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 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

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Scientific reports. - 10 (2020) , 1457, ISSN: 2045-2322

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2025
• Creator: Wiener, Julius ; Kokotek, Daniel M. U. ; Rosowski, Simon ; Lickert, Heiko ; Meier, Matthias

Further information

• DOI: 10.1038/s41598-020-58238-6
• URN: urn:nbn:de:bsz:25-freidok-2595594
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:22 AM CEST