Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples
The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validati...
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creator | Klafki, Hans W. Rieper, Petra Matzen, Anja Zampar, Silvia Wirths, Oliver Vogelgsang, Jonathan Osterloh, Dirk Rohdenburg, Lara Oberstein, Timo J. Jahn, Olaf Beyer, Isaak Lachmann, Ingolf Knölker, Hans-Joachim Wiltfang, Jens |
description | The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set. |
doi_str_mv | 10.3390/ijms21186564 |
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Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.</description><identifier>ISSN: 1422-0067</identifier><identifier>ISSN: 1661-6596</identifier><identifier>EISSN: 1422-0067</identifier><identifier>DOI: 10.3390/ijms21186564</identifier><identifier>PMID: 32911706</identifier><language>eng</language><publisher>Basel: MDPI AG</publisher><subject>Alzheimer's disease ; Amyloid precursor protein ; Antibodies ; Binding sites ; Biological properties ; Biomarkers ; Blood plasma ; Brain ; Cell culture ; Dementia ; Dementia disorders ; Immunoassay ; Immunohistochemistry ; Immunoprecipitation ; Isoelectric focusing ; Monoclonal antibodies ; Mutation ; N-Terminus ; Peptides ; Plasma ; Precursors ; Proteins ; Selectivity ; Transgenic animals</subject><ispartof>International journal of molecular sciences, 2020-09, Vol.21 (18), p.6564</ispartof><rights>2020. 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Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.</description><subject>Alzheimer's disease</subject><subject>Amyloid precursor protein</subject><subject>Antibodies</subject><subject>Binding sites</subject><subject>Biological properties</subject><subject>Biomarkers</subject><subject>Blood plasma</subject><subject>Brain</subject><subject>Cell culture</subject><subject>Dementia</subject><subject>Dementia disorders</subject><subject>Immunoassay</subject><subject>Immunohistochemistry</subject><subject>Immunoprecipitation</subject><subject>Isoelectric focusing</subject><subject>Monoclonal antibodies</subject><subject>Mutation</subject><subject>N-Terminus</subject><subject>Peptides</subject><subject>Plasma</subject><subject>Precursors</subject><subject>Proteins</subject><subject>Selectivity</subject><subject>Transgenic animals</subject><issn>1422-0067</issn><issn>1661-6596</issn><issn>1422-0067</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2020</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>eNpdkUtOHDEQhq0oKDySXQ5gKRsiMcSvtrs3kSaQECSkIPHYWh53mfHIbQ_tbiQWSCyzhpvkIByCk8QEiEhWVaX_01_1qxB6T8k25w355BddZpTWspLiFVqjgrEJIVK9ftGvovWcF4QwzqrmDVrlrKFUEbmGrnbhAkJadhAHbGKLj8HOo7cm4FMTfGsGnyJOrmh4v-vGmEzO5hK71ONhDngXBrDPzPTwUMrm_vpWUYo3p3e_7n_e8DIK8hH7iL_4FNLZH-8j0y0D5LdoxZmQ4d1T3UAn374e73yfHPzY29-ZHkwsJ0JMoKmpVVLVonXCCFeWUCba1s6s5MJRomgJZJywligGvJkR6epZA6y1zpmab6DPj77LcdZBa0vY3gS97H1n-kudjNf_KtHP9Vm60KqqKsVkMdh8MujT-Qh50J3PFkIwEdKYNROCSsoKXdAP_6GLNPaxxHugGGGVqlShth4p26ece3B_j6FEP_xVv_wr_w1HlJc-</recordid><startdate>20200908</startdate><enddate>20200908</enddate><creator>Klafki, Hans W.</creator><creator>Rieper, Petra</creator><creator>Matzen, Anja</creator><creator>Zampar, Silvia</creator><creator>Wirths, Oliver</creator><creator>Vogelgsang, Jonathan</creator><creator>Osterloh, Dirk</creator><creator>Rohdenburg, Lara</creator><creator>Oberstein, Timo J.</creator><creator>Jahn, Olaf</creator><creator>Beyer, Isaak</creator><creator>Lachmann, Ingolf</creator><creator>Knölker, Hans-Joachim</creator><creator>Wiltfang, Jens</creator><general>MDPI AG</general><general>MDPI</general><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>K9.</scope><scope>M0S</scope><scope>M1P</scope><scope>M2O</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-4369-6875</orcidid><orcidid>https://orcid.org/0000-0002-8672-9032</orcidid><orcidid>https://orcid.org/0000-0001-9326-8193</orcidid><orcidid>https://orcid.org/0000-0003-0134-0974</orcidid><orcidid>https://orcid.org/0000-0002-9631-5239</orcidid><orcidid>https://orcid.org/0000-0002-4115-0334</orcidid><orcidid>https://orcid.org/0000-0003-1492-5330</orcidid><orcidid>https://orcid.org/0000-0002-3397-8924</orcidid></search><sort><creationdate>20200908</creationdate><title>Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples</title><author>Klafki, Hans W. ; Rieper, Petra ; Matzen, Anja ; Zampar, Silvia ; Wirths, Oliver ; Vogelgsang, Jonathan ; Osterloh, Dirk ; Rohdenburg, Lara ; Oberstein, Timo J. ; Jahn, Olaf ; Beyer, Isaak ; Lachmann, Ingolf ; Knölker, Hans-Joachim ; Wiltfang, Jens</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3044-e981c76784df4a4f669124ddcbc634f1071291af4cc072e39b06f8b9e2dcffa83</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2020</creationdate><topic>Alzheimer's