A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.
Sci Rep
; 6: 22675, 2016 Mar 07.
Article
em En
| MEDLINE
| ID: mdl-26947912
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 12. 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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Proteínas
/
Reação em Cadeia da Polimerase
/
Anticorpos
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
Holanda