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Finnish Researchers Leveraging Fluorescence to Develop Fast, High-Volume SARS-CoV-2 Antigen Test


NEW YORK ─ Researchers at the University of Helsinki are developing technology that they believe could make it possible to test up to 500 samples in one hour using one computer-sized instrument.

The group has developed a proof-of-concept antigen test that leverages time-resolved fluorescence resonance energy transfer, or TR-FRET, involving the transfer of energy between two light-sensitive molecules, or fluorophores.

The performance of a prototype device described in a recent study published in mBio demonstrates the potential to develop a commercial SARS-CoV-2 test using TR-FRET technology, said Jussi Hepojoki, who is leading the development of the test and is an Academy of Finland research fellow at the University of Helsinki.

"In past studies, we had already demonstrated the use of TR-FRET to detect antibodies in clinical samples for different medical conditions, including antibodies against SARS-CoV-2, but this was our first study for antigen detection as an indicator of active disease," Hepojoki said.

The group plans to commercialize a test the size of a benchtop computer for deployment in small clinical laboratories and to enable the completion of up to 4,000 antigen tests in an eight-hour shift, Hepojoki said, but for that to happen the researchers will need to attract interest and investment from a diagnostic company to help them bring the test to market.

TR-FRET involves the transfer of energy between fluorophores when they are in close proximity — about 1 to 5 nanometers from each other. Fluorescence resonance energy transfer is also called Förster resonance energy transfer, named after Theodor Förster, a German scientist, for his contribution to the field.

The University of Helsinki's TR-FRET assay uses donor- and acceptor-labeled polyclonal rabbit antibodies that bind to SARS-CoV-2 nucleoproteins and spike proteins. When a sample is positive, polyclonal antibodies that are connected to donor fluorophores move close to polyclonal antibodies connected to acceptor fluorophores, and both versions of the antibodies bind to SARS-CoV-2 nucleoproteins or spike proteins in the sample.

The close proximity of the fluorophores enables a transfer of energy from the acceptor to the donor, and the resulting change in the TR-FRET signal is measured by the assay. The test system uses ultraviolet light for fluorophore excitation and measures an emitted signal after a time delay, which is implemented to remove background noise from biological matter in the sample.

The technique "is both quick and high throughput and could be developed for use in point-of-care settings," said Kristina Hotakainen, an associate professor at the University of Helsinki and director of laboratory services at Finland-based Mehiläinen, which operates a central laboratory in Helsinki and approximately 75 outpatient clinics that provide point-of-care testing among other medical services.

Hotakainen, who is also a physician, has not been involved in the development of the diagnostic test but plans to collaborate with Hepojoki on its future development. 

From a laboratorian's perspective, the TR-FRET test does not require extensively trained personnel or sophisticated equipment, and it has the potential to be deployed in field settings such as for drive-in testing at the point of care, Hotakainen said.

In the mBio study, the University of Helsinki researchers sought to demonstrate the viability of using TR-FRET to detect SARS-CoV-2 in 48 clinical nasopharyngeal swab specimens that were positive for the virus and 96 samples that were negative by RT-PCR.

The TR-FRET assay detected 37 out of 38 specimens deemed positive by RT-PCR and from which the researchers also isolated SARS-CoV-2 using cell culturing.

For the 10 remaining samples that tested positive by RT-PCR, the researchers were unable to isolate the virus, and none of the samples yielded a positive result using the researchers' antigen test. Hepojoki said the most likely cause of this was the detection of remnant nucleic acids by RT-PCR, reflecting the presence of a virus that was no longer contagious, while the new method detected the virus at contagious levels.

Hotakainen said that when the antigen test is positive and the virus can also be isolated and cultivated, "the test explicitly detects viable viruses, not remnants of nucleic acids," which can happen with RT-PCR testing.

When a positive result is confirmed by cultivating the virus it "is also likely to be a sign of contagious disease, which is important to detect quickly in order to prevent spreading," she added.

Overall, the University of Helsinki test's sensitivity was 97.4 percent and its specificity was 100 percent compared to viral cultivation.

The group believes that its technology could be developed to diagnose infections other than SARS-CoV-2. In internal studies, the researchers previously applied TR-FRET for the rapid detection of SARS-CoV-2 antibodies and have shown that it can he used to detect Zika and hantaviruses as well as autoantibodies indicative of celiac disease, Hepojoki said.

If a SARS-CoV-2 commercial test can be developed and brought to market, a laboratory instrument could be made available for $5,000, or less, Hepojoki said, adding, "Reagent costs would be in the range of 10 cents per reaction."

However, getting a test to market may be its biggest challenge. It would require a collaboration with a diagnostic company interested in working with the University of Helsinki group and able to design, develop, and manufacture an instrument, as well as help it take the test through clinical validation and regulatory approvals, Hepojoki said.

If that materializes, the group could spin out a startup company to produce the test's reagents, and develop dried reagents that would enable easier storage and distribution, he said, adding, "An instrument similar to the one we use in the lab — which is about the size of a tabletop computer — could very easily be built."

However, a timeline for commercialization is highly uncertain and largely dependent on the group being able to collaborate with a diagnostic industry partner, Hepojoki added.