. We previously reported a five-channel WM sensor with a size of 27 12 7.5 cm3 (23). At present, the minimal WM sensor is 15 7 5 cm3, equipped with four-channel chips (C and I KK (Tsukuba, Japan), personal communications). Therefore, in addition to immunochromatography and a portable SPR sensor, a WM sensor could be used as an on-site portable diagnostic device in emergency settings, particularly in large-scale disasters. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2017.01.004.
ACKNOWLEDGMENTS The authors thank Ms. Risa Kono for expert technical assistance, Dr. Mami Yamamoto, Dr. Alena Lagumdzija, Ms. Ayumi Sato, and other members of the Makishima laboratory for helpful comments and assistance, the Advanced Functional Materials Research Center of Shin-Etsu Chemical Co. Ltd., for supplying sensing plates, and Dr. Andrew I. Shulman for editorial assistance. This work was supported by SENTAN program of Japan Science and Technology Agency (2012e2014) and Japan Agency for Medical Research and Development (2015e2016; grant numbers 15hm0102001h0004 and 16hm0102001s0205).
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7. Fujimaki, M., Rockstuhl, C., Wang, X., Awazu, K., Tominaga, J., Ikeda, T., Koganezawa, Y., and Ohki, Y.: Biomolecular sensors utilizing waveguide modes excited by evanescent fields, J. Microsc., 229, 320e326 (2008). 8. Fujimaki, M., Rockstuhl, C., Wang, X., Awazu, K., Tominaga, J., Koganezawa, Y., Ohki, Y., and Komatsubara, T.: Silica-based monolithic sensing plates for waveguide-mode sensors, Opt. Express, 16, 6408e6416 (2008). 9. Fujimaki, M., Nomura, K., Sato, K., Kato, T., Gopinath, S. C., Wang, X., Awazu, K., and Ohki, Y.: Detection of colored nanomaterials using evanescent field-based waveguide sensors, Opt. Express, 18, 15732e15740 (2010). 10. Campagnolo, C., Meyers, K. J., Ryan, T., Atkinson, R. C., Chen, Y. T., Scanlan, M. J., Ritter, G., Old, L. J., and Batt, C. A.: Real-time, label-free monitoring of tumor antigen and serum antibody interactions, J. Biochem. Biophys. Methods, 61, 283e298 (2004). 11. Ladd, J., Lu, H., Taylor, A. D., Goodell, V., Disis, M. L., and Jiang, S.: Direct detection of carcinoembryonic antigen autoantibodies in clinical human serum samples using a surface plasmon resonance sensor, Colloids Surf. B. Biointerfaces, 70, 1e6 (2009). 12. Xu, H., Gu, D., He, J., Shi, L., Yao, J., Liu, C., Zhao, C., Xu, Y., Jiang, S., and Long, J.: Multiplex biomarker analysis biosensor for detection of hepatitis B virus, Biomed. Mater. Eng., 26(suppl. 1), S2091eS2100 (2015). 13. Gopinath, S. C. B., Awazu, K., Fujimaki, M., and Shimizu, K.: Evaluation of anti-A/Udorn/307/1972 antibody specificity to influenza A/H3N2 viruses using an evanescent-field coupled waveguide-mode sensor, PLoS One, 8, e81396 (2013). 14. Tanaka, M., Yoshioka, K., Hirata, Y., Fujimaki, M., Kuwahara, M., and Niwa, O.: Design and fabrication of biosensing interface for waveguide-mode sensor, Langmuir, 29, 13111e13120 (2013). 15. Fujimaki, M., Wang, X., Kato, T., Awazu, K., and Ohki, Y.: Parallel-incidencetype waveguide-mode sensor with spectral-readout setup, Opt. Express, 23, 10925e10937 (2015). 16. Ikeda, M., Abe, K., Dansako, H., Nakamura, T., Naka, K., and Kato, N.: Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system, Biochem. Biophys. Res. Commun., 329, 1350e1359 (2005). 17. Park, J. Y. and Kricka, L. J.: Prospects for the commercialization of chemiluminescence-based point-of-care and on-site testing devices, Anal. Bioanal. Chem., 406, 5631e5637 (2014). 18. Liedberg, B., Nylander, C., and Lunström, I.: Surface plasmon resonance for gas detection and biosensing, Sens. Actuators, 4, 299e304 (1983). 19. Chuang, T. L., Wei, S. C., Lee, S. Y., and Lin, C. W.: A polycarbonate based surface plasmon resonance sensing cartridge for high sensitivity HBV loopmediated isothermal amplification, Biosens. Bioelectron, 32, 89e95 (2012). 20. Bolduc, O. R., Live, L. S., and Masson, J. F.: High-resolution surface plasmon resonance sensors based on a dove prism, Talanta, 77, 1680e1687 (2009). 21. Horiuchi, T., Tobita, T., Miura, T., Iwasaki, Y., Seyama, M., Inoue, S., Takahashi, J., Haga, T., and Tamechika, E.: Floating chip mounting system driven by repulsive force of permanent magnets for multiple on-site SPR immunoassay measurements, Sensors, 12, 13964e13984 (2012). 22. Ashiba, H., Fujimaki, M., Awazu, K., Fu, M., Ohki, Y., Tanaka, T., and Makishima, M.: Rapid detection of hemagglutination using restrictive microfluidic channels equipped with waveguide-mode sensors, Jpn. J. Appl. Phys., 55, 027002 (2016). 23. Ashiba, H., Fujimaki, M., Awazu, K., Tanaka, T., and Makishima, M.: Microfluidic chips for forward blood typing performed with a multichannel waveguide-mode sensor, Sens. Bio-Sensing Res., 7, 121e126 (2016).
