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

JAGL van Buggenum, JP Gerlach, S Eising… - Scientific reports, 2016 - nature.com
JAGL van Buggenum, JP Gerlach, S Eising, L Schoonen, RAPM van Eijl, SEJ Tanis
Scientific reports, 2016nature.com
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 …
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.
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