Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy
We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody–antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl...
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| Veröffentlicht in: | Journal of the American Society for Mass Spectrometry 2018-05, Vol.29 (5), p.961-971 |
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| creator | Lin, Margaret Krawitz, Denise Callahan, Matthew D. Deperalta, Galahad Wecksler, Aaron T. |
| description | We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody–antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody–antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA.
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| doi_str_mv | 10.1007/s13361-017-1883-9 |
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Graphical Abstract
ᅟ</description><identifier>ISSN: 1044-0305</identifier><identifier>EISSN: 1879-1123</identifier><identifier>DOI: 10.1007/s13361-017-1883-9</identifier><identifier>PMID: 29512051</identifier><language>eng</language><publisher>New York: Springer US</publisher><subject>Analytical Chemistry ; Antigens ; Binding ; Bioinformatics ; Biotechnology ; Carboxyl group ; Chemistry ; Chemistry and Materials Science ; Crystal structure ; Data analysis ; Diamonds ; Glycine ; Hydroxyl radicals ; Labeling ; Mapping ; Mass spectrometry ; Monoclonal antibodies ; Organic Chemistry ; Oxidation ; Proteins ; Proteomics ; Quality control ; Reagents ; Research Article ; Scientific imaging ; Spectroscopy ; Transmission electron microscopy</subject><ispartof>Journal of the American Society for Mass Spectrometry, 2018-05, Vol.29 (5), p.961-971</ispartof><rights>American Society for Mass Spectrometry 2018</rights><rights>Journal of The American Society for Mass Spectrometry is a copyright of Springer, (2018). All Rights Reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c372t-8e6c22e3186dd5977ff193ca4f87c62fe346f980afd2a24e4fbcf7c6aa46613e3</citedby><cites>FETCH-LOGICAL-c372t-8e6c22e3186dd5977ff193ca4f87c62fe346f980afd2a24e4fbcf7c6aa46613e3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s13361-017-1883-9$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s13361-017-1883-9$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27924,27925,41488,42557,51319</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29512051$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lin, Margaret</creatorcontrib><creatorcontrib>Krawitz, Denise</creatorcontrib><creatorcontrib>Callahan, Matthew D.</creatorcontrib><creatorcontrib>Deperalta, Galahad</creatorcontrib><creatorcontrib>Wecksler, Aaron T.</creatorcontrib><title>Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy</title><title>Journal of the American Society for Mass Spectrometry</title><addtitle>J. Am. Soc. Mass Spectrom</addtitle><addtitle>J Am Soc Mass Spectrom</addtitle><description>We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody–antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody–antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA.
Graphical Abstract
ᅟ</description><subject>Analytical Chemistry</subject><subject>Antigens</subject><subject>Binding</subject><subject>Bioinformatics</subject><subject>Biotechnology</subject><subject>Carboxyl group</subject><subject>Chemistry</subject><subject>Chemistry and Materials Science</subject><subject>Crystal structure</subject><subject>Data analysis</subject><subject>Diamonds</subject><subject>Glycine</subject><subject>Hydroxyl radicals</subject><subject>Labeling</subject><subject>Mapping</subject><subject>Mass spectrometry</subject><subject>Monoclonal antibodies</subject><subject>Organic Chemistry</subject><subject>Oxidation</subject><subject>Proteins</subject><subject>Proteomics</subject><subject>Quality control</subject><subject>Reagents</subject><subject>Research Article</subject><subject>Scientific imaging</subject><subject>Spectroscopy</subject><subject>Transmission electron microscopy</subject><issn>1044-0305</issn><issn>1879-1123</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>8G5</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>GUQSH</sourceid><sourceid>M2O</sourceid><recordid>eNp1kUGPEyEUx4nRuOvqB_BiSLx4QXnADDPHpulqk64eup4JZWCcTQsVmE3qx_ATC9tVExNPvITf-_F4f4ReA30PlMoPCThvgVCQBLqOk_4JuoRO9gSA8aelpkIQymlzgV6kdEcLSHv5HF2wvgFGG7hEP5ffdNQm2zj90HkKHgeHV5v1doEXPk-7MJxILUbr8doXrLCVmtPkR3wdQj7Gqdz7kdzolPD2aE2O4WBzPGHtB_zZjsV7b_E268nXptuofTpMKVXPav_Ae3wzmRiSCcfTS_TM6X2yrx7PK_T1enW7_EQ2Xz6ul4sNMVyyTDrbGsYsh64dhqaX0jnoudHCddK0zFkuWtd3VLuBaSascDvjyo3Wom2BW36F3p29xxi-zzZlVYYydr_X3oY5KUYBWhBM9AV9-w96F-boy3QPlKCsk02h4EzVn6RonSq7Oeh4UkBVDUydA1MlB1UDU9X85tE87w52-NPxO6ECsDOQ6qJHG_8-_X_rL702o40</recordid><startdate>20180501</startdate><enddate>20180501</enddate><creator>Lin, Margaret</creator><creator>Krawitz, Denise</creator><creator>Callahan, Matthew D.</creator><creator>Deperalta, Galahad</creator><creator>Wecksler, Aaron T.