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor Takenori Shimizu,1 Torahiko Tanaka,1 Shigeyuki Uno,1 Hiroki Ashiba,2 Makoto Fujimaki,2 Mutsuo Tanaka,3 Koichi Awazu,2 and Makoto Makishima1, * Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan,1 Electronics and Photonics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan,2 and Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan3 Received 31 October 2016; accepted 10 January 2017 Available online xxx
In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion. However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect antigeneantibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus (HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant changes in the reflectance spectra, which indicate specific antigeneantibody interaction for anti-HBs antibody and antiHCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for antiHBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be applied to on-site blood testing in emergency settings. Ó 2017, The Society for Biotechnology, Japan. All rights reserved. [Key words: Waveguide-mode sensor; Hepatitis B virus; Hepatitis C virus; Antibody; Horseradish peroxidase; Aminoethyl carbazole]
During large-scale disasters, such as significant earthquakes, many victims need surgical treatment and/or blood transfusion. In this setting, clinical laboratories cannot work effectively for blood typing and screening of infectious diseases due to physical damage, disrupted electric supply and transport system, and damage or injury to laboratory workers (1). Although volunteer blood donors can contribute greatly to saving the lives of victims, blood typing in the ABO and Rh(D) systems for donors and recipients is necessary before transfusion, and viral examinations of donor blood are required to decrease the risk of transfusion-transmissible viral infections, such as hepatitis B virus (HBV) and hepatitis C virus (HCV) (2,3). We have developed waveguide-mode (WM) sensors for Influenza virus detection and blood typing (4,5). A WM sensor utilizes electric field enhancement in a sensor chip, similar to a surface plasmon resonance (SPR) sensor, and is more sensitive than a reflectance absorption spectrometer (6,7). A WM sensor detects target proteins on a monolithic sensing chip with a layer of silicon sandwiched with SiO2 layers (8). Excitation of the waveguide mode in a sensing chip decreases light reflectance at a specific wavelength, and a change in surface conditions, such as a change in the refractive index caused by binding of large molecules, results in
altered reflectance spectra (6). Biological interactions, including antigeneantibody reactions for the detection of Influenza virus, can be observed on a sensing chip as a change in reflectance spectra (4). Utilization of conjugates with gold particles or dyes efficiently increases detection sensitivity of biological interactions, because the presences of such molecules on a chip surface influence reflectance property, wavelength and depth of the change in reflectance (6,9). A SPR sensor has been successfully used for detection of specific antibodies in human serum samples (10e12). Although a WM sensor is based on a principle similar to that of a SPR sensor, there are several differences between WM and SPR sensing systems. A WM sensor uses waveguide modes, instead of SPR (6,8). While the material used to induce the SPR restricts the wavelength of incident light for a SPR sensor, there is no such restriction for a WM sensor. In addition, a stable sensing surface made of glass is available for a WM sensor. Thus, the wavelength of a WM sensor is widely controllable over the visible light region by adjusting the thicknesses of the top silicon oxide and embedded silicon layers. A WM sensor has sensitivity similar to enzyme-liked immunosorbent assay (ELISA) and consumes shorter experimental time than ELISA (13). Although a WM sensor can detect leptin diluted in human serum by immunoassay using anti-leptin antibody (14), it has been unclear whether a WM sensor can be applied for detection of endogenous antibodies in blood samples.
