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With 96-plex Proseek Assay Optimized, Olink Now Aiming to Develop 384-plex Version

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NEW YORK (GenomeWeb) – Researchers from Olink Biosciences have completed an optimization study of the company's Proseek protein assay, demonstrating the feasibility of performing the assay in a 96-plex format while achieving significant improvements in precision.

Having successfully optimized the 96-plex version of the assay, Olink is now aiming to expand to a 384-plex version, President and CEO Simon Fredriksson told ProteoMonitor this week.

The company detailed its optimization of the assay in a paper published last month in PLOS One. Proseek is based on Olink's proximity elongation assay technology, which uses pairs of antibodies attached to unique DNA sequences to detect proteins of interest. When the antibodies bind their targets, the attached DNA strands are brought into proximity and ligate, forming a new DNA amplicon that can then be quantified using real-time PCR. The quantity of the DNA corresponds to the quantity of the target protein.

Olink began selling Proseek in a single-plex format in 2011, and in March 2013 it launched the 96-plex version described in the PLOS One paper. In August, the company signed a co-marketing deal with Fluidigm under which it offers the 96-plex assay on Fluidigm's BioMark HD real-time PCR platform, which allows researchers to measure in a single run the levels of up to 92 proteins in as many as 96 samples.

With development of the 96-plex Proseek, Olink has moved into the protein biomarker discovery panel business, launching in the last year two 92-biomarker panels – one aimed at oncology and the other at cardiovascular disease. Within the next several months, the company plans to launch a third panel, this one targeted at inflammation, Fredriksson said.

He declined to provide information on revenues from the 96-plex Proseek assay, but said that in the 14 months since it launched, "there have been around 15,000 samples run using the technology."

Demand has come primarily from clinical and translational researchers, typically those working in an academic setting with access to biobanked clinical samples, he said, adding that pharma companies "with an agenda in the biomarker discovery area" have also been among the product's early adopters.

Clinical research is a particularly promising space for the assay, Fredriksson noted, due to its low sample requirements. While biomarker discovery panels like those offered by Myriad RBM require 50 μL and 750 μL of sample, depending on the panel, the Proseek 96-plex requires 1 μL, which, he said, allows it to use a variety of sources ill-suited to more sample-intensive methods.

"It opens up a lot of new avenues in terms of [using] dried blood spots, xenograft mice, tissue lysates, fine needle biopsies, and so on," he said. Dried blood spots, in particular, have lately become an area of interest within proteomics with a variety of researchers touting their potential as a convenient, stable format for collecting and storing samples.

They could prove particularly useful for purposes like in-home testing and monitoring, Fredriksson noted. "If you want to get into products that [for instance] enable wellness monitoring in the home, home sampling is going to be an issue," he said. "And dried blood spots are the only solution I know of where a person can take a sample, put it in an envelope, and send it to a central lab for the analysis."

"I'm quite certain that in the future dried blood spots will be part of the biomarker discovery and diagnostics area, driven by technology that enables highly multiplexed assays in small sample volumes," he added.

Given the potential for denaturation of proteins in dried blood spot samples, there are some concerns regarding the effectiveness of antibody-based methods for measuring proteins collected in this format. However, Fredriksson said, the company has thus far found such samples "to be surprisingly stable even across a large range of protein analytes."

He noted, as well, the high levels of preanalytical variability associated with conventional sample collection methods.

"Preanalytical variability is a huge issue in diagnostics, and it really precludes certain analytes from being analyzed," he said. "You handle these [collection] tubes in many different ways — some are frozen, some are at room temperature, some are shipped to central labs — it's a big mess. So, if we can standardize that with the dried blood spot procedure, that will really enable new diagnostic avenues in the future."

As for the present, however, the approach's scope for protein biomarker researcher is limited due to the lack of biobank samples collected this way, he said.

In the PLOS One study detailing development of the 96-plex Proseek assay, Olink researchers adjusted a variety of components to maintain precision and specificity at the higher level of multiplexing.

Among the most important changes to the assay, Fredriksson said, was the replacement of the T4 DNA polymerase used in the initial version of the assay to unite the two PEA probes with a different enzyme, Pwo Hypernova, that is inactive at room temperature. This change allowed for all the reagents required for the assay to be added in a single pipetting step at room temperature, without creating background due to unwanted DNA polymerase activity.

Streamlining the assay to a single pipetting step helped increase its precision, Fredriksson said. Also key to improving precision was modifying the proximity probe oligonucleotides to work with universal PCR primers as opposed to using multiple different primer pairs. This, the authors noted, reduced bias associated with differential amplification of different primers and potential saturation of primers to particularly high abundant analytes.

Intraplate coefficients of variation for the 96-plex Proseek assay are all below 10 percent, with interplate CVs all coming in below 20 percent, Fredriksson said. This, he said, represents a more than two-fold improvement in precision compared to the previous version of the assay.

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