NEW YORK – McGill University spinout Nplex is targeting an early 2021 launch for its highly multiplexed protein immunoassay system.
The company last week announced that it received a $1 million grant through the Canadian biopharma research consortium CQDM to fund development of the system, with McGill, the Québec government, and drugmaker GlaxoSmithKline funding the award. Nplex is also testing the system, which it calls nELISA, in work with GSK and the Montreal Neurological Institute.
Based on technology developed in the lab of David Juncker, professor of biomedical engineering at McGill, nELISA uses a miniaturized, bead-based-ELISA format to multiplex hundreds to thousands of immunoassays rapidly and without the problem of antibody cross-reactivity, said Milad Dagher, the company's CEO and co-founder, and former postdoc in Juncker's lab.
"What we have done is essentially miniaturized the classic ELISA technology," Dagher said. "And by miniaturizing it we have been able to scale it up in a very high-content, high-throughput manner."
In addition to miniaturizing the format, the company has changed the way the antibodies used for protein detection are applied, which is key to eliminating cross-reactivity. A traditional sandwich ELISA uses a capture antibody bound to some sort of surface and a detection antibody that can be made to produce a signal indicating the presence of the target protein. In this system, the capture antibody is incubated with the sample of interest, binding to any target protein present, and then the detection antibody is added. In some cases, these detection antibodies can cross-react with proteins other than their target, producing a signal that is non-specific for the protein they are meant to detect. This issue becomes more challenging as the number of ELISAs multiplexed together increases, which has limited the multiplexing capabilities of the approach.
Nplex has tackled this problem by preassembling the capture and detection antibodies so that they are localized together before the sample is added. This way the detection antibody never has the opportunity to bind non-specifically.
"We have color-coded beads and we have preassembled antibody pairs on every bead," Dagher said. "So each bead contains all the reagents you need for an assay."
The two antibodies are bound together using a DNA linker. When a sample is added, the capture antibody and detection antibody both bind to their target protein if it is present, forming a complex of the three molecules. On the other hand, if the target protein is not present, the capture and detection antibodies remain apart. A user can then unbind the DNA linker connecting the two proteins. Detection antibodies unbound to a target protein can then be washed away while detection antibodies bound to a target protein will remain.
This reaction can be done in a microtiter plate containing thousands of different beads each hosting a different nELISA reaction.
"And because the reagents don't interact, you maintain the performance and the specificity that a single-plex ELISA has always provided," Dagher said.
Each bead is fluorescently barcoded according to the nELISA it hosts and those barcodes are read using a flow cytometer. The company is using a barcoding approach based on multicolor Förster resonance energy transfer (mFRET) that Dagher, Juncker, and colleagues detailed in a 2018 paper in Nature Nanotechnology. Dagher said that nplex recently demonstrated it could manage more than 2,000 barcodes on a three-laser flow cytometer.
"In terms of multiplexing, we are really only limited by the barcoding capability and the availability of the reagents," he said, noting that there are currently around 2,500 ELISA antibody pairs on the market and as many as 5,000 to 10,000 if you use polyclonal antibodies.
"So there is quite a bit of content that we can onboard," he said.
Dagher said that nplex is currently building a 150-plex cytokine panel focused on the cell-based screening market and that it plans to steadily expand the assays available on the platform based on customer feedback and demand for new targets. He added that the company expects to have a panel with assays to more than 1,000 proteins in less than two years.
While the method offers a much higher level of multiplexing than is possible using traditional ELISA, it falls well short of platforms like like SomaLogic's Somascan and Olink's proximity extension assay system. Somascan currently measures around 5,000 proteins, and Olink is planning to expand its assay from its current count of around 1,500 proteins to 4,500 proteins by 2022.
Dagher said he believes that nplex would compete favorably with existing options on throughput and cost. He said that a lab could run between 1,000 and 2,000 samples per day using the nELISA approach and that nplex is currently running around 1,000 a day out of its lab.
Nplex is currently focused primarily on the pharma cell screening market, Dagher said, noting that the flexibility of the system allows it to be applied at several points in the drug screening process.
"They can use our platform all the way from secondary screens running [for example] 100,000 compounds, all the way to hit-to-lead optimization that they are doing at a lower throughput or more regular basis," he said. "So we can hit several applications there with a single panel."
As nplex scales up its content it also plans to tackle the plasma proteome discovery market, he said. "As we scale up our plex from 150 to 500 in the coming year, we are going to be offering our platform as more of a discovery service, running plasma assays at high throughput and enabling customers to do really big and well-powered studies."
The company has raised funds in a pre-seed round with investors including Real Ventures, First Star Ventures and 2048 Ventures, though Dagher declined to say how much it has raised. It has fewer than 10 employees, he said.