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 1536, both as part of the assay selection process, and afterwards 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 (1469/1472) in Olink Explore 1536 exhibited no cross-reactivity according to the tests described. FOLR3 showed a non-specific signal against the related FOLR2 protein, CCL3 against the related CCL4 protein, and LHB against CGB. The signal contribution at endogenous levels were further investigated and more details are noted on the specific biomarker assay pages concerned.

The example graph above shows the specificity testing approach. Each assay is exposed to samples containing either subsets or all of the selected antigens. A given assay should only generate a signal when the corresponding antigen is included in the sample. The lack of unspecific signal indicates that each assay is specific for its target antigen, demonstrating the outstanding specificity of the PEA method at high multiplex degree
Questions?
If you have any questions or comments about how we validate our assays, or if there is anything else we can help you with, please contact us using the link below:
