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Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure

  • Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficientImmunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagentsmare expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via Nhydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.zeige mehrzeige weniger

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Metadaten
Autoren/innen:Peter Carl, Inês I. Ramos, Marcela A. Segundo, Rudolf J. Schneider
Dokumenttyp:Zeitschriftenartikel
Veröffentlichungsform:Verlagsliteratur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):PLoS ONE
Jahr der Erstveröffentlichung:2019
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.8 Umweltanalytik
Veröffentlichende Institution:Bundesanstalt für Materialforschung und -prüfung (BAM)
Verlag:Public Library of Science
Verlagsort:San Francisco, California, USA
Jahrgang/Band:14
Ausgabe/Heft:6
Erste Seite:e0218686, 1
Letzte Seite:18
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Bead injections; Biomarkers; Carbamazepine; ELISA; Mesofluidics
Themenfelder/Aktivitätsfelder der BAM:Analytical Sciences
Analytical Sciences / Sensorik
DOI:https://doi.org/10.1371/journal.pone.0218686
URN:urn:nbn:de:kobv:b43-483919
URL:http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=Alerting&SrcApp=Alerting&DestApp=WOS_CPL&DestLinkType=FullRecord&UT=WOS:000482883600046
ISSN:1932-6203
Verfügbarkeit des Dokuments:Datei für die Öffentlichkeit verfügbar ("Open Access")
Lizenz (Deutsch):License LogoCreative Commons - CC BY - Namensnennung 4.0 International
Datum der Freischaltung:03.07.2019
Referierte Publikation:Ja
Datum der Eintragung als referierte Publikation:03.07.2019
Schriftenreihen ohne Nummerierung:Wissenschaftliche Artikel der BAM