Scientists develop rapid-fire tests for botulinum toxin

Scientists at the University of Wisconsin-Madison have developed a pair of rapid-fire tests for botulinum toxin, a feat that could underpin new technologies to thwart bioterrorism and spur the development of agents to blunt the toxic action of the world's most poisonous substance.

Writing this week in the Proceedings of the National Academy of Sciences (PNAS), the Wisconsin group, led by UW-Madison physiologist Edwin R. Chapman, describes the development of two assays for botulinum toxin - one a real-time test - that vastly improve on current technologies to detect the deadly poison.

"We needed a real-time assay," says Chapman, suggesting that the technology could potentially be deployed to protect the food supply, soldiers on the battlefield or used by emergency responders dealing with an unknown agent. "The old test takes days."

In addition to the real-time assay, which could be deployed in a kit and used in the field, the Wisconsin team also developed a cell-based assay that helps provide a glimpse of the toxin doing its dirty work in living cells. This technology promises a rapid screen for millions of chemicals to see which might inhibit the paralyzing effects of the toxin, according to Min Dong, a UW-Madison postdoctoral fellow and the lead author of the PNAS report.

"The primary application is to conduct cell-based, large-scale screening for toxin inhibitors," Dong says. "A cell-based assay has the potential to reveal molecules that may inhibit various toxin action pathways."

Botulinum toxin is made by a bacterium that causes food poisoning. The poison is the most toxic substance known - 6 million times more potent than rattlesnake venom. It works by binding to nerve endings. The toxin is taken up by the nerves, where it blocks chemical signals from reaching muscles. With enough blocked nerve endings, the toxin can cause paralysis and death.

In recent years, the nerve toxin has been used therapeutically to treat nerve disorders and help calm the muscles of cerebral palsy and stroke patients. It is best known to the public by the trade name Botox, which, in minute doses, is widely used in cosmetic procedures to smooth frown lines and wrinkles.

Last year, Chapman's group identified the mechanism by which the toxin enters cells. Inside the cell, the toxin targets three key proteins, which are essential for mediating the release of chemical signals from neurons and that govern how messages are sent from brain to muscles.

"The toxins are smart," Chapman notes. "They know where to go" inside cells to do the most damage.

The newest work, says Chapman, helps give scientists an inside-the-cell view of the toxin at work. The toxin employs a four-step process - from cell entry to blocking the release of chemical messengers from nerve endings - and interfering with any of the steps in the process can inhibit the poison's toxic action.

"We can screen for [agents] to block any one of those steps," explains Chapman. "We could screen 1 million drugs at a time, and you can do all the screening using live cells."

The potential upshot of such a screening technology could be the development of drugs that act like a prophylactic to confer protection from botulinum poisoning.

The new tests, according to Chapman, can be conducted with ordinary lab equipment. They work by introducing into cells bioluminescent proteins whose glow is extinguished in the presence of the toxin. The tests are capable of detecting all seven variants of the poison.

Currently, the most sensitive and common test for toxin activity is exposing mice to an agent. The process takes time, and many animals are used and die in the process.

The Wisconsin Alumni Research Foundation has patented the new botulinum toxin technology. In addition to Chapman and Dong, co-authors of the new PNAS paper include William H. Tepp and Eric A. Johnson, both of the UW-Madison department of food microbiology and toxicology.