FAQ

We tested cross-reactivity with the human homologue proteins and could show that only one assay detects its human homologue (Cxcl12) with a cross-reactivity of 13%.

Target 48 Mouse Cytokine is validated for plasma and serum samples and detectability of the respective proteins is described in the validation data document at olink.com. Other samples such as BAL, tissue lyzates and conditioned media have been tested.

Olink offers the Target 48 Cytokine panel targeting human proteins. There is a 42% overlap with the murine homologue protein in Target 48 Mouse Cytokine. Olink’s Flex library covers 75% of the corresponding murine proteins that can be quantified by Target 48 Mouse Cytokine.

Olink offers two products targeting murine proteins. The Target 96 Mouse Exploratory and Target 48 Mouse Cytokine panel. It has been shown that Target 96 Mouse Exploratory panel is also suitable to analyse rat proteins. For more information, contact our support team and refer to our Technical note “Extending Olink’s Mouse Exploratory panel to studies in rats” which is found on the documents page.

Apart from the above mentioned mouse panels, all Olink assays are developed using antibodies targeted against human proteins. There is of course a certain level of antibody cross-reactivity between human proteins and their homologues in other species. Consequently, a certain proportion of assays in our panels generate signals in samples of non-human origin and some of our customers have used them in animal studies.

Olink’s human panels are only validated for use with human samples, but you may of course use them with samples from non-human species at your own discretion.

A sample will get a QC warning (not pass the quality control) if Incubation Control 2 or Detection Control (corresponding to that specific sample) deviates more than a pre-determined value (+/- 0.3) from the median value of all samples on the plate.

QC warnings may occur due to operator error, for example, or because of the properties/quality of a specific sample. Samples with QC warnings due to operator error are always rerun at Analysis Service. Samples that get QC warnings due to sample quality/ properties are included in the data set but should be handled with care in statistical analysis.

If more than 1/6 of the samples per run get a QC warning, there is a high probability that the entire run is unreliable and should therefore be rerun.

The plasma free Calibrator in the Target 48 Mouse Cytokine panel consists of a buffer with a fixed concentration of recombinant proteins targeted by the panel. The buffer composition is tested with serum/plasma samples to ensure linear behavior at different concentrations.

The concentration of the antigens was chosen to be in the linear range of the respective standard curve.

Target 96 Mouse Exploratory and Target 48 Mouse Cytokine have 14 biomarkers in common. Results from using the same samples with both products correlate well.

Olink Target 96 and Olink Explore panels use relative quantification and the delivered data is in the Olink’s own arbitrary unit NPX (Normalized Protein eXpresstion) . These products are developed for high throughput protein profiling where changes in protein expression in one set of a group or population are quantified relative to another, e.g. patients vs. controls, or samples before vs. samples after treatment.

Olink Target 48, Olink Flex and Olink Focus panels can deliver data both with both relative (NPX) and absolute quantification (pg/mL) depending on the needs of our customers.

A high dose hook effect is a state of antigen excess relative to the antibody probes, resulting in falsely lowered values. If overlooked, a significantly lower value can be reported and lead to misinterpretation of results.

This is because these proteins consist of several subunits and each subunit has its own Uniprot number.

Olinks products are optimized for the sample volumes stated in the respective manuals. It is not recommended to increase sample volume since this will affect assay performance.

Please contact our support team.

The absolute NPX-values should not be expected to be the same across panels. However, we do expect ratios to be the same between samples for analytes measured by multiple panels.

The absolute NPX-values will differ across panels. However, ratios between samples should still be the same. Olink recommends to check the correlation between the overlapping analytes/proteins, select one of them for the statistical analysis and thereafter use the results from the duplicated analyte/protein for verification of the first results.

Some of the proteins addressed by our panels have a very broad range of expression levels in vivo, which makes it difficult to develop an assay in multiplex that can measure both healthy and diseased individuals.

For example, some of the assays on the CVD-panels are developed mainly to measure the elevated levels that can be seen after or during cardiovascular disease. If you would still like to use the results from an assay with low detectability you can use non-parametric statistics (i.e. detected vs. not detected in your groups).

To reduce technical variation between plates and studies, Olink recommends that samples are randomized to allow for intensity normalization before statistical analysis.

How do you normalize between plates of randomized samples?

Olink recommend to apply intensity normalization when samples are randomized. This is a robust and high performing normalization method accounting for any plate-to-plate technical variation. In brief, this normalization strategy is based on using the plate median per assay to make assay medians equal across plates that is why sample randomization is a requirement for this normalization strategy. N.B. this normalization is conducted in the NPX software prior to data delivery and does not need to be done by the end user.

Intensity normalization is performed in the following way:

For each assay the plate median is calculated and subtracted for each sample per assay, this centralizes the median of the plates to 0.

For Target 48 Cytokine and Flex the Sample Control is composed of a pooled human plasma sample, with spiked in recombinant antigens for some proteins, to ensure that all 45 proteins are detected. For Target 48 Mouse Cytokine, Target 48 Immune Surveillance and Target 48 Neurodegeneration we use a mixture of recombinant proteins for all biomarker targeted. These products are plasma-free. Sample controls are included in triplicate on each plate. The Sample Control is used to assess potential variation between runs and plates, as well as quality control of the quantification of data in pg/mL. Validation data for all panels is available on the Olink website.

The Interplate Control (IPC) contains antibodies coupled to the two complementary DNA-tags used for each assay in the panel, thus having them already in proximity. This is a powerful control used for normalization and compensates for potential sequence biases and variation between runs/plates.

Pooled plasma is recommended as sample control. It is not delivered with the reagent kit. The sample control is used as external control to estimate inter and intra precision for each assay.