Olink proteomics in drug development

Olink’s Proximity Extension Assay (PEA) technology
Olink’s Proximity Extension Assay (PEA) technology is a proprietary method. This innovative technology enables highly sensitive and specific protein profiling for thousands of proteins per sample, accommodating nearly hundreds of samples per run.

Detection sensitivity
Unlike traditional immunoassays, like sandwich ELISA, which rely on immunofluorescence reporters limited by the number of detectable fluorescent signals, PEA technology uses antibodies conjugated with unique oligonucleotide (oligos) reporters.

Using oligos as reporters offers greater versatility and an almost indefinite range of detectable markers. These oligos are quantified through next-generation sequencing (NGS) or quantitative PCR (qPCR), which provide far higher sensitivity than immunofluorescence.

Detection specificity
The outstanding specificity of the PEA technology is achieved by binding two different antibodies to detect a protein. Each antibody pair carries unique oligonucleotides (oligos) that are complementary to each other. These oligos can only hybridize with each other if both antibodies are in close proximity (i.e., bound to the same protein), significantly enhancing specificity and reducing cross-reactivity.

Validated assays for scalable multiplexing
The PEA technology supports scalable multiplexing without compromising specificity or sensitivity. Each assay in the Olink panels is rigorously tested and validated to ensure minimal non-specific antigen binding, even for proteins with high homology within the same protein family. The analytical performance of each panel, including sensitivity, dynamic range, specificity, precision, and scalability, is thoroughly validated and documented in the data validation documents available on Olink’s website.

All panels include internal controls for the incubation/immunoreaction, extension, and amplification/detection.

Validated assays for scalable multiplexing
The PEA technology supports scalable multiplexing without compromising specificity or sensitivity. Each assay in the Olink panels is rigorously tested and validated to ensure minimal non-specific antigen binding, even for proteins with high homology within the same protein family. The analytical performance of each panel, including sensitivity, dynamic range, specificity, precision, and scalability, is thoroughly validated and documented in the data validation documents available on Olink’s website.

All panels include internal controls for the incubation/immunoreaction, extension, and amplification/detection.

All Olink platforms are designed for plasma and blood but are also compatible with various other tissues and biofluids. Protocols are available for these alternative matrices:

• Cell lysates
• Microvesicles
• Interstitial fluids
• Urine
• Synovial fluids
• Dried blood spots
• Fine needle biopsies
• Cerebrospinal fluids
• Microdialysis fluids
• Plaque extracts
• Bronchial aspirates
• Cell culture media
• Extracellular vesicles
• Saliva
• Tissue lysates
• Plasma
• Serum

The data generated is reported as Normalized Protein eXpression (NPX) levels, which are log2-transformed and normalized. This process allows for easy comparison across different samples and conditions, enhancing the reliability of the data. NPX is a quantification unit used by Olink, where raw values are normalized through a series of computations designed to minimize technical variations and improve the interpretability of the results.

NPX values for each protein are obtained using Olink’s analysis software: Olink NPX Signature software for the Target panels and Olink NPX Explore software for the Explore platform. These software tools include built-in quality control steps and generate comprehensive reports. To avoid batch effects and ensure data consistency and reliability across different runs, Olink randomizes samples, which is crucial for large-scale studies over time.

For qPCR readouts, NPX is calculated from the Ct values, with normalization performed to minimize intra- and inter-assay variation. This process enables the identification of changes in individual protein levels across all sample sets. A high NPX value indicates a high protein concentration. Since NPX is on a log2 scale, a 1 NPX difference corresponds to doubling the protein concentration.

It is important to note that NPX values are relative, meaning they can only be compared for the same protein across the samples analyzed in the project. Direct comparison of NPX values between different proteins is not possible.

Most protein assays in the Olink Explore 3072 panel are compatible with non-human primate (macaque) plasma and have passed rigorous quality control measures. For further advice on NHP studies, please get in touch with us.

Olink offers the Target 48 Cytokine panel for mouse studies and the Olink Target 96 Mouse Exploratory panel for translational research.