disease</topic><topic>Amyloid precursor protein</topic><topic>Antibodies</topic><topic>Binding sites</topic><topic>Biological properties</topic><topic>Biomarkers</topic><topic>Blood plasma</topic><topic>Brain</topic><topic>Cell culture</topic><topic>Dementia</topic><topic>Dementia disorders</topic><topic>Immunoassay</topic><topic>Immunohistochemistry</topic><topic>Immunoprecipitation</topic><topic>Isoelectric focusing</topic><topic>Monoclonal antibodies</topic><topic>Mutation</topic><topic>N-Terminus</topic><topic>Peptides</topic><topic>Plasma</topic><topic>Precursors</topic><topic>Proteins</topic><topic>Selectivity</topic><topic>Transgenic animals</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Klafki, Hans W.</creatorcontrib><creatorcontrib>Rieper, Petra</creatorcontrib><creatorcontrib>Matzen, Anja</creatorcontrib><creatorcontrib>Zampar, Silvia</creatorcontrib><creatorcontrib>Wirths, Oliver</creatorcontrib><creatorcontrib>Vogelgsang, Jonathan</creatorcontrib><creatorcontrib>Osterloh, Dirk</creatorcontrib><creatorcontrib>Rohdenburg, Lara</creatorcontrib><creatorcontrib>Oberstein, Timo J.</creatorcontrib><creatorcontrib>Jahn, Olaf</creatorcontrib><creatorcontrib>Beyer, Isaak</creatorcontrib><creatorcontrib>Lachmann, Ingolf</creatorcontrib><creatorcontrib>Knölker, Hans-Joachim</creatorcontrib><creatorcontrib>Wiltfang, Jens</creatorcontrib><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>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>ProQuest Central Essentials</collection><collection>ProQuest Central</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>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>Publicly Available Content Database</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><collection>PubMed Central (Full Participant titles)</collection><jtitle>International journal of molecular sciences</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Klafki, Hans W.</au><au>Rieper, Petra</au><au>Matzen, Anja</au><au>Zampar, Silvia</au><au>Wirths, Oliver</au><au>Vogelgsang, Jonathan</au><au>Osterloh, Dirk</au><au>Rohdenburg, Lara</au><au>Oberstein, Timo J.</au><au>Jahn, Olaf</au><au>Beyer, Isaak</au><au>Lachmann, Ingolf</au><au>Knölker, Hans-Joachim</au><au>Wiltfang, Jens</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples</atitle><jtitle>International journal of molecular sciences</jtitle><date>2020-09-08</date><risdate>2020</risdate><volume>21</volume><issue>18</issue><spage>6564</spage><pages>6564-</pages><issn>1422-0067</issn><issn>1661-6596</issn><eissn>1422-0067</eissn><abstract>The ratio of amyloid precursor protein (APP)669–711 (Aβ−3–40)/Aβ1–42 in blood plasma was reported to represent a novel Alzheimer’s disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aβ−3–x and the development and “fit-for-purpose” technical validation of a sandwich immunoassay for the measurement of Aβ−3–40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aβ immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aβ−3–40 with no appreciable cross reactivity with Aβ1–40 or N-terminally truncated Aβ variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aβ−3–40 with a quantitative assay range of 22 pg/mL–7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aβ42/Aβ−3–40 and Aβ42/Aβ40 ratios were lower in patients with dementia of the Alzheimer’s type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aβ42/Aβ−3–40 ratio as a novel biomarker candidate for Alzheimer’s disease has been set.</abstract><cop>Basel</cop><pub>MDPI AG</pub><pmid>32911706</pmid><doi>10.3390/ijms21186564</doi><orcidid>https://orcid.org/0000-0002-4369-6875</orcidid><orcidid>https://orcid.org/0000-0002-8672-9032</orcidid><orcidid>https://orcid.org/0000-0001-9326-8193</orcidid><orcidid>https://orcid.org/0000-0003-0134-0974</orcidid><orcidid>https://orcid.org/0000-0002-9631-5239</orcidid><orcidid>https://orcid.org/0000-0002-4115-0334</orcidid><orcidid>https://orcid.org/0000-0003-1492-5330</orcidid><orcidid>https://orcid.org/0000-0002-3397-8924</orcidid><oa>free_for_read</oa></addata></record> |
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subjects | Alzheimer's disease Amyloid precursor protein Antibodies Binding sites Biological properties Biomarkers Blood plasma Brain Cell culture Dementia Dementia disorders Immunoassay Immunohistochemistry Immunoprecipitation Isoelectric focusing Monoclonal antibodies Mutation N-Terminus Peptides Plasma Precursors Proteins Selectivity Transgenic animals |
title | Development and Technical Validation of an Immunoassay for the Detection of APP669–711 (Aβ−3–40) in Biological Samples |
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