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

Martin Lundberg; Stine Buch Thorsen; Erika Assarsson; Andrea Villablanca; Bonnie Tran; Nick Gee; Mick Knowles; Birgitte Egedie 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 Brünner; Jan Stenvang; Simon Fredriksson

doi:10.1074/mcp.m110.004978

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

Martin Lundberg, Stine Buch Thorsen, Erika Assarsson, Andrea Villablanca, Bonnie Tran, Nick Gee, Mick Knowles, Birgitte Egedie 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 Brünner, Jan StenvangSimon Fredriksson

Department of Clinical Medicine

54 Citations (Scopus)

Abstract

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.

Original language	English
Journal	Molecular and Cellular Proteomics
Volume	10
ISSN	1535-9476
DOIs	https://doi.org/10.1074/mcp.m110.004978
Publication status	Published - Apr 2011

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Lundberg, M., Thorsen, S. B., Assarsson, E., Villablanca, A., Tran, B., Gee, N., Knowles, M., Nielsen, B. E. S., Couto, E. G., Martin, R., Nilsson, O., Fermer, C., Schlingemann, J., Christensen, I. J., Nielsen, H. J., Ekstrom, B., Andersson, C., Gustafsson, M., Brünner, N., ... Fredriksson, S. (2011). Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material. Molecular and Cellular Proteomics, 10. https://doi.org/10.1074/mcp.m110.004978

Lundberg, M, Thorsen, SB, Assarsson, E, Villablanca, A, Tran, B, Gee, N, Knowles, M, Nielsen, BES, Couto, EG, Martin, R, Nilsson, O, Fermer, C, Schlingemann, J, Christensen, IJ, Nielsen, HJ, Ekstrom, B, Andersson, C, Gustafsson, M, Brünner, N , Stenvang, J & Fredriksson, S 2011, 'Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material', Molecular and Cellular Proteomics, vol. 10. https://doi.org/10.1074/mcp.m110.004978

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title = "Multiplexed homogeneous proximity ligation assays for high throughput protein biomarker research in serological material",

abstract = "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.",

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AU - Lundberg, Martin

AU - Thorsen, Stine Buch

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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

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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.

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DO - 10.1074/mcp.m110.004978

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VL - 10

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

ER -

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

Abstract

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