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Multiplexed protein detection by proximity ligation for cancer biomarker validation

Abstract

We present a proximity ligation–based multiplexed protein detection procedure in which several selected proteins can be detected via unique nucleic-acid identifiers and subsequently quantified by real-time PCR. The assay requires a 1-μl sample, has low-femtomolar sensitivity as well as five-log linear range and allows for modular multiplexing without cross-reactivity. The procedure can use a single polyclonal antibody batch for each target protein, simplifying affinity-reagent creation for new biomarker candidates.

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Figure 1: Schematic outline of multiplexed proximity ligation.
Figure 2: Multiplex proximity ligation assays.
Figure 3: Multiplex proximity ligation of six proteins and subsequent detection by qPCR in plasma samples from 20 patients with pancreatic cancer and 14 matched healthy controls.

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Acknowledgements

This work was supported by the Swedish Research Council, the Canary Foundation, the Liu Bie Ju Cha and Family Fellowship in Cancer and the US National Institutes of Health (Center Grant 2P01HG000205 and Glue Grant U54GM062119).

Author information

Authors and Affiliations

Authors

Contributions

S.F., M.M. and R.W.D. initiated the project; S.F. and R.W.D. designed experiments and methods; S.F. and W.D. performed experiments; A.C.K. supplied clinical samples; S.F., H.J., A.C.K. and R.W.D. analyzed data; S.F. drafted the initial manuscript and all authors contributed to subsequent revisions.

Corresponding authors

Correspondence to Simon Fredriksson or Ronald W Davis.

Ethics declarations

Competing interests

S.F. is a shareholder in Olink AB.

Supplementary information

Supplementary Figs. 1

Performance improvement of the proximity ligation protocol. (PDF 149 kb)

Supplementary Figs. 2

Single proximity ligation assays using polyclonal antibody based probes linked with oligonucleotides via biotin/streptavidin. (PDF 33 kb)

Supplementary Figs. 3

Plasma levels for all nine proteins in cases and controls. (PDF 77 kb)

Supplementary Figs. 4

Correlation between multiplex proximity ligation and sandwich ELISA. (PDF 107 kb)

Supplementary Figs. 5

Inter- and intra-assay variation for proximity ligation. (PDF 115 kb)

Supplementary Table 1

Summary of each assay performance within the multiplex panel. (PDF 275 kb)

Supplementary Table 2

Proximity probe sequence design. (PDF 79 kb)

Supplementary Methods (PDF 57 kb)

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Fredriksson, S., Dixon, W., Ji, H. et al. Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nat Methods 4, 327–329 (2007). https://doi.org/10.1038/nmeth1020

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  • DOI: https://doi.org/10.1038/nmeth1020

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