High Throughput Protein Biomarker Discovery

Protein Biomarker Investigation

Olink Proteomics

Mosquito LV during Olink Assay

Accurate multichannel pipetting of nanolitre volumes on a Mosquito LV as part of Olink assay streamlined and automated workflow.

Why an Olink Assay?

The Olink Explore, Olink Target, and Olink Flex assays represent a groundbreaking platform that empowers scientists engaged in diverse fields, such as drug development, clinical research, and fundamental life science research. Targeted protein screening by Olink panels offers a unique way of investigating a selected part of the proteome in the search for novel biomarkers. The Olink Explore assays are particularly appealing to scientists undertaking comprehensive and large-scale proteomic studies.

All Olink protein biomarker analysis tools are based on Olink’s proximity extension assay (PEA) technology. The PEA is an antibody-based assay. Recognition of protein antigens in the assay is twofold, so for each target two individual antibodies are required. The detection mechanism is facilitated by single stranded DNA (ssDNA) bound to these detection antibodies. The antibodies bind in close proximity on the surface of the target protein antigen. This allows the ssDNA to hybridise, forming a double strand. The assay takes place in solution, and many assays can be performed in parallel, in the same reaction volume.

Olink Assay Technology in Short

In all PEA assays the basic principle behind the signal generation is the same. When two antibodies linked to appropriate DNA oligos (with complementary sequence) find the same target, a double stranded DNA (dsDNA) will form. The presence in the reaction mix of a polymerase and other components will allow for elongation and subsequent amplification In this way, each antigen will lead to a unique DNA code. The sequence uniquely identifies the target antigen, and the abundance correlates with protein antigen concentration.

In the most comprehensive Olink assay, the number of target proteins in each reaction is over 5400. Assays of this type belong to the Olink Explore series and are processed using next generation sequencing (NGS). More focused assays, such as those in the Olink Target series, are processed using quantitative PCR (qPCR).

When generating qPCR readout a microfluidic chip is employed. This enables physical separation of the qPCR readout for unique sample and target combinations. Each chip will accommodate up to 88 samples, and each sample is assayed for concentrations of 92 target proteins. In this way more than 8000 data points can be generated from a single chip.

The NGS readout is based on counting individual sequences. A reaction is performed for each sample, assaying for thousands of target proteins at once. A plate of 172 samples will produce close to one million protein concentration datapoints. After PCR amplification of target sequences, the DNA is loaded directly onto a flow cell and sequenced. The raw data from the sequencing is then processed to determine protein expression of thousands of protein targets in each individual sample.

The readout for most Olink assays is in units of normalised protein expression (NPX). This data format allows users to identify changes for individual protein levels across their sample set, and then use this data to establish protein signatures. A high NPX value equals a high protein concentration.

The science behind the PEA technology. The video also describes the two methods of generating the protein concentration data: qPCR and NGS. The two data acquisition methods are used in the Olink Target and Olink Explore assays respectively.

Olink Explore HT

Best Possible Coverage

The latest development regarding the PEA assay, is the launch of the Olink Explore HT panel. This panel offers the highest possible multiplex capacity so far, allowing the simultaneous analysis of over 5400+ targets. The HT panel is based on NGS readout and delivers data in the form of normalised protein expression (NPX) for each protein assay. The assay requires a sample volume of just 2 µL. A complete list of targets included in the panel can be found below:

Olink Explore HT stands out as the most comprehensive and cost-effective approach for protein analysis in complex biological samples. The panel has been assembled focusing on functional, actionable, druggable, and circulating proteins. Compared to the Olink Explore 3072 (which is described further below), The Olink Explore HT coverage is increased, particularly in the areas of tissue-specific proteins, as well as the coverage of exploratory level biomarkers.

Importantly, every individual assay within the Olink HT panel undergoes rigorous evaluation for sensitivity, specificity, precision, and dynamic range. At BioXpedia, we add our own layer of quality control, as every dataset is carefully reviewed by our internal team of data scientists before release. Olink Explore is fully validated for human plasma and serum samples. If you have samples of other types, such as CSF, don’t hesitate to contact us for information about how to proceed.

