 Markus Kalkum and Karine Bagramyan |
City of Hope scientists have developed an assay that could protect the public from a bioterrorist attack as well as improve food safety.
Markus Kalkum, Ph.D., assistant professor in the Division of Immunology, and Karine Bagramyan, Ph.D., a research fellow in the division, have devised a new method for detecting botulinum toxin, the substance responsible for botulism, in an array of substances including food products. Findings were published in the April 30 issue of PLoS ONE.
Botulinum toxin is produced by the bacterium Clostridium botulinum. When ingested, the toxin disables nerve function, resulting in paralysis and even death.
Botulism normally results when a person eats food tainted with C. botulinum bacteria or if a wound is infected by the bacterium. Infants, whose digestive systems are not yet fully developed, also are susceptible to the disease if the bacterium gains a foothold in their intestinal tract.
In addition, government agencies indicate that botulinum toxin may be a tool in a potential terrorist attack. The poison is so potent, just one gram could kill more than a million people, according to a 2001 study in the Journal of the American Medical Association.
The new assay developed by Kalkum and Bagramyan is less expensive, faster and easier to perform than the current standard, and it does not require the use of mice for testing, unlike the current standard. It also is far more sensitive.
“We can detect about 300 molecules [of toxin] in a one milliliter sample volume,”
making the test 12,000 times more sensitive than the standard, said Kalkum.
Added Bagramyan: “There are other assays that are similarly sensitive, to those use deionized water or buffers. In contrast, our assay can use virtually any solution,” including milk and juices, making the test more effective and flexible.
To create the assay, the researchers bound antibodies against botulinum toxin to microscopic beads. Scientists can test a solution by mixing the beads into it. The antibodies grab any toxin molecules floating about. The beads are then washed and exposed to photoreactive chemicals. The chemicals glow under ultraviolet light when the botulinum toxin is present.
The scientists tested the assay by mixing controlled amounts of botulinum toxin in a variety of substances, including milk, gelatin solutions and carrot juice. Kalkum and Bagramyan also successfully tested the assay using blood serum. That is
encouraging because they want to adapt the technology to test transplant and other immune-compromised patients for possible fungal infections such as aspergillosis.
Research indicates the risk of death three months after a diagnosis of aspergillus infection in a bone marrow transplant patient is extremely high, ranging from 54 percent to 85 percent depending on the type of transplant.
 The new assay uses antibodies bound to microscopic beads to capture botulinum toxin. Special molecules react to the captured toxin and become flourescent. |
“I see no reason why we can’t adapt this technology to benefit transplant patients,” said Kalkum. The mechanism would be very similar, “and we’ve already been able to detect other biomolecules associated with fungal infection,” he said.
City of Hope’s DNA/RNA & Peptide Synthesis Lab, under the direction of Bruce Kaplan, Ph.D., is creating novel molecules to help advance these new assay applications.
In addition, the assay could be used to better understand how the human body reacts to and metabolizes medicines such as Botox, making use of the cosmetic drugs safer, according to Kalkum.
The research was funded under the auspices of the Pacific Southwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases, one of 10 nationally funded centers that support the National Institute of Allergy and Infectious Diseases’ Biodefense and Emerging Infectious Diseases Research Agenda. The center recently announced a program to support research on an automated device that uses the new assay.
Jason R. Barash, C.L.S., M.T., and Stephen S. Arnon, M.D., of the California Department of Public Health’s Infant Botulism Treatment and Prevention Program, collaborated on the research.
