Vol.8 No.2 2015

Research paper : Detection of influenza viruses with the waveguide mode sensor (K. AWAZU et al.)−102−Synthesiology - English edition Vol.8 No.2 (2015) the virus subtypes other than H3N2 were used as detection targets. Since the handling of the H5N1 virus was difficult in Japan, HA proteins were used for all samples. The results are shown in Fig. 8. The dashed line shows the reaction with the virus alone, while the solid line shows the reaction of antibodies highly sensitized with gold nanoparticles. The virus used in the measurement was 1 g. Figures 8 (a), (b), (c), and (d) show the results of the HA detection test for A/Wisconsin/67/2005(H3N2), A/chicken/India/NIV33487/2006(H5N1), A/California/07/2009(H1N1), and A/Japan/305/1957, respectively. For H3N2, as shown in (a), spectrum change was seen just for the HA. However, no spectrum change was detected in the HAs of other sub-species. This means that the HAs of different sub-species did not bond with the fixed antibodies. Therefore, when the detection plate onto which the H3N2 antibodies were fixed was used, the H3N2 virus could be detected while other sub-species could not be detected, and it was possible to identify the sub-species using the waveguide mode sensor.We shall introduce another identification method for the influenza virus subtypes.[21] The schematic diagram of the various reactions and the experiment results are shown in Figs. 9 and 10. The detection targets were the virus particles of human influenza type A H3N2, and the HA of avian influenza H5N1. To identify each virus, two types of gold nanoparticles coated with 2,6-sialic acid and 2,3-sialic acid were used as labels. High sensitivity was achieved by using such gold nanoparticles as labels. The HA protein of the human influenza H3N2 virus reacted with the 2,6-sialic acid on the surface of gold nanoparticles, and the spectrum changed [Fig. 9(a) and (c)], while there was no spectrum change since it did not react with the 2,3-sialic acid on the gold nanoparticle surface [Fig. 9(b) and (d)]. In contrast, the HA protein of avian influenza H5N1 did not react with the 2,6-sialic acid on the gold nanoparticle surface and the spectrum did not change [Fig. 10(a) and (c)], while it reacted with the 2,3-sialic acid on the gold nanoparticle surface and the spectrum changed [Fig. 10(b) and (d)]. Hence, simple identification of the HAs of human influenza H3N2 and avian influenza H5N1 viruses became possible.Next, type A influenza virus H3N2 Udorn was used for the detection sensitivity test using viruses. Here, comparison was conducted using the plaque forming unit (pfu), which is the index of infectivity, and the results are summarized in Table 2. In the detection test using the waveguide mode sensor, the virus and the antibodies labeled with gold nanoparticles were mixed, and left for 10 min. The mixture was dropped onto the sensor, and signals 30 min later were measured. The data indicated that the detection limit of the waveguide mode sensor would be 8 × 102 pfu/ml. For immuno-chromatography, the detection limit was 8 × 104 pfu/ml using the same sample. The H3N2 virus detection limit using the Sandwich ELISA method was 2 × 103 pfu/ml. Fig. 8 Results of the HA detection for various subtypes using the detection plate to which the H3N2 antibodies were fixedThe subtypes were (a) A/Wisconsin/67/2005 (H3N2), (b) A/chicken/India/NIV33487/2006 (H5N1), (c) A/California/07/2009 (H1N1), and (d) A/Japan/305/1957 (H2N2).Table 2. Comparison of the detection limit concentration of the waveguide mode sensor, immuno-chromatography, and ELISA, using the H3N2 Udorn strain(b) A/chicken/India/NIV33487/2006 (H5N1) (c) A/California/07/2009 (H1N1) Wavelength (nm) (d) A/Japan/305/1957 (H2N2) Wavelength (nm) Reflectivity (%) Wavelength (nm) Reflectivity (%) (a) A/Wisconsin/67/2005 (H3N2) Wavelength (nm) Reflectivity (%) Reflectivity (%) HA HA HA HA 5805605405205004805060708090100580560540520500480506070809010058056054052050048050607080901005805605405205004807080901002 x 103ELISA8 x 104Immuno-chromatography800Waveguide mode sensorpfu/mlH3N2Method


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