title: 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.
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Developmental Biology, Nijmegen, the Netherlands.
authors.forename: Jessie A G L
authors.initials: JA
authors.lastname: van Buggenum
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Developmental Biology, Nijmegen, the Netherlands.
authors.forename: Jan P
authors.initials: JP
authors.lastname: Gerlach
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Biomolecular Chemistry, Nijmegen, the Netherlands.
authors.forename: Selma
authors.initials: S
authors.lastname: Eising
authors.affiliation: Radboud University, Faculty of Science, Institute for Molecules and Materials, Department of Bio-organic Chemistry, Nijmegen, the Netherlands.
authors.forename: Lise
authors.initials: L
authors.lastname: Schoonen
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Developmental Biology, Nijmegen, the Netherlands.
authors.forename: Roderick A P M
authors.initials: RA
authors.lastname: van Eijl
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Developmental Biology, Nijmegen, the Netherlands.
authors.forename: Sabine E J
authors.initials: SE
authors.lastname: Tanis
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Developmental Biology, Nijmegen, the Netherlands.
authors.forename: Mark
authors.initials: M
authors.lastname: Hogeweg
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Biology, Nijmegen, the Netherlands.
authors.forename: Nina C
authors.initials: NC
authors.lastname: Hubner
authors.affiliation: Radboud University, Faculty of Science, Institute for Molecules and Materials, Department of Bio-organic Chemistry, Nijmegen, the Netherlands.
authors.forename: Jan C
authors.initials: JC
authors.lastname: van Hest
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Biomolecular Chemistry, Nijmegen, the Netherlands.
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Bonger
authors.affiliation: Radboud University, Faculty of Science, Radboud Institute for Molecular Life Sciences, Department of Molecular Developmental Biology, Nijmegen, the Netherlands.
authors.forename: Klaas W
authors.initials: KW
authors.lastname: Mulder
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D000906:Antibodies; D011506:Proteins; D004247:DNA