* Corresponding author. Tel./fax: þ81 3 3972 8199. E-mail address: [email protected] (M. Makishima).
1389-1723/$ e see front matter Ó 2017, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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In this study, we evaluated the potential of a WM sensor as a method for detection of antibodies against HBV and HCV in blood samples. Since a WM sensor-based method can utilize a portable, small-sized instrument and work with battery power source, it should be useful during large-scale disasters. We increased the detection sensitivity for anti-HBs antibody more than 300-fold by introducing a peroxidase reaction in the detection process and succeeded in detecting endogenous anti-hepatitis B surface (HBs) antibody in human plasma. MATERIALS AND METHODS WM sensors Optical arrangement of a WM sensor device was based on the Kretschmann configuration as reported previously (6). When light enters through a prism at a particular angle of incidence, light of a specific wavelength is propagated in a slab waveguide of a sensing chip, a phenomenon known as excitation of the WM, which causes decrease in the reflectance of light (Fig. 1A). Sensing chips were composed of a glass substrate, an embedded Si layer, and a top SiO2 layer. The Si and SiO2 layers function as the slab waveguide. We used sensing chips for colorless and colored targets as reported previously (8,9). When a spectrum of reflected light from a white LED is measured by the spectrometer, a specific wavelength appears as a dip in reflectance spectra. When target molecules (particles) are captured on a sensing chip, the interaction influences the reflective property of the waveguide surface and the complex refractive index near the waveguide surface can be evaluated as a change in the depth and/or wavelength of the dip in reflectance spectra (Fig. 1B). The interaction of colorless target molecules on a chip preferentially induces a shift of the dip peak position (Dl) (6). Dl levels are dependent on both the amount and size of bound molecules. For detection of colored target molecules, a change in the depth (DR) on a chip is also useful to obtain high sensitivity (6,15). The detection limit was defined as the lowest concentration of antibody that showed a statistically significant difference from control. All measurements were performed at room temperature with a WM sensor device EVA-001 (Optex, Otsu, Japan). Antigens and antibodies HBs adr antigen was purchased from ProsPec (East Brunswick, NJ, USA), and HCV core domain 1 protein was generated using an expression plasmid of core domain 1, which was made from the pON/C-5B/KE plasmid harboring HCV strain O genome (kindly provided by Prof. Nobuyuki Kato of Okayama University, Japan) (16). Mouse monoclonal antibodies, anti-HBs Hs33 and anti-HCV core protein H6-29, were purchased from HyTest Ltd. (Turku, Finland) and BioAcademia Inc. (Osaka, Japan), respectively. Immobilization of antigens on sensing chips surfaces To capture antibodies, HBs antigen and HCV core antigen were immobilized on chips. For antigen immobilization, a silica surface of a sensing chip was chemically modified with