The Olink Explore HT panel is essentially an expansion of the Explore 3072. Regarding proteins unique to each assay the reference list below can be helpful:

Olink Explore 3072 vs Olink Explore HT

All in all, both the Olink Explore HT and the Olink 3072 assay offers is a great choice for high throughput proteomics research. Get in touch with us to hear more about our capacity and prices.

Sample Requirements

Both Olink Explore HT and Olink Explore 3072 are designed for high sample throughput and minimal sample consumption. The Explore HT consumes just 2 µL of sample, and the full Explore 3072 panel consumes only 6 µL of sample. Olink Explore assays have undergone thorough validation for use with human plasma and serum samples. Many other biofluids (such as cell supernatants, cerebrospinal fluid, dried blood spots, skin tape strips, urine, synovial fluid) are compatible with the assay. Please contact our support team via mail or telephone for additional guidance regarding sample compatibility.

Olink Explore Workflow

Olink Explore workflow

Workflow of the Olink Explore assays. Samples are first diluted to adjust target protein concentrations to assay dynamic range. Antibodies attached to oligo linkers are added. Target proteins with matched antibody pairs will result in DNA oligo hybridisation. A PCR reaction will elongate the double stranded DNA at both ends. The DNA product is detected and sequenced using NGS. The quantity of a specific DNA product correlates with the number of antibody binding events to the target protein.

NPX units

Readout from the Olink Explore assays and from the Olink Target 96 assays (further below) is provided in units of normalised protein expression (NPX). NPX is relative protein concentration unit and is on a log2 scale. NPX values from Explore assays are calculated from the number of matched counts (i.e. the number of sequence reads from a NovaSeq 6000 run). The data for each sample is normalised to internal controls or to reference samples within the same experiment, and the protein concentration provided is relative to a reference expression level.

Supporting Your Olink Explore Projects

We welcome projects of any scale at BioXpedia.

Our laboratory pipeline accommodates from 88 to 50,000+ samples. Contact us to hear about open slots and get a non-binding quote for your current project.

Olink Explore 3072

Adaptable Multiplex Proteomics

The Olink Explore 3072 is designed for high multiplex proteomics, with a total target list of 2900+ protein biomarkers. The assay uses PEA technology coupled to NGS readout. The Explore 3072 panel is made up of eight individual Explore 384 sub-panels. There is virtually no overlap between the panels; thus the subpanels are suitable for running individually or in combinations of up to eight panels. The Explore panels are divided into four broad categories representing selected themes of medical research. Two panels are dedicated to each theme:

The proteins included in the Olink Explore 3072 panel have been chosen with a particular focus on inflammation related proteins. actively secreted proteins, protein drug targets, organ-specific proteins, and discovery stage biomarkers

Olink Explore 3072 and HT

The Olink Explore HT and the Olink Explore 3072 are built with the same basic focus on likely biomarker proteins. However, the Olink Explore HT protein target repertoire is almost twice as large as that of the Explore 3072. In the illustration above, the Olink Explore 3072 assay is represented by the inner pie chart. The outer portion of the pie represents the expansion of coverage in each assay category with the introduction of Explore HT.

Charlotte Busch Ahler, Sr. Business Manager

Olink Target Panels

Validation and verification of protein biomarkers.

Our service includes more than just discovery of biomarkers. We will guide you through validation using a targeted approach, as well as integration with other omics, such as RNAomics and genomics.

