[LABORATORY]
by Linda L. Leake
Food Allergen Test Kits
T
he prevalence of food allergies worldwide
has nearly doubled during the past 20 years,
doctors and scientists observe. Approximately
4% of the U.S. population is believed to be suffering
from one or more food allergies, according to
Wesley Burks, Chief of Pediatric Allergy and
Immunology at Duke University Medical Center,
Durham, N.C. (wesley.burks@duke.edu). With the
current population estimated by the U.S. Census
Despite stringent cleaning protocols embraced by manufacturing facilities,
there are constant industry concerns about contamination with trace amounts of food allergens.
Bureau to be 299,682,000, that means that nearly
12 million people suffer from food allergies.
The number of individuals affected by this dis-
ease is virtually immeasurable when family, friends,
caregivers, associates, and health care providers
of sufferers, along with food allergy researchers,
foodservice providers of various types, and even
all the players in the entire food industry from
farm to fork are taken into account, Burks says.
Eight foods or food groups are thought to
pg 68 10.06 www.ift.org
The Alert test kit is a screening test that compare up to five
samples at a time against a known level of allergen.
Photo courtesy of Neogen Corp.
account for more than 90% of all immunoglobulin
Emediated (true) food allergies. Known as the
Big Eight, they are milk, eggs, finfish, crustacean
shellfish, peanuts, soybeans, tree nuts, and wheat.
The foods most likely to cause anaphylaxis,
the most severe form of allergic reaction,
are peanuts, tree nuts, and shellfish.
Almost any food has the potential to
trigger an allergy, Burks emphasizes. More
than 160 other foods have been documented
as causing food allergies less frequently.
The only way to avoid experiencing allergic
reactions is to completely avoid the offending
foods. Knowing that some consumers have
food allergies can complicate commercial food
production significantly. Despite stringent cleaning
protocols embraced by manufacturing facilities,
there are constant industry concerns about con-
tamination with trace amounts of food allergens.
Manufacturers are responsible for fol-
lowing the requirements of the Food Allergen
Labeling and Consumer Protection Act of 2004
(FALCPA), which became effective on January
1, 2006. The bill requires food manufacturers
to state clearly if a product contains any
of the Big Eight foods/food groups.
Domestic or imported packaged foods that are
regulated by the Food and Drug Administra-
tion are required to make changes to their
labels that list the type of allergene.g., the
type of tree nut (almond, walnut) or the type of
crustacean shellfish (crab, shrimp)as well
as any ingredient that contains a protein from
the group of eight major food allergens. The
labels also must include any allergens found
in flavorings, colorings, or other additives.
Use of Test Kits Increases
Food manufacturers are using commercial
enzyme-linked immunoassay (ELISA) tests more
than ever to ensure the absence of undeclared
allergens in food and to validate allergen control
measures, says Tong-Jen Fu, an FDA research
scientist at the National Center for Food Safety
and Technology, Div. of Food Processing and

Decagon
Packaging, Summit, Ill. (tong.fu@fda.hhs.gov).
In fact, according to the Food Allergy Research
& Resource Program (FARRP) at the University
of Nebraska, Lincoln, lawyers who specialize in
food law consider using allergen residue test kits
to be the standard of care in the United States.
Many of these tests are designed to allow
quantitative determination of food allergens

1/2 ISL 4C
where the presence of the target protein is
detected by binding with specific antibodies, Fu
points out. The concentration of the antigens
is then determined from a standard curve
generated with reference protein standards.
Because quantitation is achieved via
measurement of antigenicity, any changes in
the antigenic property of the target proteins
may greatly influence assay results, she
says. For example, thermal processing
can change the antigenic property.
Heat often affects the solubility and immunore-
activity of proteins, but how the thermal treatment
of food affects the quantitation of allergens by
ELISA test kits remains to be determined, she says.
In a recent study, Fu compared the
performance of two major types of test kits,
one that employs antibodies that are reactive to
all protein components in allergenic foods and
one that uses antibodies that are specific for a
single allergenic compound or marker protein.