J. BIOSCI. BIOENG., triethoxysilane derivatives, C12Es (silane bearing succinimide ester moiety) and M3EG (silane bearing methoxytriethylene glycol moiety), as described previously (14). Chips were immersed in a mixture of C12Es and M3EG toluene solutions for 72 h at 50 C, where the molar ratio of C12Es and M3EG was 1:10. M3EG and C12Es were used for blocking and immobilization of proteins, respectively. The modified chip was rinsed with acetone and dried. The succinimide ester group of C12Es was reacted to form a covalent bond with amine residues of antigens. Detection of mouse monoclonal anti-HBs antibody and anti HCV core antibody diluted in plasma with a WM sensor Before antigeneantibody reaction, phosphate buffered saline (PBS) was placed on a chip immobilized with HBs antigen or HCV core antigen at room temperature and a baseline spectrum was measured. Each chip has a material-dependent characteristic baseline spectrum. To develop a detection system for anti-HBs antibody on a WM sensor, human plasma containing the mouse anti-HBs antibody Hs33 was utilized. Antibody was diluted in plasma from HBs antibody-negative volunteers at final concentrations from 0.03 to 100 nM. Antibody containing plasma was added for antigeneantibody reaction on a chip for 30 min. The chip surface was washed with PBS five times and a spectrum was measured. To develop a detection system for anti-HCV core antibody, anti-HCV core antibody H6-29 was diluted in plasma from HCV antibody-negative persons, and was incubated on a chip immobilized with HCV core domain 1 antigen for 30 min. The chip surface was washed with PBS five times and a spectrum was measured. Measurement of anti-HBs antibody with a WM sensor and signal enhancement method For enhancement of detection sensitivity, a method utilizing a peroxidase reaction was evaluated. For this reaction, after incubation of a chip immobilized with HBs antigen with a test sample as described above for a method without enhancement, HRP-conjugated anti-mouse or anti-human IgG antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in Can Get Signal Solution 2 (Toyobo Co., Osaka, Japan) was incubated on a chip for 30 min. The chip surface was washed with PBS-0.1% Tween 20 (PBST) instead of PBS, and then washed with PBS. Finally, aminoethyl carbazole (AEC) solution (Enzo Life Sciences, Farmingdale, NY, USA) was incubated as substrate of peroxidase reaction on a chip for 5 min. HRP catalyzes the conversion of AEC to red water-insoluble precipitates. The chip surface was washed with PBST and with PBS to remove unreacted AEC, and a spectrum was measured. Human plasma from healthy vaccinated and non-vaccinated volunteers were also examined with the peroxidase method for anti-HBs antibody detection. Anti-HBs antibody-positive blood was taken from a medical worker, who was inoculated with recombinant hepatitis B vaccine three times and was confirmed to be immunized by a hospital laboratory. We also utilized a polymerized secondary antibody, MAHG-SZ (Anti-human IgG [H, g-chain specific]-Strongzyme HRP Conjugate, Mouse-Mono; Stereospecific Detection Technologies, Baesweiler, Germany), which harbors multiple molecules of an anti-human IgG antibody and HRP on dextran polymers instead of the conventional HRP-conjugated antihuman IgG. This study was approved by Research Ethics Committee of Nihon University School of Medicine (reference number 25-6-0).
FIG. 1. A schematic diagram of a WM sensor for detection of antigeneantibody complexes. (A) A frame format of a WM sensor. A sensing chip is composed of 3 layers of glass (SiO2 and Si). (B) Illustration of dips detected in reflectance spectra. Interaction of target molecules on a chip changes a dip shape as DR in depth and/or Dl in wavelength.
