TNF/IFN-gamma upgrades
On this page you will find information showing how Olink improved the key assays for tumor necrosis factor and interferon-gamma (Q2, 2019). These assays were introduced into both our Immuno-Oncology and Inflammation panels, and the data shown below was generated during validation of the Inflammation panel.
Tumor Necrosis Factor (TNF) Uniprot number P01375.
Our previous assay for this analyte, (also known as TNF-alpha) used a monoclonal antibody that can only recognize one epitope per full-length monomer of the target protein. PEA technology requires two matched antibody/ oligonucleotide probes to bind in close proximity for the complementary DNA sequences to hybridize and generate a readout signal, and hence the previous assay required a multimeric TNF molecule for detection. In vivo, TNF exists as both monomers and biologically active trimers (see reference), and the trimeric form has been shown to be unstable at physiological concentrations (see reference). It is extremely likely therefore that the previously low detectability of TNF in human plasma (relative to the sensitivity estimated using recombinant trimeric antigen in vitro) reflected the low abundance of TNF trimers in vivo combined with the inability of our assay to measure the monomers present in the samples.
Since the majority of commercially available assays also detect TNF monomers and our customers have expressed a wish to be able to measure all forms of the protein , we have developed a new assay using polyclonal antibodies that addresses this issue. In our validation experiments, the new assay exhibits significantly higher detectability in human plasma than the previous version, and also correlates very well with a commercially available high-sensitivity ELISA, as shown in the figures below. In our validation experiments, the new assay was able to measure TNF (signal >LOD/LLOQ) in 100% of all tested plasma samples, from both healthy controls and patients with a range of inflammation-related diseases.
The detectability of the TNF assay in the Inflammation panel in samples from healthy controls & multiple different diseases is shown, with the results from the old assay displayed in the left-hand plot and those from the new assay shown to the right. Click the image to expand (shown in a new tab).
Correlation with high-sensitivity ELISA
The new TNF assay was also compared to a commercially available high-sensitivity ELISA for TNF (Human TNF-alpha Quantikine HS ELISA from R&D Systems). The plot below compares measurements taken from the same samples using the new version of the Olink INFLAMMATION panel (i.e. measured as part of the 92-plex assay, values expressed in NPX units) and the commercial single-plex ELISA (expressed as log2 values of the absolute concentration measurements).
Comparison plot of TNF values measured with the new Inflammation panel and a commercial ELISA. Click the image to expand (opens in a new tab).
Interferon-gamma (IFN-gamma) Uniprot number P01579.
Based on customer feedback that a higher level of detectability would be appreciated for this important marker, we have worked hard to optimize and improve this assay. As the data from our validation experiments below shows, the new assay exhibits significantly higher detectability in plasma taken from healthy controls and a range of different disease types. The new assay was able to measure IFN-gamma (signal >LOD/LLOQ) in 100% of all tested plasma samples, from both healthy controls and patients with a range of inflammation-related diseases.
The detectability of the IFN-gamma in the Inflammation panel in samples from healthy controls & multiple different diseases is shown, with the results from the old assay displayed in the left-hand plot and those from the new assay shown to the right. Click the image to expand (opens in a new tab).