Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability

PLoS One. 2014 Apr 22;9(4):e95192. doi: 10.1371/journal.pone.0095192. eCollection 2014.


Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Proteins / metabolism
  • Cross Reactions
  • DNA-Directed DNA Polymerase / metabolism
  • Dried Blood Spot Testing
  • Enzyme Stability
  • Female
  • Heterografts
  • Humans
  • Immunoassay / methods*
  • Mice, Nude
  • Oligonucleotides / metabolism
  • Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Temperature


  • Blood Proteins
  • Oligonucleotides
  • DNA-Directed DNA Polymerase

Grants and funding

This work was funded by the EU FP7-Health-2007-B under the PROACTIVE project, FP7- HEALTH-2010 under the DiaTools project, and VINNOVA within the Forska&Väx program (www.vinnova.se). Olink AB provided support in the form of salaries for all authors (besides SBT and JS), but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section.