Brief Communication | Published:

Multiplexed protein detection by proximity ligation for cancer biomarker validation

Nature Methods volume 4, pages 327329 (2007) | Download Citation

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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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

Affiliations

  1. Stanford Genome Technology Center, Bio-X, 318 Campus Dr., Stanford, California 94305, USA.

    • Simon Fredriksson
    • , William Dixon
    • , Hanlee Ji
    • , Michael Mindrinos
    •  & Ronald W Davis
  2. Department of Radiation Oncology, Stanford, California 94305, USA.

    • Albert C Koong

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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.

Competing interests

S.F. is a shareholder in Olink AB.

Corresponding authors

Correspondence to Simon Fredriksson or Ronald W Davis.

Supplementary information

PDF files

  1. 1.

    Supplementary Figs. 1

    Performance improvement of the proximity ligation protocol.

  2. 2.

    Supplementary Figs. 2

    Single proximity ligation assays using polyclonal antibody based probes linked with oligonucleotides via biotin/streptavidin.

  3. 3.

    Supplementary Figs. 3

    Plasma levels for all nine proteins in cases and controls.

  4. 4.

    Supplementary Figs. 4

    Correlation between multiplex proximity ligation and sandwich ELISA.

  5. 5.

    Supplementary Figs. 5

    Inter- and intra-assay variation for proximity ligation.

  6. 6.

    Supplementary Table 1

    Summary of each assay performance within the multiplex panel.

  7. 7.

    Supplementary Table 2

    Proximity probe sequence design.

  8. 8.

    Supplementary Methods

About this article

Publication history

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DOI

https://doi.org/10.1038/nmeth1020

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