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Japanese Researchers Develop One-Minute Bioluminescent COVID-19 Test


NEW YORK – Japanese researchers have developed a one-minute COVID-19 test leveraging a bioluminescent molecule found in some small crustaceans that glows when it reacts with the SARS-CoV-2 virus spike protein in a saliva sample.

In an article published in ACS Central Scienceresearchers found that Cypridina luciferin glows when it reacts with the wild-type SARS-CoV-2 spike protein but doesn't react with six human proteins found in saliva. They developed a bioluminescent assay that provides results in saliva without pretreatment and wrote that the assay could serve as a rapid and convenient test to complement laboratory-based RT-PCR testing for COVID-19.

Ryo Nishihara, senior researcher at the Health and Medical Research Institute within Japan's National Institute of Advanced Industrial Science and Technology, said by email that that the methods he and his colleagues developed could be used to create a "mix-and-read" test that could be used with commercially available handheld luminometers at the point-of-care. The researchers have pending patent applications for some of those techniques and are planning to reach out to potential partner companies for joint research that could lead to a commercial test.

Luciferin substrates produce an oxidative luminescent reaction in the presence of their corresponding luciferase enzymes. Luciferases are used as light-emitting reporters for the detection of gene expression, proteins, and cells through the glow emitted during that reaction, and researchers have long used firefly luciferase as a reporter for drug screening.

While bioluminescent diagnostic assays typically require genetic modification to incorporate luciferase genes into the test, Nishihara's team reported that the Cypridina luciferin substrate will react in the presence of the SARS-CoV-2 virus without modifications.

Nishihara noted that the Cypridina luciferin also reacted to the SARS-CoV-2 Omicron variant, but the luminescence of that reaction is relatively low, so developing a luciferin that is suitable for detection of the variant would be necessary.

He and his colleagues noted that prior research indicates the imidazopyrazinone-type (IPT) luciferins that are found in marine organisms can exhibit luminescence reactions in response to other proteins and biomolecules such as human alpha-1 acid glycoprotein, bovine serum albumin, and insulin. Nishara's team reported in 2020 in Bioconjugate Chemistry that a synthetic Cypridina luciferin, dubbed HuLumino1, was catalyzed by human serum albumin in serum samples but not by the other proteins tested, including bovine serum albumin.

In the ACS Central Science article published this month, the team wrote, "This discovery suggested that protein assays based on the 'pseudoluciferase activity' of the targeted protein itself may potentially serve as a novel platform for protein analysis and alternative luminescence assays that do not rely on luciferase derived from luminous organisms."

Because prior research suggested that the luminescence reaction of IPT luciferins required a hydrophobic pocket for an oxidative reaction, Nishihara's team hypothesized that they could catalyze such a reaction by finding a luciferin that fit into the hydrophobic pockets of the SARS-CoV-2 spike protein.

The team investigated 36 IPT luciferins and found that only the IPT luciferin from crustaceans of the genus Cypridina glowed in a pseudo-luciferase reaction, or biomolecule-catalyzing chemiluminescence (BCL), with the SARS-CoV-2 spike protein.

Nishihara said Cypridina luciferin likely emits light when catalyzed by a structure unique to the SARS-associated coronavirus spike protein that helps the virus enter human cells using the receptor-binding domain. The light from the reaction could be detected using a commercial luminescence reading instrument, but it was not visible to the naked eye.

The team also tested whether similar coronaviruses would catalyze reactions in the Cypridina luciferase and found that the substrate molecule had a slight reaction to the SARS-CoV spike protein but no response to the MERS-CoV spike protein. The team is now developing luciferin analogs with a different chemical structure that could improve on the specificity of the Cypridina luciferin and distinguish between SARS-CoV-2 and SARS-CoV, Nishihara said but added that the researchers had not tested their method for reactions with other viruses, such as influenza and respiratory syncytial viruses.

Nishihara said the research team hopes to develop the technology into diagnostic tests for use in Japanese and US markets, but it's not yet clear where else they could commercialize those tests.

Maarten Merkx, a professor of biomedical engineering at Eindhoven University of Technology in the Netherlands, said the authors of the ACS Central Science article seemed to demonstrate well that Cypridina luciferin is reacting to the SARS-CoV-2 virus with good specificity and that the assay produces measurable luminescence more quickly than a lateral flow or PCR assay.

Merks has also been developing infectious disease testing methods that employ bioluminescent proteins, and he was not connected with the study by Nishihara's team.

He also said the article describes well how the SARS-CoV-2 virus spike protein appears to have the right structure to catalyze a luminescent reaction from the luciferin substrate.

However, he said the test falls short of the sensitivity that he would expect in comparison with a commercial test that includes virus amplification steps. A good ELISA, for example, has greater sensitivity by three orders of magnitude, he said, and he would expect an improvement of at least two if not three orders of magnitude to produce a commercial test that would be useful for disease detection.

"It's not easy to foresee how they could really improve that sensitivity by more than one order of magnitude," he said.

While he said it was already surprising that the researchers were able to achieve such sensitivity, he thinks that tweaking the luciferin to improve the efficiency of its oxidation when catalyzed by the spike protein would be difficult.

Nishihara's team found that the Cypridina luciferin produced luminescence in response to the SARS-CoV-2 spike protein in 10 percent human saliva as well as in buffer, with a linear response to spike protein concentrations in the range of 2.5 μg/mL to 30 μg/mL. The test had a limit of detection of 2.1 nM in buffer and 2.2 nM in the saliva test. The assay also could detect the spike protein in 50 percent human saliva without any observed inhibition by the saliva components.

Nishihara said the limit of detection value of the assay is comparable to that of assays with spike protein-binding materials, including lateral flow assays with sialic acid and aptamer-linked immobilized sorbent assays with the aptamer. While he acknowledged that some lateral flow assays can exhibit an LOD value that is orders of magnitude lower, he said that preparing perfectly matched paired antibodies to the spike protein is time-consuming and costly, and the time to receive results from an antibody-based lateral flow assay is higher, typically about 16 minutes.

Nishihara thinks the luciferin-based spike protein-based detection method may find use as a SARS-CoV-2 test that is cheaper and faster than existing antibody-based assays. It's difficult to estimate the costs of a commercial kit, he said, but his team found in its testing that the costs per test were miniscule — about $.0012 per assay.