Proximity Extension Assay
Immuno-PCR for targeted biomarker discovery
The protein biomarker panels developed by Olink Proteomics is a unique technology (Proximity Extension Assay) enabling high-throughput, multiplex immunoassay-qPCR that measure 92 proteins across 96 samples simultaneously using only one microliter of serum, plasma, or equivalent amounts of almost any other type of biological sample. The innovative dual recognition, DNA-coupled methodology provides exceptional readout specificity, enabling high multiplex, rapid throughput protein biomarker analysis without compromising on data quality assay robustness.
Determine 92 proteins simultanously
The 1165 immuno-PCR-assays developed are divided into 15 disease-focused panels of 92 proteins. Samples may be analysed against one or more panels using only 1µl of sample per panel.
See the full list of proteins in all panels here:
PROTEIN BIOMARKER PANELS
The content of each panel can be found here, including proteins mapped to disease area and biological process:
Highly sensitive protein quantification
The main steps involved in each protein quantification assay are outlined to the left. A pair of oligonucleotide-labelled antibodies, Proseek probes, are allowed to pair-wise bind to the target protein present in the sample in a homogeneous assay, with no need for washing. When the two Proseek probes are in close proximity, a new PCR target sequence is formed by a proximity-dependent DNA polymerization event. The resulting sequence is subsequently detected and quantified using the Fluidigm Biomark HD real-time PCR system that allows for the analysis of 96 samples against 96 assays in a nanoliter scale within 3 hours.
Real-Time qPCR Technology
Microfluidic high-throughput analysis
The integrated fluidic circuits empower life science research by automating molecular biology in nanoliter volumes. This means using less sample and reagent, and a single microfluidic device, to achieve high quality, consistent results. Fluidigm microfluidic technology supports genomics-based applications such as gene expression (RNA and miRNA), high-throughput SNP genotyping, protein expression analysis, digital PCR, mutant detection and more.
Efficient and reproducible analysis
The integrated fluidic circuits (IFCs) – often referred to as chips or plates – offer rapid, efficient, highly parallel and reproducible multistep sample preparation and analysis of up to hundreds of genetic markers across thousands of samples in just hours, rather than days or weeks. This allows for analysis of DNA, RNA, miRNA, protein and epigenetics at a single cell level.
96 PCR assays in one analysis
The Fluidigm 96.96 Dynamic Array™ IFC enables 9,216 parallel RT PCR reactions using 96 samples and 96 assays. On one side of the array-frame are 96 wells to accept the samples and, on the other, 96 wells to accept the probe and primer pairs. Once in the wells, the components are pressurized into the chip using an IFC controller. The components are then systematically combined into the 9,216 reactions.
BioMark™ HD System is unique among analytical instrumentation as a multi-application platform without compromise, providing high-quality results for every experimental approach.
Real-time PCR gene expression
The system includes the optical, thermal cycling, and software components necessary to perform real-time PCR gene expression (RNA, miRNA), genotyping and digital analysis, as well as end-point analysis for genotyping and digital applications. The BioMark™ HD System provides orders of magnitude higher throughput for real-time PCR compared to conventional platforms due to its integrated fluidic circuits (IFCs). This innovative solution for real-time PCR provides experiment densities far beyond what is possible with microplate platforms, and significantly reduces the number of liquid-handling steps and volumes per reaction.
Extraction of DNA, RNA and proteins
BioXpedia provides sample preparation and high-quality extraction of DNA, RNA, and protein for any downstream analysis. To ensure maximum reproducibility and efficiency, automated workflow and robotic pipetting is used whenever possible.
Types of clinical samples
DNA and RNA can be isolated from fresh, frozen and fixed tissue along with any biofluid sample. Additionally, we offer isolation of free-circulating nucleic acids and isolation of extracellular vesicles like exosomes from plasma, serum and other biofluid for nucleic acid and protein analysis. Extraction of proteins are optimized for the specific sample type, e.g. cells or tissue, to ensure extraction of native proteins and avoid degradation of proteins prior to analysis.
Automated sample preparation and quality analysis
The concentration and quality of extracted DNA and RNA is assessed by state-of-the-art technologies (4200 TapeStation, QIAxpert) to assure high quality input material for our downstream analysis. For automated extractions and robotic pipetting BioXpedia employ BioRobot Universal System and QIASymphony (Qiagen) whenever possible to optimize throughput, hands-on time and efficacy.
