A
better germ-detecting mousetrap
— By Kevin T. Higgins, Senior Editor
A quick test designed for combat soldiers under attack from biological weapons could make life easier for food companies concerned about pathogens in their plants and products.
Lab mice, with the assistance
of biologists and Lincoln Laboratory engineers at Massachusetts Institute of
Technology, are providing biosensors that yield a fast, sensitive and specific
test for the presence of the most deadly pathogenic bacteria in food. And a
College Park, MD, firm is cranking up commercial production of those engineered
cells, based on B lymphocytes from mice, to deliver a rapid diagnostic system
for beef processors and other food companies. Known as CANARY™ (cellular
analysis and notification of antigen risks and yields), the technology grew
out of work begun in 1997 by Todd H. Rider, a biologist with Lincoln Lab’s
biosensor and molecular technologies group. Funding came from the Defense Advanced
Research Projects Agency, which wanted to equip US soldiers with a portable,
easy-to-use field test for biological warfare agents such as anthrax and smallpox.
Rider’s genius was to leverage white blood cells’ ability to detect
bacteria and viruses. When antibodies in the cell bind to a germ, they trigger
a calcium signal within seconds. Rider and his colleagues infused white blood
cells from a mouse with bioluminescent protein from a glowing jellyfish. The
protein glows in response to calcium, and that light becomes the signal noise
of detected bacteria and viruses. The white blood cells have been further engineered
to produce antibodies only to specific cells, rather than all foreign agents.
In a paper published in the July 2003 issue of Science magazine, researchers
reported detection of as few as 50 colony forming units (CFU) of Yersinia pestis,
the bacterium that causes plague, in less than three minutes. The probability
of detection for 200 CFU was 99%, with a false-positive rate of 0.4%.
In October 2003, Innovative Biosensors Inc. (IBI) licensed CANARY and related
hardware—a centrifuge, computer with analytical software and a luminometer
engineered by MIT staffers—with the intention of tapping the estimated
$500 million food pathogen testing market and the $3 billion health diagnostic
area. Spearheading the commercialization effort is Thomas Hazel, senior director
of research and development. Dr. Hazel joined IBI last summer. The Michigan
native was a graduate student at Northwestern University Hospital in Chicago
and received his doctorate in genetics from the University of Illinois College
of Medicine. He completed postdoctoral work at the National Institutes of Health.
Dr. Hazel has held senior research positions in molecular genetics and cell
structure. His areas of expertise include engineering of human cell lines and
the manufacture and storage of cell-culture based reagents. Food Engineering
recently spoke with him about CANARY and the rapid detection of food pathogens.
A researcher at Innovative
Biosensors’s lab prepares an assay for the firm’s rapid diagnostic
system. The company is targeting meat processors with an E. coli 0157:H7
test. Source:
IBI.
__________________________________________
Thomas
Hazel, senior director of R&D,
Innovative
Biosensors Inc., College Park, MD
FE: How does CANARY work?
Hazel: It’s a B cell-based detection system that is engineered
to take advantage of the human immune system’s ability to respond to pathogens.
Instead of secreting a general antibody to any bacteria or virus, these cells
are engineered to respond only to a specific strain. The antibodies are tethered
to the cell membrane and are engineered to express a calcium sensitive bioluminescent
protein. An enzymatic cascade is produced when the antibody comes in contact
with a specific pathogen, resulting in light emission.
The test protocol is very simple and does not require a high level of technical
expertise, since it was designed to have the capability to be conducted in the
field. A centrifuge is used to spin a sample’s cellular material to the
bottom of a test tube. Liquid is removed, the B cells are added, and the cells
are spun again to speed the reaction. The luminometer then detects the emitted
light. CANARY can detect as few as 50 colony-forming bacteria in three minutes.
In a complex medium like ground beef, we can detect less than 1,000 CFU.
If zero tolerance is required, an enriched culture has to be grown, which is
why ground beef processors typically hold finished product for 48 hours while
the culture is enriched at an outside lab. We can shorten the enrichment time
to one shift. The assay protocol is simple enough to be done in-house by non-scientists.
