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Novel antibody tagging strategy using lanthanide loaded NHS-dota-ester for the application in highly selective LA-ICP-MS-based immunoassays

  • Using metal tagged antibodies for multiplex immunoassays is an important aspect in the research field of bioanalytics. Various tagging strategies have been published, however the formed antibody conjugates were described only rudimentarily. We present an inexpensive, simple and fast DOTA-NHS-ester tagging strategy and the characterization of the conjugates. The monoclonal anti-beta-actin-antibody was used as model system and was modified with the bifunctional ligand DOTA-NHS-ester complexing monoisotopic lanthanide ions. In contrast to the antibody tagging with other tags based on maleimide chemistry the DOTA-NHS-ester reacts with unprotonated amino groups instead of free sulfhydryl groups. The great advantage of this is that a partially reduction of the antibody is not required and therefore the antibody remains intact. The characterization of the conjugates by different analytical techniques demonstrates that no antibody fragments are formed. A mean tagging degree of 8 tags/antibodyUsing metal tagged antibodies for multiplex immunoassays is an important aspect in the research field of bioanalytics. Various tagging strategies have been published, however the formed antibody conjugates were described only rudimentarily. We present an inexpensive, simple and fast DOTA-NHS-ester tagging strategy and the characterization of the conjugates. The monoclonal anti-beta-actin-antibody was used as model system and was modified with the bifunctional ligand DOTA-NHS-ester complexing monoisotopic lanthanide ions. In contrast to the antibody tagging with other tags based on maleimide chemistry the DOTA-NHS-ester reacts with unprotonated amino groups instead of free sulfhydryl groups. The great advantage of this is that a partially reduction of the antibody is not required and therefore the antibody remains intact. The characterization of the conjugates by different analytical techniques demonstrates that no antibody fragments are formed. A mean tagging degree of 8 tags/antibody was determined. The applicability of this tagging strategy is shown in first results of an Alzheimer’s disease study in a mouse model. We have chosen three different NHS-DOTA-ester tagged antibodies for multielement LA-ICP-MS based bioimaging of mice brain tissue. The artificial introduced lanthanides as well as naturally occurring elements were analyzed simultaneously and differences between transgenic and wild-type animals are presented.zeige mehrzeige weniger

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  • 2017.08.16 Metallomics Antje Herrmann.pdf
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Metadaten
Autoren/innen:Antje Jutta Wanka
Koautoren/innen:Karima Schwab, Franz Theuring, Larissa Müller, Norbert Jakubowski, Dilyara Lauer
Dokumenttyp:Vortrag
Veröffentlichungsform:Präsentation
Sprache:Englisch
Jahr der Erstveröffentlichung:2017
Organisationseinheit der BAM:1 Analytische Chemie; Referenzmaterialien
1 Analytische Chemie; Referenzmaterialien / 1.1 Anorganische Spurenanalytik
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Freie Schlagwörter:Antibody tagging; LA-ICP-MS based immunoassays
Themenfelder/Aktivitätsfelder der BAM:Analytical Sciences
Veranstaltung:Metallomics
Veranstaltungsort:Wien, Austria
Beginndatum der Veranstaltung:13.08.2017
Enddatum der Veranstaltung:17.08.2017
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:11.09.2017
Referierte Publikation:Nein