• Proximity Extension Assay (PEA) technology

    Innovation for targeted protein biomarker discovery

    The unique technology behind our Olink® Target 96 panels enables high-throughput, multiplex immunoassays that measure 92 proteins across 96 samples simultaneously using only one microliter of serum, plasma, or almost any other type of biological sample. Here we will show you how our technology delivers this scale of multiplexing and sample throughput without compromising on data quality or assay robustness.

    Olink’s PEA technology – the movie

    The short video below overviews the Proximity Extension Assay (PEA) technology that lies behind our Olink Target 96 and Olink® Explore 1536 platforms, with qPCR or NGS readout, respectively.  92-plex immunoassay panels for human protein biomarker research. The animation explains how our innovative dual recognition, DNA-coupled methodology provides exceptional readout specificity, enabling high multiplex, rapid throughput biomarker analysis without compromising on data quality.


    Olink assay overview

    The main steps involved in an assay are outlined below. A pair of oligonucleotide-labeled antibodies (“probes”) are allowed to pair-wise bind to the target protein present in the sample in a homogeneous assay, with no need for washing. When the two probes are in close proximity, a new PCR target sequence is formed by a proximity-dependent DNA polymerization event. With our Target 96 panels, the resulting sequence is subsequently detected and quantified using standard real-time PCR.


    Overview of the PEA technology for Olink Target 96 panels. (A) 92 Antibody pairs, labelled with DNA oligonucleotides, bind target antigen in solution. (B) Oligonucleotides that are brought into proximity hybridize, and are extended by a DNA polymerase. (C) This newly created piece of DNA barcode is amplified by PCR. (D) The amount of each DNA barcode is quantified by microfluidic qPCR.


    June 3, 2020 – please note: for information related to the new Olink® Explore 1536 platform with PEA on NGS, please visit the Olink Explore page

    You can view an instructional video of how to run an Olink Target 96 assay in the video below:

    Overcoming the specificity issues of multiplexed immunoassays

    Each of the 96 oligonucleotide antibody-pairs contains unique DNA sequences allowing hybridization only to each other. Subsequent proximity extension will create 96 unique DNA reporter sequences which are amplified by real-time PCR. A limiting factor of multiplexed immunoassays is the antibody cross-reactivity which restricts the degree of multiplexing of most assays to below 10-plex. Cross-reactive events will not be detected with Olink’s panels since only matched DNA reporter pairs are amplified with real-time PCR, see figures below. This allows for scalable multiplexing without loss of specificity and sensitivity.


    A) Conventional immunoassays: cross-reactivity due to unspecific binding of antibodies limits the degree of multiplexing.


    B) Olink’s technology: unique DNA oligo sequences report only matched DNA-pairs (e.g. 1A+1B). Cross-reactive events are not detected.