TY - JOUR

T1 - Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material

AU - Lundberg, Martin

AU - Thorsen, Stine Buch

AU - Assarsson, Erika

AU - Villablanca, Andrea

AU - Tran, Bonnie

AU - Gee, Nick

AU - Knowles, Mick

AU - Nielsen, Birgitte Egedie Sander

AU - Couto, Eduardo Golzalez

AU - Martin, Roberto

AU - Nilsson, Olle

AU - Fermer, Christian

AU - Schlingemann, Jorg

AU - Christensen, Ib Jarle

AU - Nielsen, Hans Jørgen

AU - Ekstrom, Bjorn

AU - Andersson, Claes

AU - Gustafsson, Mats

AU - Brünner, Nils

AU - Stenvang, Jan

AU - Fredriksson, Simon

PY - 2011/4

Y1 - 2011/4

N2 - 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.

AB - 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.

U2 - 10.1074/mcp.M110.004978

DO - 10.1074/mcp.M110.004978

M3 - Journal article

C2 - 21242282

VL - 10

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

ER -