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The Capital Region of Denmark - a part of Copenhagen University Hospital
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Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

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  • Martin Lundberg
  • Stine Buch Thorsen
  • Erika Assarsson
  • Andrea Villablanca
  • Bonnie Tran
  • Nick Gee
  • Mick Knowles
  • Birgitte Sander Nielsen
  • Eduardo Golzalez Couto
  • Roberto Martin
  • Olle Nilsson
  • Christian Fermer
  • Jorg Schlingemann
  • Ib Jarle Christensen
  • Hans Jørgen Nielsen
  • Bjorn Ekstrom
  • Claes Andersson
  • Mats Gustafsson
  • Nils Brunner
  • Jan Stenvagn
  • Simon Fredriksson
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A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays (PLA) in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub pM sensitivity each consuming only 1 micro Litre of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex PLA thereby converts multiple target analytes into real-time PCR amplicons that are individually quantificatied using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer (CRC) using a multivariate signature.
Original languageEnglish
JournalMolecular & cellular proteomics : MCP
Volume10
DOIs
Publication statusPublished - Apr 2011

ID: 32508465