</creator><general>Springer US</general><general>Springer Nature B.V</general><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FG</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>P5Z</scope><scope>P62</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope></search><sort><creationdate>20180501</creationdate><title>Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy</title><author>Lin, Margaret ; Krawitz, Denise ; Callahan, Matthew D. ; Deperalta, Galahad ; Wecksler, Aaron T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c372t-8e6c22e3186dd5977ff193ca4f87c62fe346f980afd2a24e4fbcf7c6aa46613e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Analytical Chemistry</topic><topic>Antigens</topic><topic>Binding</topic><topic>Bioinformatics</topic><topic>Biotechnology</topic><topic>Carboxyl group</topic><topic>Chemistry</topic><topic>Chemistry and Materials Science</topic><topic>Crystal structure</topic><topic>Data analysis</topic><topic>Diamonds</topic><topic>Glycine</topic><topic>Hydroxyl radicals</topic><topic>Labeling</topic><topic>Mapping</topic><topic>Mass spectrometry</topic><topic>Monoclonal antibodies</topic><topic>Organic Chemistry</topic><topic>Oxidation</topic><topic>Proteins</topic><topic>Proteomics</topic><topic>Quality control</topic><topic>Reagents</topic><topic>Research Article</topic><topic>Scientific imaging</topic><topic>Spectroscopy</topic><topic>Transmission electron microscopy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lin, Margaret</creatorcontrib><creatorcontrib>Krawitz, Denise</creatorcontrib><creatorcontrib>Callahan, Matthew D.</creatorcontrib><creatorcontrib>Deperalta, Galahad</creatorcontrib><creatorcontrib>Wecksler, Aaron T.</creatorcontrib><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>ProQuest Central Essentials</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Research Library</collection><collection>Research Library (Corporate)</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of the American Society for Mass Spectrometry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lin, Margaret</au><au>Krawitz, Denise</au><au>Callahan, Matthew D.</au><au>Deperalta, Galahad</au><au>Wecksler, Aaron T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy</atitle><jtitle>Journal of the American Society for Mass Spectrometry</jtitle><stitle>J. Am. Soc. Mass Spectrom</stitle><addtitle>J Am Soc Mass Spectrom</addtitle><date>2018-05-01</date><risdate>2018</risdate><volume>29</volume><issue>5</issue><spage>961</spage><epage>971</epage><pages>961-971</pages><issn>1044-0305</issn><eissn>1879-1123</eissn><abstract>We describe epitope mapping data using multiple covalent labeling footprinting-mass spectrometry (MS) techniques coupled with negative stain transmission electron microscopy (TEM) data to analyze the antibody–antigen interactions in a sandwich enzyme-linked immunosorbant assay (ELISA). Our hydroxyl radical footprinting-MS data using fast photochemical oxidation of proteins (FPOP) indicates suppression of labeling across the antigen upon binding either of the monoclonal antibodies (mAbs) utilized in the ELISA. Combining these data with Western blot analysis enabled the identification of the putative epitopes that appeared to span regions containing N-linked glycans. An additional structural mapping technique, carboxyl group footprinting-mass spectrometry using glycine ethyl ester (GEE) labeling, was used to confirm the epitopes. Deglycosylation of the antigen resulted in loss of potency in the ELISA, supporting the FPOP and GEE labeling data by indicating N-linked glycans are necessary for antigen binding. Finally, mapping of the epitopes onto the antigen crystal structure revealed an approximate 90° relative spatial orientation, optimal for a noncompetitive binding ELISA. TEM data shows both linear and diamond antibody–antigen complexes with a similar binding orientation as predicted from the two footprinting-MS techniques. This study is the first of its kind to utilize multiple bottom-up footprinting-MS techniques and TEM visualization to characterize the monoclonal antibody-antigen binding interactions of critical reagents used in a quality control (QC) lot-release ELISA.
Graphical Abstract
ᅟ</abstract><cop>New York</cop><pub>Springer US</pub><pmid>29512051</pmid><doi>10.1007/s13361-017-1883-9</doi><tpages>11</tpages></addata></record> |
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| subjects | Analytical Chemistry Antigens Binding Bioinformatics Biotechnology Carboxyl group Chemistry Chemistry and Materials Science Crystal structure Data analysis Diamonds Glycine Hydroxyl radicals Labeling Mapping Mass spectrometry Monoclonal antibodies Organic Chemistry Oxidation Proteins Proteomics Quality control Reagents Research Article Scientific imaging Spectroscopy Transmission electron microscopy |
| title | Characterization of ELISA Antibody-Antigen Interaction using Footprinting-Mass Spectrometry and Negative Staining Transmission Electron Microscopy |
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