Olink Target 96 Panels

Olink Target panels rely on PEA assay technology, same as the Explore panels. However, the detection technology is fundamentally different. In the Target panel series, multiplexing is achieved through microfluidics-based qPCR. The combined use of the qPCR and microfluidics allows for each single sample and target protein combination to be assayed separately

Like the Olink Explore 3072 assays, the Olink Target 96 panels cover various different areas of research. The list of Explore 96 assays can be found below:

Furthermore, a single Target 96 panel is dedicated to preclinical or translational research in mice: 

Target 48 Panels

The technology for the Target 48 panels closely resembles that of the Target 96 panels, except for a few key differences. Firstly, each Target 48 panel comprises 43 to 45 assays, all specifically targeting cytokines (small proteins important in cell signalling). One of the Target 48 panels is designed for mouse cytokines, and one is for human cytokines. Importantly, both the Target 48 assays offer the flexibility of providing readouts in either absolute concentration units (pg/mL) or NPX units. The details of each assay can be found in the documents linked below:

The lower limit of detection for the Target 48 assays is defined as three standard deviations above background and is reported in pg/mL. For the majority of the included assays, this value is below 1 pg/mL.

Olink Flex Panel

The Olink Proteomics Flex panel is a customisable product that allows researchers to select and combine protein targets from a list of human proteins. The assay allows measurement of 15-21 protein biomarkers simultaneously and provides readout in absolute units (pg/mL) or as normalised protein expression (NPX). Researchers can mix and match freely from a library of ~200 pre-validated protein biomarkers to create their own custom panel. The assay is designed to be highly flexible without compromising on performance. The list of available targets for building a custom Olink Flex panel can be found in the document below:

Sample Types

Olink Target panels are built for high throughput, cost efficiency, and low sample consumption. Each Target panel requires only 1 µL of sample. The assays have undergone validation for use with EDTA plasma and serum samples. Other sample matrices are compatible, please contact our team to hear about options. The Target assays have been used with success on dried blood spots and on skin tape strips.

Olink Target and Flex Workflow

Olink Target and Flex assays with PCR

Workflow of the Olink Target and Flex assays. Samples are first diluted to bring target concentration within the assay dynamic range. Each sample is combined with a pool of labelled antibodies. Matching antibodies in close proximity will result in DNA oligo hybridisation. A qPCR reaction will amplify double stranded DNA. Each qPCR reaction takes place in an individual nanolitre-size chamber on a microfluidic chip, so each sample and target pairing is assayed separately. Each chip accommodates 88 samples, each assayed for 92 biomarkers, resulting in 8092 datapoints.

Marie Holt, Project Manager

Protein Biomarkers in Mice

Biomarker screening in mice.

For mouse samples, we offer characterisation of up to 123 known mouse biomarkers using Olink Target 96 and Target 48 panels. Our in vivo facility supports this research with sample tissues.

Mouse Panels and In Vivo Facility

As described above, two of the Olink Target panels are specifically designed for mouse biomarker characterisation. These are the Target 96 Mouse Exploratory Panel and the Target 48 Mouse Cytokine Panel. The panels include assays for 92 and 43 proteins respectively. The files linked below give the details of each assay. In total 123 mouse proteins can be assayed with the Olink Target kits:

Taking advantage of our integrated in vivo facility, we are able to feed various mouse samples into our Olink assay pipeline. This means we can leverage our Olink biomarker expertise to support early toxicology studies in preclinical models. Conversely we can support biomarker discovery and validation with data and specimens from translational mouse models.

Frequently Asked Questions

Why is a special panel needed for mouse samples?

All PEA assays from Olink are based on antibody recognition, and since epitopes vary among species the assays have to be validated for each individual species. This is why the assays are considered species specific. There is a good chance that an assay will work with many related mammalian species, but only a thorough validation will guarantee it. Due to these circumstances, Olink has released two specific mouse panels. A number of the assays in the Mouse Exploratory Panel are validated specifically and exclusively on mouse samples, and make use of antibodies raised against mouse proteins.

Will Olink Explore HT work with my samples?

Olink explore HT is validated against samples of EDTA plasma and serum. The assays are guaranteed to work with these samples. This does not rule out compatibility with a range of other matrices. In fact many biofluids of human origin will produce good results. When considering an unusual sample matrix you are always welcome to reach out to our scientists. In many cases they will have access to yet unpublished data.

Can you provide me with a quote for my funding application?

Absolutely, we will be happy to. Our quotes are competitive, and they are also non-binding!