Soon after the 9-11 terrorist attacks in the U.S., letters containing anthrax spores were sent to several news media offices and democratic U.S. senators Tom Daschle and Patrick Leahy, killing five people and infecting 17 others. Now, more than seven years later, the FDA has completed a proof-of-concept study of a test designed to quickly and accurately detect the presence of even the smallest amount of the anthrax toxin.

"The FDA findings could form the basis of a test that allows earlier diagnosis of anthrax infection than currently possible," said Indira Hewlett, PhD, the senior author of the study and chief of the Laboratory of Molecular Virology, Office of Blood Research and Review, at the FDA's Center for Biologics Evaluation and Research (CBER). "The earlier those infected with anthrax can be treated, the better."

The proof-of-concept study developed by FDA researchers relies on a nanotechnology-based test platform built from tiny molecular-sized particles. According to the researchers, this assay, the europium nanoparticle-based immunoassay (ENIA), was able to detect the presence of a protein made by the anthrax bacteria known as protective antigen (PA). PA combines with another protein called lethal factor to form anthrax lethal factor toxin, the protein that enters cells and causes toxic effects.

The researchers showed that ENIA is capable of detecting PA in quantities that are 100 times lower than current tests, such as the enzyme-linked immunosorbent assay (ELISA). According to the scientists, both the ELISA and ENIA rely on antibodies that have an affinity for the anthrax protein of interest.

Hewlett told Medical Device Daily the ELISA format is good, but his team wanted to see if it could improve the detection of anthrax by incorporating nanoparticles into the assay.

"From our perspective, we're encouraged by the ability of nanotechnology to provide what we think are remarkable improvements to the existing assays," Hewlett said.

A proof-of-concept study is an initial investigation that aims to determine if a new scientific idea or concept holds promise for further development. A report on the results of this study appears in the March issue of Clinical and Vaccine Immunology.

Hewlett said this research has the potential for further development, and could be used in an emergency. Before the prototype assay can be commercialized, however, it will require further development, most likely via a technology transfer agreement, with a company that could create a device component.

"There is a device component to every assay," Hewlett said. "We are not actually set up to make products, these types of prototype devices or discoveries are what we call bench-to-bedside research; it certainly could be further developed through a technology transfer."

Hewlett also said this type of assay could be easily refined to emerge as a rapid test.

Anthrax is an infectious disease caused by the bacterium Bacillus anthracis, a bacteria that forms spores, or dormant cells, which can come to life under the right temperature, nutrients and other conditions to allow growth. Anthrax occurs in humans after exposure to an infected animal or infected animal tissue or when anthrax spores are used as a bioterrorist weapon as was the case in September 2001.

The FDA test is a modified version of ELISA, which is already commonly used to detect anthrax and other infections. The researchers call their new test europium nanoparticle-based immunoassay,' because atoms of europium are key to the assay's sensitivity.

The ENIA uses molecular spheres (called nanospheres) covered with thousands of light-emitting atoms of europium that emit light, which acts as a signal that PA is present. The CBER team further enhanced the signal by modifying the nanospheres so they held additional atoms of europium, making the test more sensitive.

The ENIA detected PA in 100% of samples of mouse plasma compared to 36.4% through ELISA.

Nanotechnology-based tests like the ENIA are rapidly emerging as convenient tools for a variety of laboratory uses, according to Shixing Tang, MD, PhD, a visiting associate scientist in the Laboratory of Molecular Virology, CBER. "ENIA has potential use in an emergency because its relatively simple design makes the technology adaptable to point-of-care uses," said Tang, the first author of the study.

According to the FDA, the researchers developed the ENIA for PA in response to the increased interest in the scientific community for new anthrax assays following the 2001 anthrax attack that killed five people.

Co-authors of the article, "Detection of Anthrax Toxin by an Ultrasensitive Immunoassay Using Europium Nanoparticles," include Jiangqin Zhao (CBER), Mahtab Moayeri, Zhaochun Chen, Haijing Hu, Robert Purcell, and Stephen Leppla (National Institute of Allergy and Infectious Diseases, National Institutes of Health), and Harri Harma (University of Turku, Finland).

Hewlett said his team is studying biomarkers of anthrax infections to improve the current assay. The research is limited, however. "With anthrax it is not easy to get specimens to validate the test."

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