Publications (82) View all
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Article: Nanocapsule-based probe for evaluating the orientation of antibodies immobilized on a solid phase.
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ABSTRACT: The orientation of sensing molecules on solid phase biosensors has to be optimized to facilitate efficient binding of analytes. Since conventional observation methods (e.g., electron microscopy, atomic force microscopy, time-of-flight secondary ion mass spectrometry) require exaggerated machines and possess insufficient resolution for single molecule analyses, functional assays based on the reactivity to analytes have thus far been used for this optimization. However, it is not clear whether these assays can judge whether sensing molecules are fixed in an oriented-immobilization manner or not. Here, we describe that bio-nanocapsules of about 30 nm diameter, displaying approximately 120 molecules of a tandem form of the immunoglobulin (Ig) G Fc-binding Z domain (ZZ-BNCs), can discriminate between the Fc regions of IgGs fixed in an oriented-immobilization manner and those fixed randomly, thus facilitating the evaluation of the orientation of IgGs in immunosensors. Furthermore, in sandwich immunoassays, ZZ-BNCs can bind specifically to detection-IgGs fixed in an oriented-immobilization manner by antigen-capture IgG complexes, rather than to capture-IgGs fixed randomly onto a solid phase, allowing the simultaneous use of the same IgG as capture- and detection-IgGs. Thus, we demonstrate that ZZ-BNCs are a unique probe for evaluating the orientation of IgGs on a solid phase.The Analyst 05/2013; · 4.23 Impact Factor -
Article: A bio-nanocapsule containing envelope (E) protein domain III of Japanese encephalitis virus (JEV) protects mice against lethal JEV infection.
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ABSTRACT: An engineered bio-nanocapsule (BNC), comprising modified hepatitis B surface antigen L protein, was used as a physical scaffold for envelope (E) protein domain III (D3) of Japanese encephalitis virus (JEV). At the N-terminus, the BNC contained a two-tandem repeat of the Z domain (ZZ) derived from Staphylococcus aureus protein A (ZZ-BNC). The lysine-rich ZZ moiety exposed on the surface of ZZ-BNC was used for chemical conjugation with the JEV D3 antigen, which was expressed and purified from Escherichia coli. Immunization of mice with D3 loaded on the surface of ZZ-BNC (ZZ-BNC:D3) augmented serum IgG responses against JEV, and conferred increased protection against lethal JEV infection. These results suggest that the surface display of weakly immunogenic inert recombinant antigens, which by themselves cannot form virus-like particles, can be transformed to immunogenic antigens by the exploitation of ZZ-BNC.Microbiology and Immunology 04/2013; · 1.30 Impact Factor -
Article: Bio-Nanocapsules for Signal Enhancement of Alkaline Phosphatase-Linked Immunosorbent Assays.
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ABSTRACT: The bio-nanocapsules displaying about 240 molecules of immunoglobulin G Fc-binding Z domains (ZZ-BNCs) enhanced the signals of enzyme-linked immunosorbent assay by tethering the Fc regions of secondary antibodies (Abs), which were eliminated using high-molecular mass enzymes (e.g., alkaline phosphatase). By way of optimizing the distance between enzymes and Abs, ZZ-BNCs improved sensitivity independently of enzymes.Bioscience Biotechnology and Biochemistry 04/2013; · 1.28 Impact Factor -
Article: Cell surface-fluorescence immunosorbent assay for real-time detection of hybridomas with efficient antibody secretion at the single-cell level.
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ABSTRACT: For establishing cells that secrete antibodies most efficiently (e.g., hybridomas, CHO (Chinese hamster ovary) cells), the screening and subsequent breeding of promising cells have been performed at the single-colony level, which requires several weeks to propagate a substantial number of cells by forming colonies from single cells for evaluation by the conventional assays. However, this screening process lacks high-throughput performance in time and colony numbers. Therefore, development of novel methods is expected to identify single cells secreting higher amounts of antibodies in real-time and in a nondestructive manner without colony formation. In this study, we prepared lipid-labeled anti-mouse IgG Fc antibodies (capture molecules) that were uniformly displayed on the surface of candidate cells. Secreted nascent antibodies were subsequently sandwiched between capture molecules and fluorescence-labeled anti-mouse IgG F(ab')(2) F(ab')(2) (detection molecules). This newly developed method is hereinafter referred to as a cell surface-fluorescence immunosorbent assay (CS-FIA). The fluorescence intensity of each cell was found to correlate well with the amount of sandwiched antibodies (from 6.25 fg/cell to 6.40 pg/cell). When about 4 × 10(3)cells of mouse hybridomas were subjected to CS-FIA, we isolated 28 hybridomas showing the highest fluorescence intensity within a day. Furthermore, after propagation of single cells to about 10(5) cells (after 2 weeks), 20 hybridomas were still able to secrete higher amounts (up to 7-fold) of antibodies than parental hybridomas. Our results demonstrate that CS-FIA is a powerful method for the single-cell-based establishment of cells that secrete most efficiently not only antibodies but also various biomaterials.Analytical Chemistry 01/2013; · 5.86 Impact Factor -
Article: An automated system for high-throughput single cell-based breeding.
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ABSTRACT: When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.Scientific Reports 01/2013; 3:1191.