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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Measurement of anti-HBs antibody with a WM sensor To evaluate a detection system for anti-HBs antibody on a WM sensor, we chose anti-HBs monoclonal antibody Hs33 after comparing several commercial anti HBs antibodies for the sensitivity of antigeneantibody interaction. First, we examined whether we can detect interaction of anti-HBs antibody suspended in human plasma with HBs antigen immobilized on a chip using a WM sensor without a enhancing method. Although plasma used for dilution of antibody contain immunoglobulin, we used control mouse monoclonal antibody at the same concentrations to verify specificity of anti-HBs antibody. While addition of control antibody did not, incubation with anti-HBs antibody resulted in a dip shape spectra (Fig. 2A). An anti-HBs antibody-dependent Dl change was observed at 100 nM, although it was not significant at 10 nM (Fig. 2B). A Dl value obtained from anti-HBs antibody at 100 nM was larger than that at 10 nM (Fig. 2B). The difference between Dl values from control antibody at 10 nM and 100 nM was not significant. Measurement of anti-HCV antibody with a WM sensor Similarly to detection of anti-HBs antibody, anti-HCV core antibody H6-29 was dissolved in human plasma and was detected with a WM sensor. We observed a dip for 10 nM antiHCV antibody, which was not statistically significant (Fig. 3A). This method significantly detected anti-HCV antibody at 100 nM in a WM sensor (Fig. 3B). Enhanced sensitivity in detection of anti-HBs antibody We used chips modified with M3EG-C12Es, which were reported to show the highest nonspecific adsorption-resistant effect in human serum (14). Although we detected antigeneantibody interactions for anti-HBs antibody and anti-HCV antibody with a WM sensor (Figs. 2 and 3), we investigated how to enhance detection sensitivity and/or to reduce nonspecific signals. We
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could decrease background signals by washing a tip surface with PBST instead of PBS. Furthermore, in order to increase detection sensitivity, we examined a method using peroxidase-conjugated secondary antibody with a peroxidase reaction on AEC. This signal enhancement method successfully detected anti-HBs antibody at 0.03 and 0.1 nM (Fig. 4A). The color change was not recognized with naked eye. We observed a concentrationdependent change from 0.01 nM to 10 nM, and the detection limit was 0.03 nM (Fig. S1). Compared to the detection sensitivity without enhancement (Fig. 2), the method using peroxidaseconjugated secondary antibody and AEC staining increases detection sensitivity of anti-HBs antibody in a WM sensor more than 300-fold. Finally, we applied a WM sensor with peroxidase enhancement to detect anti-HBs antibody in human blood samples. Using a chip immobilized with HBs antigen and HRP-conjugated anti-human IgG antibody with peroxidase reaction, we observed a significant difference in DR between plasma from non-vaccinated and vaccinated persons (Fig. 4B). Interestingly, when we used the polymerized secondary antibody MAHG-SZ and the peroxidase reaction, the difference in DR became larger (Fig. 4C). Therefore, a WM with enhanced by conjugated secondary antibody and dye reaction can be applied to detection of anti-HBs antibody in human blood samples. DISCUSSION We report here a novel method to detect anti-viral antibodies in human blood samples using a WM sensor. This is the first report describing the use of a WM sensor for detection of endogenous antibody in human blood. First, we evaluated a detection system by using mouse monoclonal antibody (anti-HBs antibody or anti-HCV core antibody) diluted in human plasma, and found that anti-HBs antibody (100 nM) and anti-HCV core antibody (100 nM) can be detected in a WM sensor. Next, we developed a signal enhancement
FIG. 2. Detection of anti-HBs antibody with a WM sensor. (A) Spectral changes by antigeneantibody reaction on a sensor chip. Left, control antibody. Right, anti-HBs antibody (Hs33). (B) Anti-HBs antibody is detected with a WM sensor. *p < 0.05; **p < 0.01; n.s., not significant (one-way ANOVA followed by Tukey’s multiple comparisons). Data are presented as means S.D. (n ¼ 3).