Both kits were used to detect egg that had
been subjected to moist or dry heat. Elevated heat
treatment resulted in a lower level of proteins
being extracted. While the decrease in the amount
of extractable protein in dry-heated samples
was generally reflected in the readings observed
using both test kits, the change in the amount of
extractable proteins in boiled samples was not
proportionately indicated by either test kit.
The test kit that was reactive to all protein
components in egg underestimated the amount

10.06 www.ift.org 69 pg
[LABORATORY]
Food Allergen Test Kits continued...
The Aller-tect swab test is used for the of residual protein in
detection of protein residues. boiled samples. The kit
Photo courtesy of Biotrace International PLC
that employed antibodies
specific to a heat-stable
marker protein (ovomucoid)
overestimated the amount
of protein remaining.
These results suggest
that heat-induced changes
in the solubility and
structure of proteins need
to be considered when com-
mercial ELISA test kits are
used for the quantification
of allergens in thermally
processed foods, Fu says.
Besides providing
food manufacturers with
a useful tool for testing
ingredients, food allergen
test kits have other uses.
Manufacturers of Food We need to be able
Allergen Test Kits to accurately and reliably
detect food allergens from
the perspective of what the
T
he following is a list of some consumer may purchase or
companies that manufacture food eat, says Eric A.E. Garber,
allergen test kits that are not Lead for Food Allergen
already mentioned in this article. Methods at FDAs Center
Biotrace International PLC, for Food Safety and Applied
Bothell, Wash. (phone 425-398-7993, Nutrition, College Park, Md.
www.biotraceamericas.com) (eric.garber@fda.hhs.gov).
r-Biopharm Inc., Marshall, For that reason, there is
Mich. (phone 877-789-3033, a need to conduct detailed
www.r-biopharm.com) evaluations of the test
SafePath Laboratories LLC, kits with the ultimate goal
Carlsbad, Calif. (phone 760-929- being to have validated
7744, www.safepath.com) methods, Garber explains.
Tepnel BioSystems, Stamford, Conn. Through validation, the
(phone 802-849-2948, www.tepnel.com) results obtained using a test
kit are defined relative to a
pg 70 10.06 www.ift.org
recognized reference mate-
rial and the performance
characterized statistically.
If you use different brands
of test kits for the same
allergen with the same
sample, you should obtain
the same result at a defined
level of confidence. Thats
the goal of validation.
Ongoing Research Collaboration
To address the critical need
for validated methods for the
detection of food allergens
in food products, research
scientists from FDA, Health
Canada, FARRP, the Food
Products Association, and
the European Commissions
Institute for Reference
Materials and Measure-
ments began in 2005 a
collaboration to maximize
the use of available scientific
expertise and laboratories
for validation trials.
By employing the
best science possible and
working with the test kit
manufacturers, potential
problems are identified and
troubleshot, and the test kits
validated, Garber relates.
The collaborating research-
ers want to facilitate the
successful validation of
test kits. This provides
more analytical tools for
any user concerned with
detecting food allergens.
For their initial project,
the group tackled the
validation of egg allergen
detection test kits. This
required three phases.
First, the five col-
laborating laboratories
evaluated the performance
of six different test kits with
two potential reference
materials, Garber says.
Based on the results
obtained, a spray-dried,
whole egg powder was
chosen to be the reference
material for this study. This
material has been deposited
with the National Institute of
Standards and Technology
(NIST) as a reference
material and represents the
first NIST reference material
specifically designed for
food allergen detection.
Currently, in the second
phase, test kits are being
evaluated with food samples
representative of those
that will be used in the
multi-laboratory validation.
Finally, in the third
phase, after identifying and
sharing the results with the
test kit manufacturers, a
multi-laboratory validation
will be conducted at the 95%
confidence level for four
matrices containing egg at
three concentrations (one
concentration is 0 and two
other concentrations, one
representing a target level
and the second encompass-
ing the calibration curve),
using a minimum of 10
laboratories. The validation
study will also examine the
specificity/cross-reactivity

of the test kits with other foods.
