The Experts below are selected from a list of 13668 Experts worldwide ranked by ideXlab platform
Hiroo Iwata - One of the best experts on this subject based on the ideXlab platform.
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Antibody microarray for correlating cell phenotype with surface marker
Biomaterials, 2005Co-Authors: In Kap Ko, Koichi Kato, Hiroo IwataAbstract:Abstract To correlate cell surface markers with the cell phenotype, an Antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the Immobilized Antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (β-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.
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Antibody microarray for correlating cell phenotype with surface marker
Biomaterials, 2005Co-Authors: Koichi Kato, Hiroo IwataAbstract:To correlate cell surface markers with the cell phenotype, an Antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the Immobilized Antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (beta-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.
Kazuhiko Ishihara - One of the best experts on this subject based on the ideXlab platform.
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Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel
Langmuir, 2017Co-Authors: Akihisa Otaka, Kazuhiko Ishihara, Kazuki Kitagawa, Takahiko Nakaoki, Mitsuhi Hirata, Kyoko Fukazawa, Atsushi Mahara, Tetsuji YamaokaAbstract:When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently Immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the Immobilized-Antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These resu...
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Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA‑1 Antibody Immobilized Microfluidic Channel
2017Co-Authors: Akihisa Otaka, Kazuhiko Ishihara, Kazuki Kitagawa, Takahiko Nakaoki, Mitsuhi Hirata, Kyoko Fukazawa, Atsushi Mahara, Tetsuji YamaokaAbstract:When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently Immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the Immobilized-Antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state
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single step diagnosis system using the fret phenomenon induced by Antibody Immobilized phosphorylcholine group covered polymer nanoparticles
Sensors and Actuators B-chemical, 2008Co-Authors: Junji Watanabe, Kazuhiko IshiharaAbstract:Abstract We report here a single step molecular diagnosis system using phosphorylcholine group-covered nanoparticles (PC-NPs). The most favorable characteristics were (i) the suppression of non-specific protein adsorption by the PC group-covered surface and (ii) molecular diagnosis by the capture of the target antigen. The fundamental strategy was based on antigen–Antibody reactions on the PC-NPs. In particular, we installed a fluorescence resonance energy transfer (FRET) system on the Immobilized antibodies. The Immobilized Antibody was labeled by donor or acceptor molecules separately, which were fluorescent molecules. These molecules induce the FRET phenomenon if they are close to each other upon capture of the target antigen by the PC-NPs. Therefore, the resulting fluorescence was readable using the FRET phenomenon. In this paper, C-reactive protein (CRP) was used as the target antigen, and the anti-CRP Antibody was labeled by fluorescent molecules. The labeled Antibody was then Immobilized onto the PC-NPs. The resulting fluorescence intensity was well correlated with the change in the concentration of CRP. The protocol for molecular diagnosis is quite simple, and only involves mixing the FRET-installed NPs and target molecules such as CRP. Single step molecular diagnosis via FRET may be of great importance for designing sensor devices.
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Antibody immobilization to phospholipid polymer layer on gold substrate of quartz crystal microbalance immunosensor
Colloids and Surfaces B: Biointerfaces, 2007Co-Authors: Shigeru Kurosawa, Jongwon Park, Madoka Takai, Kazuhiko IshiharaAbstract:Abstract To modify gold electrode for immunosensor to construct an artificial cell membrane structure, water-soluble amphiphilic phospholipid polymer, poly[2-methacryloyloxyehtyl phosphorylcholine- co - n -butyl methacrylate- co - p -nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (PMBN)] was applied. The polymer had active ester groups for immobilization of biomolecules and it was converted partially to thiol groups for binding to gold substrates. The partially thiolated PMBN was adsorbed on a gold electrode of quartz crystal microbalance (QCM). Surface characterization of adsorbed PMBN layers was thoroughly investigated with reflectance anisotropy spectroscopy, ellipsometry spectroscopy, dynamic contact angle and X-ray photoelectron spectroscopy measurements. Among several PMBN, having different degree of thiolation, it was concluded that 21.5% thiolated PMBN layer had the most well-ordered phosphorylcholine groups in its outer surface. The proteins adsorption test revealed that the phosphorylcholine group on the outer side of PMBN layers, which was substituted their active ester groups by glycine, showed suppress the non-specific adsorption of proteins, such as bovine serum albumin and γ-globulin. Also, through antigen–Antibody binding evaluation, the anti-C-reactive protein Antibody Immobilized on the PMBN surface worked well and it was confirmed that denaturation of the Antibody on the PMBN layers was hardly occurred in spite of 60 days storage at 4 °C. The Antibody conjugated phospholipid polymer layer with well-ordered phosphorylcholine group could be outstanding functional membrane for biomedical diagnostic devices without non-specific binding and reduction of immunologic activity of Immobilized Antibody.
