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Sci Rep. 2020 Jan 29;10(1):1457. doi: 10.1038/s41598-020-58238-6.

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

Author information

1
Microfluidic and Biological Engineering, Helmholtz Pioneer Campus, Helmholtz Zentrum München, Ingolstaedter Landstr. 1, 85764, Neuherberg, Germany.
2
Microfluidic and Biological Engineering, IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110, Freiburg, Germany.
3
Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, D-85764, Neuherberg, Germany.
4
German Center for Diabetes Research (DZD), D-85764, Neuherberg, Germany.
5
Institute of Stem Cell Research, Helmholtz Zentrum München, D-85764, Neuherberg, Germany.
6
Technical University of Munich, School of Medicine, Munich, Germany.
7
Microfluidic and Biological Engineering, Helmholtz Pioneer Campus, Helmholtz Zentrum München, Ingolstaedter Landstr. 1, 85764, Neuherberg, Germany. matthias.meier@helmholtz-muenchen.de.

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.

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