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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FIG. 3. Detection of anti-HCV core antibody with a WM sensor. (A) Spectral changes by antigeneantibody reaction on a sensor chip. Left, control plasma. Middle and right, anti-HCV core antibody (H6-29). (B) Anti-HCV antibody induces a concentration-dependent dip shift. **p < 0.01 (one-way ANOVA followed by Tukey’s multiple comparisons). Data are presented as means S.D. (n ¼ 3).
method and obtained improved sensitivity for detection of antiHBs antibody by more than 300-fold (0.03 nM) using a HRP reaction on AEC dye. The peroxidase reaction generates red-colored water-insoluble precipitates from AEC, which exhibit optical absorption and strongly influence light waves in a WM sensor. Thus, the WM spectra reveal a change in the depth of a dip. Comparing between before and after the HRP reaction, the following two factors are considered to contribute to the sensitivity enhancement: (i) The changes in the extinction coefficient, k, caused by a colored substance induce a larger change in the spectra than that in the refractive index, n, caused by a colorless substance (9), and (ii) The HRP reaction continuously generates the colored product until the reaction is stopped by washing, and the change in k is enhanced by the progress of the reaction. Finally, our enhanced detection method could be applied for detection of human anti-HBs antibody in blood samples from a vaccinated individual. These results have encouraged us to develop a WM sensor as a portable blood test device. The limitation of the current enhancement method is a
reaction time of about 90 min, because it needs repeated washing to decrease nonspecific effects of plasma proteins. We are further developing an improved detection method using conjugated secondary molecules to increase sensitivity and to shorten reaction time. Usual methods to detect antibodies, including anti-HBs antibody and anti-HCV core antibody, are hemagglutination assay, chemiluminescent immunoassay and chemiluminescent enzyme immunoassay. These methods are sensitive and quantitative methods, but usually require a clinical laboratory and become impractical during large-scale disasters. Several portable devices for chemiluminescent reactions have been developed and may be useful for on-site testing (17). Immunochromatography is a handy method and can be used in cases of emergency. A method using SPR is similar to a WM sensor in detection of molecules on a chip and is useful in biochemical analysis (18e20). A portable SPR sensor has been developed for immunoassay (21). We have successfully developed microfluidic chips on a WM sensor for blood typing
FIG. 4. HRP-conjugated secondary antibody and HRP reaction increase detection sensitivity. (A) Increased sensitivity in detection of anti-HBs antibody (Hs33). HRP-conjugated antimouse IgG was added after antigeneantibody interaction and HRP reacted on AEC dye. (B, C) Anti-HBs antibody in human blood samples is detected with a WM sensor in combination with enhancing methods. While HRP-conjugated anti-human IgG was used in panels B and C, HRP reactions were performed on AEC in panel B like in panel A and on the combination of MAHG-SZ and AEC reactions in panel C. Plasma A and B were derived from a non-vaccinated individual and a vaccinated individual, respectively. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons for panel A; unpaired two-group Student’s t test for panels B and C). Data are presented as means S.D. [(A, C) n ¼ 3; (B) n ¼ 6].
Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004
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(5,22,23). A method using a multichannel WM will be useful for both blood typing and screening of virus antibodies. In this study, we used a single-channel WM sensor, which is 30 cm wide, 15 cm tall and 20 cm deep. We previously reported a five-channel WM sensor with a size of 27 12 7.5 cm3 (23). At present, the minimal WM sensor is 15 7 5 cm3, equipped with four-channel chips (C and I KK (Tsukuba, Japan), personal communications). Therefore, in addition to immunochromatography and a portable SPR sensor, a WM sensor could be used as an on-site portable diagnostic device in emergency settings, particularly in large-scale disasters. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2017.01.004.
ACKNOWLEDGMENTS The authors thank Ms. Risa Kono for expert technical assistance, Dr. Mami Yamamoto, Dr. Alena Lagumdzija, Ms. Ayumi Sato, and other members of the Makishima laboratory for helpful comments and assistance, the Advanced Functional Materials Research Center of Shin-Etsu Chemical Co. Ltd., for supplying sensing plates, and Dr. Andrew I. Shulman for editorial assistance. This work was supported by SENTAN program of Japan Science and Technology Agency (2012e2014) and Japan Agency for Medical Research and Development (2015e2016; grant numbers 15hm0102001h0004 and 16hm0102001s0205).
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Please cite this article in press as: Shimizu, T., et al., Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2017.01.004