Tetsuji Yamaoka - One of the best experts on this subject based on the ideXlab platform.
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Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel
Langmuir, 2017Co-Authors: Akihisa Otaka, Kazuhiko Ishihara, Kazuki Kitagawa, Takahiko Nakaoki, Mitsuhi Hirata, Kyoko Fukazawa, Atsushi Mahara, Tetsuji YamaokaAbstract:When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently Immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the Immobilized-Antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These resu...
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Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA‑1 Antibody Immobilized Microfluidic Channel
2017Co-Authors: Akihisa Otaka, Kazuhiko Ishihara, Kazuki Kitagawa, Takahiko Nakaoki, Mitsuhi Hirata, Kyoko Fukazawa, Atsushi Mahara, Tetsuji YamaokaAbstract:When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently Immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the Immobilized-Antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state
Basar Bilgicer - One of the best experts on this subject based on the ideXlab platform.
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oriented Antibody immobilization by site specific uv photocrosslinking of biotin at the conserved nucleotide binding site for enhanced antigen detection
Biosensors and Bioelectronics, 2013Co-Authors: Nathan J. Alves, Nur Mustafaoglu, Basar BilgicerAbstract:Abstract The nucleotide binding site (NBS) is an under-utilized, highly conserved binding site found within the variable region of nearly all Antibody Fab arms. Here, we describe an NBS specific UV photocrosslinking biotinylation method (UV-NBS Biotin ) for the oriented immobilization of antibodies to streptavidin-coated surfaces, such that the antigen binding activity remains unaffected. An optimal UV exposure of 1 J/cm 2 yielded an average conjugation efficiency of ∼1 biotin per Antibody resulting in significant immobilization efficiency while maintaining maximal antigen binding activity. With the continued push for miniaturization of medical diagnostics to reduce cost and increase patient accessibility the ever shrinking on chip detection areas necessitate the highest level of Immobilized Antibody activity to maximize assay detection capabilities. The UV-NBS Biotin method yielded surfaces with significantly enhanced antigen detection capabilities, improved antigen detection sensitivity and the highest amount of active surface Immobilized Antibody when compared to other common immobilization methods including: e -NH 3 + surface conjugation, NHS–Biotin, and direct physical adsorption. Taken together, the UV-NBS Biotin method provides a universal, site-specific immobilization method that is amenable to any available assay detection modality with potential significant implications in the development of miniaturized medical diagnostics and lab on a chip technologies.
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oriented surface immobilization of antibodies at the conserved nucleotide binding site for enhanced antigen detection
Langmuir, 2012Co-Authors: Nathan J. Alves, Tanyel Kiziltepe, Basar BilgicerAbstract:The conserved nucleotide binding site (NBS), found on the Fab variable domain of all Antibody isotypes, remains a not-so-widely known and unutilized site. Here, we describe a UV photo-cross-linking method (UV-NBS) that utilizes the NBS for oriented immobilization of antibodies onto surfaces, such that the antigen binding activity remains unaffected. Indole-3-butyric acid (IBA) has an affinity for the NBS with a Kd ranging from 1 to 8 μM for different Antibody isotypes and can be covalently photo-cross-linked to the Antibody at the NBS upon exposure to UV light. Using the UV-NBS method, Antibody was successfully Immobilized on synthetic surfaces displaying IBA via UV photo-cross-linking at the NBS. An optimal UV exposure of 2 J/cm2 yielded significant Antibody immobilization on the surface with maximal relative Antibody activity per Immobilized Antibody without any detectable damage to antigen binding activity. Comparison of the UV-NBS method with two other commonly used methods, e-NH3+ conjugation and phy...
Koichi Kato - One of the best experts on this subject based on the ideXlab platform.
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Antibody microarray for correlating cell phenotype with surface marker
Biomaterials, 2005Co-Authors: In Kap Ko, Koichi Kato, Hiroo IwataAbstract:Abstract To correlate cell surface markers with the cell phenotype, an Antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the Immobilized Antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (β-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.
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Antibody microarray for correlating cell phenotype with surface marker
Biomaterials, 2005Co-Authors: Koichi Kato, Hiroo IwataAbstract:To correlate cell surface markers with the cell phenotype, an Antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the Immobilized Antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (beta-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.
