A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR

Abstract

Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.

Immuno-Detection by sequencing (ID-seq) enables large-scale high-dimensional phenotyping in cells

“…We designed the Immuno-Detection by sequencing (ID-seq) technology to simultaneously measure many proteins and post-translational modifications in high-throughput (Figure 1a). At the basis of IDseq lie antibodies that are labelled with a double-stranded DNA-tag 13 containing a 10 nucleotide antibody-dedicated barcode and a 15 nucleotide Unique Molecular Identifier (UMI, Figure S1, Supplementary note 1). Each antibody signal is now digitized and non-overlapping, allowing many antibodies to be combined and measured simultaneously.…”

Cell “hashing” with barcoded antibodies enables multiplexing and doublet detection for single cell genomics

“…Cell culture HEK293T (human) and NIH-3T3 (mouse) cells were maintained according to standard procedures in Dulbecco's Modified Eagle's Medium (Thermo Fisher, USA) supplemented with 10% fetal bovine serum (Thermo Fisher, USA) at 37˚C with 5% CO2. [clone: HI111]) and were covalently and irreversibly conjugated to HTOs by iEDDA-click chemistry as previously described 18 . In short, antibodies were washed into 1X borate buffered saline (50 mM borate, 150 mM NaCl pH 8.5) and concentrated to 1 mg/ml using an Amicon Ultra 0.5 ml 30 kDa MWCO centrifugal filter (Millipore).…”

Single-cell ID-seq identifies BMP signaling as a driver of a late stage epidermal differentiation program

“…We applied single-cell ID-seq to monitor the activity of signaling pathways and other cellular processes during epidermal differentiation using a panel of 70 antibody-DNA conjugates (Buggenum et al, 2017;Van Buggenum et al, 2016). These antibodies cover a broad range of cellular processes including cell cycle, DNA damage, epidermal self-renewal and differentiation, as well as the intracellular signalling status for the EGF, G-protein coupled receptors, calcium signalling, TNFα, TGFβ, Notch, WNT and BMP pathways (Buggenum et al, 2017 and supplemental table 1).…”

“…Antibodies and dsDNA were functionalized and conjugated as described (Van Buggenum et al, 2016). Antibody details are provided in supplemental Table 1.…”

“…Results scID-seq allows robust, reproducible and highly multiplexed protein detection in single cells. We recently described the Immuno-Detection by sequencing (ID-seq) technology to quantify >70 proteins and phospho-proteins in hundreds of cell populations simultaneously (Buggenum et al, 2017;Van Buggenum et al, 2016). In short, ID-seq entails immuno-staining with DNA-barcoded antibodies followed by quantification through high-throughput sequencing and barcode counting.…”