Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics

Abstract Oligonucleotide-conjugated antibodies have gained importance for their use in protein diagnostics. The possibility to transfer the readout signal from the protein to the DNA level with an oligonucleotide-conjugated antibody increased the sensitivity of protein assays by orders of magnitude...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Julius Wiener, Daniel Kokotek, Simon Rosowski, Heiko Lickert, Matthias Meier
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/325e7fb97ffd4a46bb5b54f873b7dae7
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:325e7fb97ffd4a46bb5b54f873b7dae7
record_format	dspace
spelling	oai:doaj.org-article:325e7fb97ffd4a46bb5b54f873b7dae72021-12-02T14:16:58ZPreparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics10.1038/s41598-020-58238-62045-2322https://doaj.org/article/325e7fb97ffd4a46bb5b54f873b7dae72020-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-58238-6https://doaj.org/toc/2045-2322Abstract Oligonucleotide-conjugated antibodies have gained importance for their use in protein diagnostics. The possibility to transfer the readout signal from the protein to the DNA level with an oligonucleotide-conjugated antibody increased the sensitivity of protein assays by orders of magnitude and enabled new multiplexing strategies. A bottleneck in the generation of larger oligonucleotide-conjugated antibody panels is the low conjugation yield between antibodies and oligonucleotides, as well as the lack of product purification methods. In this study, we combined a non-site-directed antibody conjugation technique using copper-free click chemistry with ion-exchange chromatography to obtain purified single and double oligonucleotide-conjugated antibodies. We optimized the click conjugation reaction of antibodies with oligonucleotides by evaluating crosslinker, reaction temperature, duration, oligonucleotide length, and secondary structure. As a result, we were able to achieve conjugation yields of 30% at a starting quantity as low as tens of nanograms of antibody, which makes the approach applicable for a wide variety of protein analytical assays. In contrast to previous non-site-directed conjugation methods, we also optimized the conjugation reaction for antibody specificity, confirmed by testing with knockout cell lines. The advantages of using single or double oligonucleotide-conjugated antibodies in regards to signal noise reduction are shown within immunofluorescence, proximity ligation assays, and single cell CITE-seq experiments.Julius WienerDaniel KokotekSimon RosowskiHeiko LickertMatthias MeierNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-11 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Julius Wiener Daniel Kokotek Simon Rosowski Heiko Lickert Matthias Meier Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
description	Abstract Oligonucleotide-conjugated antibodies have gained importance for their use in protein diagnostics. The possibility to transfer the readout signal from the protein to the DNA level with an oligonucleotide-conjugated antibody increased the sensitivity of protein assays by orders of magnitude and enabled new multiplexing strategies. A bottleneck in the generation of larger oligonucleotide-conjugated antibody panels is the low conjugation yield between antibodies and oligonucleotides, as well as the lack of product purification methods. In this study, we combined a non-site-directed antibody conjugation technique using copper-free click chemistry with ion-exchange chromatography to obtain purified single and double oligonucleotide-conjugated antibodies. We optimized the click conjugation reaction of antibodies with oligonucleotides by evaluating crosslinker, reaction temperature, duration, oligonucleotide length, and secondary structure. As a result, we were able to achieve conjugation yields of 30% at a starting quantity as low as tens of nanograms of antibody, which makes the approach applicable for a wide variety of protein analytical assays. In contrast to previous non-site-directed conjugation methods, we also optimized the conjugation reaction for antibody specificity, confirmed by testing with knockout cell lines. The advantages of using single or double oligonucleotide-conjugated antibodies in regards to signal noise reduction are shown within immunofluorescence, proximity ligation assays, and single cell CITE-seq experiments.
format	article
author	Julius Wiener Daniel Kokotek Simon Rosowski Heiko Lickert Matthias Meier
author_facet	Julius Wiener Daniel Kokotek Simon Rosowski Heiko Lickert Matthias Meier
author_sort	Julius Wiener
title	Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
title_short	Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
title_full	Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
title_fullStr	Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
title_full_unstemmed	Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
title_sort	preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/325e7fb97ffd4a46bb5b54f873b7dae7
work_keys_str_mv	AT juliuswiener preparationofsingleanddoubleoligonucleotideantibodyconjugatesandtheirapplicationforproteinanalytics AT danielkokotek preparationofsingleanddoubleoligonucleotideantibodyconjugatesandtheirapplicationforproteinanalytics AT simonrosowski preparationofsingleanddoubleoligonucleotideantibodyconjugatesandtheirapplicationforproteinanalytics AT heikolickert preparationofsingleanddoubleoligonucleotideantibodyconjugatesandtheirapplicationforproteinanalytics AT matthiasmeier preparationofsingleanddoubleoligonucleotideantibodyconjugatesandtheirapplicationforproteinanalytics
_version_	1718391598962704384

Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics

Ejemplares similares