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RISS 인기검색어
- 검색키워드
- 저자 : Sukyo JEONG (검색결과 5 건)
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Jeong, Sukyo,Heu, Woosung,Kim, Jong-won,Kim, Hak-Sung American Chemical Society 2016 ANALYTICAL CHEMISTRY - Vol.88 No.23
<P>An immunoassay is the most widely used method for analyzing a variety of analytes based on antigen antibody interactions in the biological and medical sciences. However, the use of secondary antibodies has certain shortcomings, such as a high cost, cross-reactivity, and loss of binding affinity during labeling. Herein, we present the development of repebodies specifically binding to immunoglobulin G with a different origin, which is a small-sized nonantibody scaffold composed of leucine-rich repeat (LRR) modules, for use in immunoassays and imaging. Repebodies specific for IgG from different species (i.e., mouse, human, and rabbit) were selected through a phage display, and their affinities were matured using a modular engineering approach. The respective repebodies were labeled with various signal generators such as horseradish peroxidase (HRP), a fluorescent dye, and quantum dots, and the resulting repebodies were used as alternatives to conventional secondary antibodies in typical immunoassays and imaging. The labeled repebodies enabled the detection of diverse target analytes with high sensitivity and specificity, showing a negligible cross-reactivity. Moreover, the repebodies labeled with different color-emitting quantum dots allowed the imaging of cell-surface receptors and proteins in a multiplex manner. The developed repebodies can be effectively used for sensitive immunoassays and multiplex imaging.</P>
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Kim, Jong-won,Heu, Woosung,Jeong, Sukyo,Kim, Hak-Sung Elsevier 2017 Analytica chimica acta Vol.988 No.-
<P><B>Abstract</B></P> <P>Molecular detection of target molecules with high sensitivity and specificity is of great significance in bio and medical sciences. Here, we present genetically functionalized ferritin nanoparticles with a high-affinity protein binder, and their utility as a signal generator in a variety of immunoassays and imaging. As a high-affinity protein binder, human IgG-specific repebody, which is composed of LRR (Leucine-rich repeat) modules, was used. The repebody was genetically fused to the N-terminal heavy-chain ferritin, and the resulting subunits were self-assembled to the repebody-ferritin nanoparticles composed of 24 subunits. The repebody-ferritin nanoparticles were shown to have a three-order of magnitude higher binding affinity toward human IgG than free repebody mainly owing to a decreased dissociation rate constant. The repebody-ferritin nanoparticles were conjugated with fluorescent dyes, and the resulting nanoparticles were used for western blotting, cell imaging, and flow cytometric analysis. The dye-labeled repebody-ferritin nanoparticles were shown to generate about 3-fold stronger fluorescent signals in immunoassays than monovalent repebody. The repebody-functionalized ferritin nanoparticles can be effectively used for sensitive and specific immunoassays and imaging in many areas.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Human IgG-specific repebody was genetically fused to N-terminus of ferritin subunit. </LI> <LI> The RbF-NPs exhibited 1000-fold higher binding affinity for human IgG than free repebody due to multivalency. </LI> <LI> The Cy3-laleled RbF-NPs generated stronger fluorescent signals than Cy3-labeled free repebody in immunoassays and imaging. </LI> <LI> The RbF-NPs can be effectively used in various types of immunoassays and imaging. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>
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Protein Binder for Affinity Purification of Human Immunoglobulin Antibodies
Heu, Woosung,Choi, Jung-Min,Lee, Joong-Jae,Jeong, Sukyo,Kim, Hak-Sung American Chemical Society 2014 ANALYTICAL CHEMISTRY - Vol.86 No.12
<P>The importance of a downstream process for the purification of immunoglobulin antibodies is increasing with the growing application of monoclonal antibodies in many different areas. Although protein A is most commonly used for the affinity purification of antibodies, certain properties could be further improved: higher stability in alkaline solution and milder elution condition. Herein, we present the development of Fc-specific repebody by modular engineering approach and its potential as an affinity ligand for purification of human immunoglobulin antibodies. We previously developed the repebody scaffold composed of Leucine-rich repeat (LRR) modules. The scaffold was shown to be highly stable over a wide range of pH and temperature, exhibiting a modular architecture. We first selected a repebody that binds the Fc fragment of human immunoglobulin G (IgG) through a phage display and increased its binding affinity up to 1.9 × 10<SUP>–7</SUP> M in a module-by-module approach. The utility of the Fc-specific repebody was demonstrated by the performance of an immobilized repebody in affinity purification of antibodies from a mammalian cell-cultured medium. Bound-antibodies on an immobilized repebody were shown to be eluted at pH 4.0 with high purity (>94.6%) and recovery yield (>95.7%). The immobilized repebody allowed a repetitive purification process more than ten times without any loss of binding capability. The repebody remained almost intact even after incubation with 0.5 M NaOH for 15 days. The present approach could be effectively used for developing a repeat module-based binder for other target molecules for affinity purification.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2014/ancham.2014.86.issue-12/ac501158t/production/images/medium/ac-2014-01158t_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ac501158t'>ACS Electronic Supporting Info</A></P>
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