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 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 μl 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 proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified 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 antibodybased 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 using a multivariate signature.
AB - A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays 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 μl 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 proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified 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 antibodybased 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 using a multivariate signature.
U2 - 10.1074/mcp.m110.004978
DO - 10.1074/mcp.m110.004978
M3 - Journal article
C2 - 21242282
SN - 1535-9476
VL - 10
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
ER -