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FE: An E. coli load of 1,000 CFU would be extremely
unusual in finished goods.What practical use, then, is there for a five minute
protocol?
Hazel: It could be used for swabs of hides or sides of beef
on the line.You can clean carcasses until you’re blue in the face, but
as soon as you eviscerate the animal, there’s danger of E. coli
0157:H7 contamination from ingesta in the gut.The test’s broadest application,
though, will be the ability to reduce the enrichment time needed to detect to
the 1 CFU level.
____________________________________________
FE: What methods do laboratories currently use for pathogen
detection?
Hazel: There are a number of tests for finished goods. The
most sensitive is PCR, which stands for polymerase chain reaction. It is an
enzyme-driven test in which the DNA signature of the bacteria is amplified in
a test tube and detected in a machine. The enzyme strings together strands of
DNA to build a detectable sample. The drawback is that the process takes a matter
of hours, whereas our test can detect the targeted organism almost immediately.
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FE: How sensitive is your test?
Hazel: The number of false positives is very low relative to
PCR, and that is the test’s strength. We run samples in tandem, which
brings the percentage of false positives down below 0.3 percent. False negatives
also are a concern, of course, and although we don’t have any hard numbers
yet, we’re confident the ratio is very competitive with PCR.
____________________________________________
FE: E.
coli 0157:H7 is the most notorious food pathogen, but many more people
die and are sickened from Listeria monocytogenes. Is there a biosensor for Listeria?
Hazel: Although an antibody has not yet been engineered to
respond specifically to Listeria, we could generate a test within a matter of
months. It’s not a long process.
At this point, we’re still determining what the food industry’s
needs are. We’re focusing initially on E. coli 0157:H7 because
of the zero-tolerance policy established by the government and the relatively
high fatality rate. Additionally, the industry has validated antibodies for
0157:H7, so we can choose the best antibodies available and engineer the cell
line for the test.
____________________________________________
FE:
Are you limited to detecting a single pathogen, or can multiple targets of concern
be flagged?
Hazel: A range of bacteria, viruses or toxins can be detected
with the test, and they can be multiplexed in a single test. If the cell was
engineered to detect three pathogens, for example, and the test yields a positive
response, you know one of the three pathogens was present, but you wouldn’t
know which one. As a practical matter, multiplexing wouldn’t be appropriate
in food applications. It would be more appropriate for human health diagnostics.
____________________________________________
FE: What
was the focus of development efforts in 2004?
Hazel: To commercialize BioFlash™, we’ve
been scaling up production of the biosensor with a quality control process that
gives us confidence that the biosensors we send to end-users will perform within
allowable parameters. Even though it’s cell-based, it’s shipped
on dry ice to end-users as a diagnostic reagent that’s essentially ready
to use. The cells live a couple of weeks refrigerated and longer when frozen.
You simply thaw them, add to the sample and quantify luminescence.
____________________________________________
FE: System hardware includes a mini-centrifuge, a luminometer
and a computer with analytical software. Is this standard-fare lab equipment?
Hazel: Yes, it’s off-the-shelf lab equipment in the first
iteration. We’re looking at opportunities for offering an integrated unit
with all the necessary components and have developed a prototype. Other system
refinements also are being developed. For example, the software is calculating
the signal to noise ratio of the emitted light by the luminometer, and that
requires some interpretation. We hope to simplify it to a yes-no response.
____________________________________________
FE: Promotional brochures for IBI’s rapid diagnostic
system include the disclaimer, “For research only. Not for use in diagnostic
procedures.” What does that mean?
Hazel: It’s simply a classification that is required
for products that are not FDA approved. Approval is not a requirement for many
food-pathogen tests.
We are in the process of gaining certification from the AOAC (the Association
of Analytical Communities that validates analytical methods and lab QA programs
and services). It takes about six months, and there’s a cost to attaining
certification that the test performs as advertised. Other voluntary validation
tests are required in the EU and Japan, but for now, we’re focusing on
the US.