• Proseek Multiplex Data Validation

    Quality, rigor and transparency are very important values for Olink Proteomics. Consequently, all of our Proseek® Multiplex assays are rigorously quality controlled and our validation data is made freely available.

    See our FAQ on the quality controls included in each Proseek assay run.

    How we validate our panels

    The analytical performance of our panels is carefully validated for sensitivity, recovery, dynamic range, specificity, precision and scalability. You can see how we perform and report on this validation below, as well as accessing download links for the complete Data Validation Document for each panel.

    Measuring ranges

    Analytical measuring range is defined by the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) and reported in pg/mL. The high dose hook effect (a state of antigen excess relative to the reagent antibodies resulting in falsely lower values) is also determined for each analyte. The figure below shows an example calibrator curve and corresponding analytical measurement data.

    Validation Fig 1

    Precision

    All assays are thoroughly validated for precision (repeatability and reproducibility). The figure below shows a representative example of a combined intra-assay and inter-assay variation validation study.

    Validation Fig 2

    Scalability: Multiplex level-independent performance

    Proseek Multiplex is able to achieve a high level of multiplexing while maintaining exceptional data quality, since unlike in many other immunoassay formats, any antibody cross-reactivity that may occur during the multiplexed assay is excluded from the detection process. This is exemplified in the figure below, comparing dCq-values from single assays for Growth Hormone (GH) and Matrix Metalloproteinase (MMP-7) with the equivalent assays performed in a full 96-plex reaction. The square of the correlation coefficient (R2) value was generated by linear regression.

    Validation Fig 3

    Specificity

    Using Proseek Multiplex, each biomarker analyte is addressed by a matched pair of antibodies, coupled to unique, partially complementary oligonucleotides and measured by quantitative real-time PCR. This dual recognition, DNA-coupled method provides exceptional readout specificity. Validation of the readout specificity for all Proseek Multiplex panels is carried out using a simple, sequential approach in which pools of protein analytes are tested with all 92 antibody probe pairs in the panel. The design of this validation study is shown below.

    Validation Fig 4v2

    A) Test ”sample” created (pool of full-length recombinant antigens corresponding to 8 of the 92 proteins targeted by the Proseek panel). B) Test sample of 8 antigens analyzed using all 92 oligo-labeled antibody pairs (Proseek probes) C) qPCR readout from all 92 Proseek probes detects only those 8 represented by the pooled recombinant antigens. The whole process is then repeated using additional pools of analytes.

     

    The figure below shows the aggregated results from such a study, clearly demonstrating that thanks to our Proximity Extension Assay technology, any antibody cross-reactivity that may occur in the high-multiplex reactions does not affect the specificity of the final readout. This is contrast to other conventional methods such as sandwich ELISA, where antibody cross-reactivity in multiplex assays contributes directly to the detection readout.

    Validation Fig 5

    Click image to enlarge

    Validation Data documents for current Proseek Multiplex panels

    The analytical performance of Proseek Multiplex panels has been carefully validated for sensitivity, recovery, dynamic range, specificity, precision and scalability, and the results are summarized in the Data Validation documents for each panel, which can be downloaded via the links below:

    Proseek Multiplex Cardiometabolic

    Note: the Cardiometabolic panel uses a 1:2025 dilution of sample. The measuring ranges shown in Figure 3 correspond to the concentrations in the original biological sample, whereas the figures quoted in Table 1 represent the actual concentration of recombinant analyte measurable in the validation assay. A multiplication factor of 2025 should therefore be taken into consideration when comparing the addressable biological concentration to the in vitro validation data.

    Proseek Multiplex Cell Regulation

    Proseek Multiplex CVD II

    Proseek Multiplex CVD III

    Note: the CVD III panel uses a 1:100 dilution of sample. The measuring ranges shown in Figure 3 correspond to the concentrations in the original biological sample, whereas the figures quoted in Table 1 represent the actual concentration of recombinant analyte measurable in the validation assay. A multiplication factor of 100 should therefore be taken into consideration when comparing the addressable biological concentration to the in vitro validation data.

    Proseek Multiplex Development

    Note: the Development panel uses a 1:100 dilution of sample. The measuring ranges shown in Figure 3 correspond to the concentrations in the original biological sample, whereas the figures quoted in Table 1 represent the actual concentration of recombinant analyte measurable in the validation assay. A multiplication factor of 100 should therefore be taken into consideration when comparing the addressable biological concentration to the in vitro validation data.

    Proseek Multiplex Immune Response

    Proseek Multiplex Immuno-Onc I

    Proseek Multiplex Inflammation I

    Proseek Multiplex Metabolism

    Note: the Metabolism panel uses a 1:10 dilution of sample. The measuring ranges shown in Figure 3 correspond to the concentrations in the original biological sample, whereas the figures quoted in Table 1 represent the actual concentration of recombinant analyte measurable in the validation assay. A multiplication factor of 10 should therefore be taken into consideration when comparing the addressable biological concentration to the in vitro validation data.

    Proseek Multiplex Oncology II

    Proseek Multiplex Neurology I

    Proseek Multiplex Organ Damage

     

    Validation Data documents for discontinued Proseek Multiplex panels

    Proseek Multiplex Oncology I v2

    Proseek Multiplex CVD I

    Biomarker Validation Data web pages

    Detailed validation data and background information for all of the 92 biomarkers in each panel are available by clicking on the individual biomarkers in the summary tables that can be accessed via the links below:

    CVD II

    CVD III

    Immune Response

    Inflammation I

    Metabolism

    Neurology I

    Oncology II

    Organ Damage