Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes
•Semi-quantitative analysis of enzyme activity in single-drop of biological fluid.•Microfluidic separation of enzymes by immunoaffinity membranes.•Antibody-captured enzymes retain partial activity. The purpose of this study was the measurement of enzyme activity within a single-drop of biological fl...
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| Veröffentlicht in: | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-05, Vol.1021, p.108-113 |
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| container_title | Journal of chromatography. B, Analytical technologies in the biomedical and life sciences |
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| creator | Shimazaki, Youji Sato, Yuki |
| description | •Semi-quantitative analysis of enzyme activity in single-drop of biological fluid.•Microfluidic separation of enzymes by immunoaffinity membranes.•Antibody-captured enzymes retain partial activity.
The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane. |
| doi_str_mv | 10.1016/j.jchromb.2015.12.054 |
| format | Article |
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The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane.</description><identifier>ISSN: 1570-0232</identifier><identifier>EISSN: 1873-376X</identifier><identifier>DOI: 10.1016/j.jchromb.2015.12.054</identifier><identifier>PMID: 26776499</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Antibodies ; Antibodies, Immobilized - chemistry ; Antibodies, Immobilized - metabolism ; Biological ; Captured-enzyme ; Enzymes ; Esterase ; Esterases ; Esterases - analysis ; Esterases - chemistry ; Esterases - metabolism ; Fluids ; Immunochromatography - instrumentation ; Immunochromatography - methods ; L-Lactate Dehydrogenase - analysis ; L-Lactate Dehydrogenase - chemistry ; L-Lactate Dehydrogenase - metabolism ; Lactate dehydrogenase ; Membranes ; Membranes, Artificial ; Micropurification ; Protein A ; Rinsing ; Single-drop analysis ; Umbelliferones - analysis ; Umbelliferones - chemistry ; Umbelliferones - metabolism</subject><ispartof>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2016-05, Vol.1021, p.108-113</ispartof><rights>2015 Elsevier B.V.</rights><rights>Copyright © 2015 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c464t-e8056f29d5ec0ca86e630a918d9762816f4624616c7641a779d05448eb88f0363</citedby><cites>FETCH-LOGICAL-c464t-e8056f29d5ec0ca86e630a918d9762816f4624616c7641a779d05448eb88f0363</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jchromb.2015.12.054$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27929,27930,46000</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/26776499$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Shimazaki, Youji</creatorcontrib><creatorcontrib>Sato, Yuki</creatorcontrib><title>Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes</title><title>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences</title><addtitle>J Chromatogr B Analyt Technol Biomed Life Sci</addtitle><description>•Semi-quantitative analysis of enzyme activity in single-drop of biological fluid.•Microfluidic separation of enzymes by immunoaffinity membranes.•Antibody-captured enzymes retain partial activity.
The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane.</description><subject>Antibodies</subject><subject>Antibodies, Immobilized - chemistry</subject><subject>Antibodies, Immobilized - metabolism</subject><subject>Biological</subject><subject>Captured-enzyme</subject><subject>Enzymes</subject><subject>Esterase</subject><subject>Esterases</subject><subject>Esterases - analysis</subject><subject>Esterases - chemistry</subject><subject>Esterases - metabolism</subject><subject>Fluids</subject><subject>Immunochromatography - instrumentation</subject><subject>Immunochromatography - methods</subject><subject>L-Lactate Dehydrogenase - analysis</subject><subject>L-Lactate Dehydrogenase - chemistry</subject><subject>L-Lactate Dehydrogenase - metabolism</subject><subject>Lactate dehydrogenase</subject><subject>Membranes</subject><subject>Membranes, Artificial</subject><subject>Micropurification</subject><subject>Protein A</subject><subject>Rinsing</subject><subject>Single-drop analysis</subject><subject>Umbelliferones - analysis</subject><subject>Umbelliferones - chemistry</subject><subject>Umbelliferones - metabolism</subject><issn>1570-0232</issn><issn>1873-376X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUFvFSEUhYnR2Fr9CRqWbmYEZgaYlTFNtSZNTIwm7gjDXFpeBngC0_pc-stl8p5um5DA4jv3XM5B6DUlLSWUv9u1O3OXop9aRujQUtaSoX-CzqkUXdMJ_uNpfQ-CNIR17Ay9yHlHCBVEdM_RGeNC8H4cz9Gfr1C0Cy7cYm2Ku3flgKPFEH4fPGSsbYGEjd6XNQHWYcbwq6SNjAE_uHLnAtY4V_kCzZzifhNPLi7x1hm9YLusbsbrBmDn_RqitrbaVRcPfko6QH6Jnlm9ZHh1ui_Q949X3y6vm5svnz5ffrhpTM_70oAkA7dsnAcwxGjJgXdEj1TOo-BMUm57znpOualfo1qIca6J9BImKS3peHeB3h7n7lP8uUIuyrtsYFnqEnHNitYZpOZCh8dRISVjfT0VHY6oSTHnBFbtk_M6HRQlamtK7dSpKbU1pShTda-qe3OyWCcP83_Vv2oq8P4IQM3k3kFS2TgIBmaXwBQ1R_eIxV8mdaj7</recordid><startdate>20160515</startdate><enddate>20160515</enddate><creator>Shimazaki, Youji</creator><creator>Sato, Yuki</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>7TB</scope><scope>8FD</scope><scope>FR3</scope></search><sort><creationdate>20160515</creationdate><title>Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes</title><author>Shimazaki, Youji ; Sato, Yuki</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c464t-e8056f29d5ec0ca86e630a918d9762816f4624616c7641a779d05448eb88f0363</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Antibodies</topic><topic>Antibodies, Immobilized - chemistry</topic><topic>Antibodies, Immobilized - metabolism</topic><topic>Biological</topic><topic>Captured-enzyme</topic><topic>Enzymes</topic><topic>Esterase</topic><topic>Esterases</topic><topic>Esterases - analysis</topic><topic>Esterases - chemistry</topic><topic>Esterases - metabolism</topic><topic>Fluids</topic><topic>Immunochromatography - instrumentation</topic><topic>Immunochromatography - methods</topic><topic>L-Lactate Dehydrogenase - analysis</topic><topic>L-Lactate Dehydrogenase - chemistry</topic><topic>L-Lactate Dehydrogenase - metabolism</topic><topic>Lactate dehydrogenase</topic><topic>Membranes</topic><topic>Membranes, Artificial</topic><topic>Micropurification</topic><topic>Protein A</topic><topic>Rinsing</topic><topic>Single-drop analysis</topic><topic>Umbelliferones - analysis</topic><topic>Umbelliferones - chemistry</topic><topic>Umbelliferones - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Shimazaki, Youji</creatorcontrib><creatorcontrib>Sato, Yuki</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Mechanical & Transportation Engineering Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><jtitle>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Shimazaki, Youji</au><au>Sato, Yuki</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes</atitle><jtitle>Journal of chromatography. B, Analytical technologies in the biomedical and life sciences</jtitle><addtitle>J Chromatogr B Analyt Technol Biomed Life Sci</addtitle><date>2016-05-15</date><risdate>2016</risdate><volume>1021</volume><spage>108</spage><epage>113</epage><pages>108-113</pages><issn>1570-0232</issn><eissn>1873-376X</eissn><abstract>•Semi-quantitative analysis of enzyme activity in single-drop of biological fluid.•Microfluidic separation of enzymes by immunoaffinity membranes.•Antibody-captured enzymes retain partial activity.
The purpose of this study was the measurement of enzyme activity within a single-drop of biological fluid after micropurification. Esterase and lactate dehydrogenase (LDH) retained their enzymatic activities after being captured by membrane-immobilized antibodies, which were prepared by non-denaturing two-dimensional electrophoresis, transferred to polyvinylidene difluoride and then stained by Ponceau S. The activities of both enzymes were also measured after being captured by antibodies and biotinylated antibodies bound to membrane-immobilized protein A or avidin, respectively. After esterase and LDH were captured from biological samples by membrane-immobilized protein A or avidin, their activities were semi-quantitatively measured on the surface of the membrane using fluorescence determination. More than 51% of enzyme activities were retained even after the enzymes were captured by biotinylated antibody bound to membrane-immobilized avidin and eluted by rinsing with 5μL of 1% Triton X-100, compared with the activities of the enzyme on the immunoaffinity membrane.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>26776499</pmid><doi>10.1016/j.jchromb.2015.12.054</doi><tpages>6</tpages></addata></record> |
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| subjects | Antibodies Antibodies, Immobilized - chemistry Antibodies, Immobilized - metabolism Biological Captured-enzyme Enzymes Esterase Esterases Esterases - analysis Esterases - chemistry Esterases - metabolism Fluids Immunochromatography - instrumentation Immunochromatography - methods L-Lactate Dehydrogenase - analysis L-Lactate Dehydrogenase - chemistry L-Lactate Dehydrogenase - metabolism Lactate dehydrogenase Membranes Membranes, Artificial Micropurification Protein A Rinsing Single-drop analysis Umbelliferones - analysis Umbelliferones - chemistry Umbelliferones - metabolism |
| title | Retaining activity of enzymes after capture and extraction within a single-drop of biological fluid using immunoaffinity membranes |
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