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

Julius Wiener,Daniel Kokotek,Heiko Lickert,Simon Rosowski,Matthias Meier

doi:10.1038/s41598-020-58238-6

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.

Highlights

Oligonucleotide-conjugated antibodies have gained importance for their use in protein diagnostics
The conjugation reaction of oligonucleotides to an antibody consists of three individual crosslinking steps: (i) functionalization of the antibody with a dibenzocyclooctyne (DBCO) click group; (ii) functionalization of the oligonucleotide with the corresponding azide click group; (iii) conjugation of the functionalized antibody and oligonucleotide via a copper-free click chemistry reaction
In order to maximize the yield of the antibody-oligonucleotide click chemistry conjugation reaction, we first sought to optimize the functionalization of the antibody and oligonucleotide

Summary

Introduction

Oligonucleotide-conjugated antibodies have gained importance for their use in protein diagnostics. Despite the vast variety of commercially-available crosslinking reagents and protocols for each of the non-site directed conjugation strategies, the oligonucleotide labeling of antibodies is not robust. The reason for this is multilayered, including problems related to a loss in the specificity of the antibody due to the masking of the antigen binding site[23], a change in polarity upon the addition of the reactive conjugation group and oligonucleotide, and a lack of purification methods for the removal of excess oligonucleotides, which increases the rate of false positive errors. We demonstrate the need of single oligonucleotide-conjugated antibodies for methods using absolute read count statistics from generation sequencing for protein quantification on the single cell level

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