NanoString nCounter Technology
Targeted multiplex expression profiling
The nCounter® Analysis System from NanoString Technologies offers single molecule detection, using unique fluorescent barcodes. The colour-coded barcodes enable direct, digital detection of hundreds (≤800) of individual target molecules in a single assay. This will allow you to see the relationship between, DNA, RNA, and protein in one single sample and compare genotyping with gene and protein expression. NanoString can detect target molecules as small as 100 base pairs and only requires a limited input amount of 100ng. This enables multiplex gene expression profiling with single cell worth of input.
Single molecule digital detection of DNA, RNA and protein – also from FFPE samples
NanoString is a fully automated technology ensuring precise and reproducible measurements, making it compatible with clinical research environments. The technology is robust across many sample types and qualities, both tissues of poor and varying quality can be used including FFPE tissue sections.
For every target of interest two adjacent 50 base pair probes complementary to the target region will be utilized: a capture probe linked to biotin for immobilization and purification, and a reporter probe connected to a unique colour coded molecular barcode. In a completely enzyme-free reaction, an excess of probe will drive the hybridization of all target-probe complexes to completion. Up to 800 different targets can be analysed in a single reaction. Once the target-probe complexes have hybridized, the complexes are captured on a streptavidin-coated surface, where they are elongated and immobilized for imaging.
Molecular barcode technology
A scanner then digitally counts the number of barcodes hybridized to a single molecule for each of the targets. One molecule equals one count. This ensures a very low false positive rate (0.1%) compared to analogue technologies that measure fluorescent intensities. The data from the digital reads are then normalized to internal controls and a set of reference genes. This will generate highly precise relative counts that can be interpreted in terms of relative change across samples. Absolute counts are not calculated using this technology.
meso scale – msd
Meso Scale Discovery
Absolute and multiplex quantification of proteins
The MSD technology offers absolute quantitation of protein markers using electrochemiluminescence immunoassays providing high sensitivity and multiplex functionality. The platform is highly flexible offering ultra-sensitive and thoroughly validated assay panels and customized assay combinations of your own choice. The assays are provided as single analyte assays or multiplex assays up to 10-plex in each well.
More than 500 assays available in panels or custom combinations
Available assays cover a wide range of biological applications, with more than 500 assays provided in panels, custom panels or reagent kits to build your own assay. The assay panels are focused on measuring proteins associated to for example inflammation, oncology, neurodegeneration, immunology, intracellular signaling, cardiovascular disease and metabolism. Assays are available for different species and compatible with an array of complex sample types e.g. serum, plasma, supernatants, cell lysates, CSF, urine, blood spots, tears, synovial fluid, tissue extracts etc.
Unique sensitivity, accuracy and dynamic range to determine protein concentration
MSD assays are based on a proprietary technology combining electrochemiluminescence and MULTI‑ARRAY technology for detection of proteins in biological samples. Analytes of interest are detected using sulfo-tag-conjugated reporter molecules and the Meso Scale instrument. Electrochemiluminescence detection offers a unique combination of sensitivity, accuracy and dynamic range for absolute quantitation of single proteins or protein profiles.
Validated protein quantification assays
V-PLEX assays are designed and produced to maximize reproducible performance, consistency and reliability in results and confidence in data. The assays and panels are fully validated in multiple matrices according to fit-for-purpose principles. Assays are organized into panels by species, analytical compatibility, clinical range and expected use to ensure optimal and consistent performance and lot-to-lot consistency is guaranteed to support long-term studies.
Build your own single protein or multiplex assays
The U-PLEX is an open platform that offers full flexibility to design and develop your own single analyte and multiplex assays targeted the proteins studied in your project. More than 160 assays are available on the U-PLEX platform, these can be used alone or combined in multiplex with your own assays.
In U-PLEX assays biotinylated capture reagents are coupled to U-PLEX linkers and bound to spots on the plate. Analytes in the sample bind to the capture reagents and detection antibodies conjugated with electrochemiluminescence labels bind to the analytes to complete the sandwich assay.
Finally, R-PLEX antibody sets can also be used for development of multiplex or single analyte immunoassays. Each R-PLEX antibody set contains a biotinylated capture antibody, sulfo-tag labeled detection antibody and a calibrator.