Limit of Detection (LOD)
In vitro sensitivity (calibrator) curves using recombinant antigen are determined in multiplex format, for each of the 384-plex panels within Olink Explore. Note that in some cases, no suitable antigen is available and no calibrator data is presented. Limit of detection (LOD) is defined as 3 standard deviations above background and reported in pg/mL. The y-axis shows NPX above background, which is defined as the median of negative control measurements and used to define the expected background level (zero).

Example calibrator curve from assay validation
High dose hook effect
The high dose hook effect is seen when there is an antigen excess relative to the reagent antibodies, resulting in falsely low results. In such cases, a significantly lower value may lead to erroneous interpretation of results.
Therefore, the hook threshold is determined for each analyte and reported in pg/mL and this value is presented on each individual biomarker page.
Measuring ranges
The analytical measuring range is defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) and reported in pg/mL. Quantification limits of LLOQ and ULOQ are calculated using relative error <30% and CV <30%.

Example analytical measuring range data from assay validation
Sample distribution
The levels of protein measured in a number of commercial plasma samples are presented in sample distribution plots. Healthy subjects (n=24) and samples obtained from patients with a range of diseases (n=48). These include inflammatory, cardiovascular (n=12) , autoimmune (n=12) & neurological diseases (n=12), as well as cancer (n=12). These data provide a general idea of the NPX range to expect but cannot cover all potential levels in clinical samples. The y-axis shows NPX above background, which is defined as the median of negative control measurements and used to define the expected background level (zero).

Example sample distribution plot from assay validation
Precision
Intra-assay variation (within-run) is calculated as the mean CV for 6 individual samples, within each of 7 separate runs during the validation studies. Inter-assay variation (between-runs) is calculated as the mean CV, for the same 6 individual samples, among 7 separate runs during the validation studies.
Across the assays of Olink® Explore 384 Inflammation, Olink® Explore 384 Oncology, Olink® Explore 384 Cardiometabolic and Olink® Explore 384 Neurology panels, the mean intra-assay and the inter-assay variations observed were 8% and 11%, respectively.
Across the assays of Olink® Explore 384 Inflammation II, Olink® Explore 384 Oncology II, Olink® Explore 384 Cardiometabolic II and Olink® Explore 384 Neurology II panels, the mean intra-assay and the inter-assay variations observed were 9% and 17%, respectively.
Reproducibility
Inter-site variation (between-site) was also investigated during the validation of Olink Explore at three sites (two service laboratory sites and Olink R&D), to estimate the expected variations in values between different laboratories running the same samples, with different operators using different equipment. The sites were trained and instructed to perform the analysis according to the same routine. Each site performed 6-7 independent runs, and the mean inter-assay variation was 6.6%
Specificity
PEA and specificity – a major problem solved
The unique features of PEA technology overcome a long-standing and well recognized issue with immunoassays. With standard assays such as ELISAs, even moderate levels of multiplexing result in cross-reactivity of antibody binding and a loss of specificity of the signals that are detected (Figure A below). By virtue of PEA’s requirement for dual antibody recognition of the target protein and high-fidelity DNA hybridization and detection, any unspecific antibody binding event that may occur will not result in a readout signal (Figure B below).
Specificity validation in Olink Explore

Rigorous specificity testing was used during the development of Olink Explore, both as part of the assay selection process, and afterward as part of the formal product validation procedure.
Assay selection
All assays have gone through a predefined protocol of at least 3 levels of specificity testing:
- First, a screen against several pools of antigens (Ag) is performed (n>100)
- After removal of poorly performing antibodies, a second screen is performed using an expanded set of Ag pools
- Validation of final product design against pools of carefully selected proteins (n=96) including,
* 15 well-known biomarkers from each of the four 384 panels
* 36 proteins with high homology within their protein families
In addition, all Explore assays with equivalent assays in Olink Target 96 panels (n=1118) have previously been tested for specificity against the proteins in the corresponding Target 96 panel.
Overview of specificity validation results
In total, 99.8% of assays (2936/2944) in Olink Explore 3072 exhibited no cross-reactivity according to the tests described.
9 assays revealed a non-specific signal to a closely related protein