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10 g/mL Immobilized anti-rabbit IgG
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1 g/mL Immobilized anti-goat IgG
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Figure 9.11. (a) Layout of a slide that was spotted and immobilized using the anti-goat IgG rabbit antibody and the anti-rabbit IgG goat antibody. (b) Fluorescent image after incubation of a Cy-5-labeled goat IgG. (c) Fluorescent image after incubation of a Cy-5-labeled rabbit IgG.
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away over a period of 10 days when they were stored at room temperature, it was maintained for B2 months when stored at 41C. This result is acceptable in terms of commercial viability, though further increases in stability would be preferable. The authors next examined the sensitivity for the immunochips. An immunochip usually has a two-dimensional (2-D) surface, so the detection limit for antigens can be estimated from the amount of immobilized antibodies that are present. It was dif cult to increase the sensitivity of a detection system that uses a photoluminescence probe. However, they succeeded in obtaining higher sensitivity for an immunochip in which they adopted a chemiluminescence detection system using an enzyme reaction. They selected adiponectin, which is a biologically active agent that is excreted from adipose cells and which prevents arteriosclerosis, as the intended biological marker, and they tried to assay it using an enzyme sandwich immunoassay on the azopolymer surface. Anti-adiponectin antibodies were photoimmobilized on the azopolymer surface and then a solution including adiponectin was reacted on the fabricated immunochip. Subsequently, the sample that had captured the adiponectin on its surface using the immobilized antibodies was treated with biotin-labeled anti-adiponectin antibodies ( rst sandwich process) and then with alkaline phosphatase (ALP)-labeled streptoavidin (second sandwich process) (Kendall et al., 1983). After introducing the chemiluminescent
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800000 700000 600000 Chemiluminescence 500000 400000 300000 200000 100000 0
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0.4 0.6 Adiponectin (ng/mL)
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Figure 9.12. Calibration curve for quantifying mouse adiponectin. Each error bar indicates the standard deviation for each data point.
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substrate onto the surface, the intensity of the chemiluminescence was measured to determine the concentration of adiponectin. The authors measured the intensity of the chemiluminescence against the concentration of adiponectin using samples with predetermined concentrations. Figure 9.12 exhibits the calibration curve that was obtained in the region of low concentration, and it shows that a linear relationship exists between intensity and concentration. Adiponectin in a sample solution can be detected down to a concentration of at least 0.1 ng/mL, which is almost the same sensitivity as that obtained with ELISA. A conventional ELISA system and the IgG chip system were compared using mouse adiponectin of culture supernatant. Figure 9.13 shows the correlation between the immunochip and conventional ELISA. A high degree of correlation exists (r2=0.97), indicating that the use of an immunochip with an azopolymer lm is a promising candidate for practical use.
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This section compares the photoinduced immobilization of IgG on two types of azopolymers (shown in Fig. 9.2) bearing various concentrations of 4-amino-4ucyanoazobenzene (CN-azopolymer) or amino azobenzene (H-azopolymer). CNazopolymer and H-azopolymer contain a push pull-type azobenzene and an amino azobenzene, respectively (Rabek, 1988). These azobenzenes have different
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1.5 Immuno chip ( g/mL)
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Figure 9.13. Correlation between the two methods, ELISA and immunochip, for quantifying mouse adiponectin at a level of 16 culture supernatant.
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adsorption spectra, and they exhibit different deformation and immobilization features under photoirradiation. Therefore, information can be obtained about the photoimmobilization mechanisms by comparing these two types of azopolymers. First, the authors examined the relationship between immobilization ef ciency and the indented depth with respect to the photoirradiation time and the speci c azobenzene moiety. Second, they compared the relationship between immobilization ef ciency and chemical structure, and elucidated how this correlated with the photoisomerization properties and the retention rate of immobilized antibodies. The photodeformation capabilities of the CN-azopolymer and the H-azopolymer were examined by determining the depth of the indents formed by polystyrene microspheres under LED irradiation. After photoirradiation and removal of the microspheres, regularly arranged indented patterns formed by the microspheres were observed on the surfaces of the azopolymers. The depths of the indents were plotted as a function of irradiation time for several kinds of azopolymers, as shown in Fig. 9.14. The indent depths in the azopolymer increased with increasing irradiation time. The depths of the indents saturated and reached a maximum after 30 min of photoirradiation for each of the azopolymers. The saturated depths were lowest in those azopolymers with the lowest content of azobenzene moieties. These results indicate that the photoresponsive moiety plays an important role in inducing photodeformation and that the indent depth is related to the content of the azobenzene in the azopolymers. There were no differences in photodeformation capabilities between the CN- and
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