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Russian Researchers Look to Commercialize New Lateral Flow Immunoassay for Heart Attack, Sepsis

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NEW YORK (360Dx) – A team of Russian scientists has developed a new assay that could be applied as a point-of-care diagnostic for indications as varied as acute myocardial infarction, sepsis, and pregnancy testing.

The immunochromatographic approach detects not only the presence of a substance in a sample, but its amount by visualizing its concentration using gold nanoparticles. The researchers described the use of nanogold labels to improve the sensitivity of lateral flow immunoassays in a paper in Nano-Micro Letters in December.

According to Alexander Osipov, a researcher at the National University of Science and Technology MISIS in Moscow, other tests that rely on immunochromatography typically rely on portable instruments to register the intensity of colors. At the same time, the use of such instruments slows the time to diagnosis.

Osipov's approach, in contrast, could be used at the point-of-care, such as an in ambulance or even at home, he said, and can produce a result in between five and 10 minutes. NUST MISIS is now looking to transfer the technology to interested partners for commercialization.

"We are now discussing the possibility to produce such tests with some Russian diagnostic companies and for the transfer of our technology," said Osipov. He noted that one potential partner, a Moscow-based firm, already produces qualitative, visual immunochromatographic tests for determining the concentrations of prostate specific antigen. "Of course, we are very interested in not only Russian but in international partners," he added.

"We have developed a simple, non-instrumental variant for quantitative immunochromatographic assay analysis, in which the concentration of the analyte is determined not by measuring the intensity of the test lines, but by the visual count of the number of test lines in [an] analytical zone of membrane," Osipov said. "The number of test lines gives one the opportunity to determine the amount of the analyte at different intervals of concentration."

Osipov, a senior fellow in the university's department for functional nanosystems and high temperature materials, has been working for several years on the platform, which combines the principles of qualitative and quantitative immunochromatography into a single test.

Osipov's team developed test strips that contain hybrid molecules labeled with gold nanoparticles or quantum dots of antibodies. Each test strip has a width of between 4 and 6 mm, and a length of about 7 cm, and consists of several porous membranes fixed on a plastic substrate and adjacent to each other.

A liquid stream containing the sample moves along these membranes by capillary action, he said. He noted that these porous membrane carriers contain reactive components immobilized in advance that allow for the detection of a particular compound in the mixture by staining an analytical test field zone, as a crossed line, of the membrane.

The higher the content of the targeted substance or marker in the sample, the greater the number of colored lines will appear. Therefore, the approach is not only qualitative, in that it answers whether or not a marker or substance is present, but also quantitative, allowing users to determine the concentration of the marker or substance in the sample.

The inventors have received a patent for the technology in Russia but have not achieved protection for it elsewhere. The patent, RU2510510C1, "Test system for semiquantitative immunochromatographic assay," describes a substrate overlapped with a test membrane that supports between two and 20 parallel test lines "formed transversally by the pre-application of … monoclonal antibodies to the test compound" where the antibody concentration "increases from line to line."

Such an approach lends itself to various applications, the most immediate of which is pregnancy testing, which already relies on immunochromatography but can currently only provide a "yes" or "no" answer. The new approach, given its ability to measure concentrations, can provide even more information.

"Using this approach to analyze human chorionic gonadotropin in urine, we can determine not only the presence but the stage of a pregnancy," he said. "For example, one line could equal one week of pregnancy, two lines could equal two weeks, et cetera."

Another application at hand is diagnosing acute myocardial infarction.

Osipov noted that fatty-acid-binding proteins are often monitored to diagnose heart attacks, since the concentration of FABPs in blood increases significantly when the myocardium is damaged. He said that by determining the presence and amount of FABP and other cardiac markers, such as troponin I, using its test, physicians should be able to "increase the efficiency of their diagnoses … and identify hidden forms of heart attacks."

Osipov said that his team has developed an immunochromatographic system for simultaneously determining the presence of FABP, linked to the early stages of heart attack, as well as troponin I, which is associated with its later stages.

One attribute of Osipov's technology is the use of nanosize gold particles or quantum dots in that conjugate with antibodies in different shapes, such as "nanoclouds," stars, popcorn, or flowers. The space created by such shapes on the test strip, allows a better quantification of a target substance in the sample.

"Depending on the conditions for making the test, it is possible to vary not only the number but also the intervals of determined concentrations," Osipov said. "The use of gold nanoparticles with different shapes … gives us the possibility to increase the sensitivity of the analysis and form the desired intervals of analyte concentrations," he said.

This particular feature is "fundamentally important" in the analysis of the markers for a number of deadly diseases, such as using procalcitonin to detect sepsis and bacterial infections, Osipov noted. "When the amount of procalcitonin corresponds to between 0.5 and 2.0 nanograms per milliliter, it's possible to detect human infection with viruses," he said. At the same time, experts believe that it is impossible to make a sepsis diagnosis based on such results, and typically wait six to 24 hours for a second test. If the level of procalcitonin is stable, then they rule out sepsis.

Osipov and colleagues have suggested a test format where multiple test lines enable the visualization of procalcitonin concentrations. For instance, no colored test lines would represent a concentration of less than 0.5 nanograms per milliliter, while results of 0.5 nanograms to 3 nanograms per milliliter would manifest as a single colored line, and results higher than 3 nanograms per milliliter would produce two colored lines.

"The level of this substance with a severe generalized parasitic, bacterial, or fungal infection grows very quickly, reaching high rates," said Osipov. "This is always confirmed by repeated analyses," he said. "If the test showed a concentration of the substance above 10 nanograms, this would indicate sepsis and reflect the patient's serious condition."

Osipov and colleagues described the use of gold nanoflowers and gold nanospheres as labels in a lateral flow immunoassay for the detection of procalcitonin in the journal Nano Hybrids and Composites in January 2017.

A number of sepsis tests that are marketed by some of the industry's biggest players and rely on procalcitonin became available for clinical use in 2017. BioMérieux, DiaZyme Laboratories, and Beckman Coulter Diagnostics all achieved US Food and Drug Administration clearance for such assays last year. BioMérieux's test, marketed as the Vidas BRAHMS PCT assay, is sold for use on its benchtop Vidas immunoassay system. Meanwhile, Roche and Thermo Fisher Scientific have joined the crowded market with tests of their own for sepsis diagnosis and management.

There also are several firms, such as DNAe, Qvella, and Biocartis, working on molecular tests for sepsis, while T2 Biosystems has FDA clearance for its T2Candida Panel and has filed for clearance of its T2 Bacteria Panel, and Accelerate Diagnostics has FDA clearance for its PhenoTest BC Kit for bloodstream infections.