In addition, FDA scientists
are conducting extensive in-house
evaluations of commercial test kits,
including those for milk, wheat/glu-
ten, egg, and soy, Garber mentions.
FDA scientists also examined
the effects of various forms of
preparation, such as baking, boiling,
and freezing, that represent the
products the consumer may acquire
and affect the utility of the test
kits. By including proper controls
and foods that have undergone
preparation in the evaluation and
validation studies, the utility of the
test kits for detecting and quantify-
ing food allergens is understood.
An Important Tool
Overall, manufacturers have
done an excellent job in design-
ing test kits, making them an
important tool in the arsenal used
to determine the presence of food
allergens, Garber points out.
By utilizing sensitive, reliable
methods that have been extensively
evaluated and validated, when
a consumer complaint is made
about a food product we can
accurately determine whether a
food allergen was present, thereby
safeguarding the consumer and
avoiding erroneous conclusions
regarding food products, he says.
Garber is quick to mention that
there are still mysteries about test
kits that need to be solved. Our
methods are focused on antigenicity
and detectability. The effects of
preparation on the bioavailability
and allergenicity of food allergens
are not fully understood.
There are two major needs that
still prevail relative to enhancing
allergen test kits as a tool, says Tim-
othy Hendra, Director of Diagnostic
Sales for Neogen Corp., Lansing,
Mich. (thendra@neogen.com).
We continue to work to make
test kits easier and quicker to
run. Waiting even 2030 minutes
for test results between runs
impacts the efficiency of the food
manufacturing process, Hendra
says. And test kits are needed
for additional allergens, such as
tree nuts like walnuts and pecans,
processed soy, and shellfish.
Neogen currently has ELISA test
kits for detection of peanuts, egg,
milk, almonds, gliadin, hazelnuts,
and soy flour. Our goal is to have
all test kits in the 5-min lateral flow
format. As these improvements
are made, I believe test kit use
will increase dramatically.
The future holds promise for the
food-allergic consumer, as allergen
tests will become more reliable,
specific, and targeted for what
they should do, says Bruce Ritter,
founder and President of ELISA
Technologies, Inc., Gainesville,
Fla. (info@elisa-tek.com).
Despite his optimism, Ritter
doesnt hesitate to mention the
kinks that need to be worked out
of allergen testing. One drawback
right now is the lack of sufficient
clinical data on thresholds for the
allergens, as well as regulatory
guidelines that would allow test
makers to target the appropriate
range of sensitivity of allergen
tests. There is such a wide range
of patient sensitivity to allergens.
We need better clinical information
and clear regulatory specifications
regarding identification of specific
allergenic target levels.
Another challenge is the food
matrix itself. The detection of
allergens in foods is affected by
the composition and processing
Waste Reservoir
Cover Strip
Membrane
Control Zone
Target Material
Zone
Reagent Pad
Sample Pad
The development of two
Backing Strip lines on a Reveal test strip
indicates a positive test
result. The development of
only one line on the strip
indicates a negative result.
Image courtesy of Neogen Corp.
of the food products, Ritter says.
If you have a protein responsible
for an allergy in five different food
products, for example, different
amounts of allergenic protein will be
recovered from the different foods
after processing. A test kit may not
see the part that has been changed.
The relevance of the modified and
undetected proteins in allergic
reactions is still largely unknown.
No one test can do it all,
Ritter says, and currently all the
commercial test kits have specific
targets and limits of detection.
While the tests produce few
false positives, no test can test down
to zero, and all methods will produce
false negatives with samples
containing allergen levels below the
detection limit, Ritter elaborates.
The differences in the allergen
threshold range among allergenic
people make food manufacturing,
testing, and regulation more difficult.
However, analytical tools are
available that can target clearly
relevant levels of allergens in
foods that can affect allergic
consumers and lead to recalls. The
food industry needs appropriate
guidelines and testing tools to
deal with these issues so they can
produce foods that consumers
with allergies can trust. FT
Linda L. Leake, Contributing
Editor, Food Safety Consultant,
Wilmington, N.C.
llleake@aol.com
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