Testing for Allergens by Levone


                                                                                                  by Neil H. Mermelstein

                        Testing for Allergens
                             ccording to the Food and Drug Administration,       1, 2006, requires that food labels identify whether a
                             more than 160 foods can cause allergic reac-        product contains any of these major food allergens
                             tions, and eight foods account for 90% of these     or ingredients containing protein derived from them.
                        reactions: milk, eggs, fish (e.g., bass, flounder, cod),       Since products not specifically formulated
                        crustacean shellfish (e.g., crab, lobster, shrimp),       with these allergenic ingredients may still contain
                        tree nuts (e.g., almonds, walnuts, pecans), peanuts,     traces of them as a result of cross-contamination
                        wheat, and soybeans. Because allergic reactions can      during processing—e.g., as a result of incomplete
                        be severe and life-threatening, Congress passed the      cleaning during changeover of equipment—food
                        Food Allergen Labeling and Consumer Protection Act       and beverage manufacturers and ingredient
                        of 2004. The law, which went into effect on January      suppliers must therefore test for the presence
                                                                                 of these allergens in their products.

                                                                                 Types of Tests
                                                                                 According to Steve Taylor (staylor2@unl.edu),
                                                                                 Professor, Dept. of Food Science & Technology,
                                                                                 University of Nebraska, Lincoln, and Director of
                                                                                 the Food Allergy Research & Resource Program
                                                                                 (www.farrp.org) located at the university, three
                                                                                 formats for enzyme-linked immunosorbent
                                                                                 assays (ELISAs) are primarily used for detec-
                                                                                 tion of allergens: quantitative, 96-well assays
                                                                                 (ELISAs), commonly conducted in laboratories,
                                                                                 and swab and lateral-flow (“dipstick”) test kits,
                                                                                 similar to pregnancy test kits, for use on-site.
                                                                                      • ELISAs require expensive equipment but
                                                                                 provide quantitative results, Taylor said. FARRP
                                                                                 has developed ELISAs for almond, egg, shrimp,
                                                                                 hazelnut, milk, peanut, sesame, soy flour, whey,
                                                                                 walnut, clam, and pecan and offers these tests at
                                                                                 its laboratory. It is currently developing ELISAs
                                                                                 for cashew, mustard, and lupine. FARRP has also
                                                                                 worked with Neogen Corp. to make commercially
                                                                                 available test kits for almond, egg, hazelnut, milk,
                                                                                 peanut, and soy flour. Gluten ELISAs are also
                                                                                 commercially available from several suppliers.
                                                                                      • Swab kits are simple and easy to use. These
                                                                                 methods yield qualitative results at 5 ppm and are

                                                                                 Technician conducts an ELISA quantitative assay for a specific allergen
                                                                                 such as peanut, milk, soy flour, or almond in a food sample. Test
                                                                                 samples and standards are added to antibody-coated microwell strips,
                                                                                 and substrates are added. After incubation, the technician adds a stop
                                                                                 reagent to end substrate color development, then spectrophotometric
                                                                                 determination of the absorption of each well is conducted to generate a
                                                                                 standard curve and determine from it the level of allergen in the sample.
                                                                                 Photo by Julie A. Nordlee, University of Nebraska, Lincoln

pg   70   09.08 • www.ift.org
often used to assess cleanliness of equipment
surfaces. These tests are designed for in-plant use.
     • Lateral-flow devices are convenient and
easy to use, and users really like the approach,
Taylor said. Where available, these tests are
supplanting swab kits. However, they generally
provide only qualitative results—indicating
whether the product contains at least a threshold

level of the allergen. For example, if a lateral-flow
test is designed to give a positive or negative
result at a level of 5 ppm and the dipstick shows
a line indicating the presence of the allergen,
you won’t know whether the allergen is present
in the sample at 5, 7, 10, or 25 ppm. The line
appears faster, however, if the level is higher.
     And test kits are not yet available for all

                                                       1/2 ISL 4C
allergens, he added. “It’s not as easy as it might
seem to develop lateral-flow tests for certain
allergens, such as milk and peanuts.” He said
that companies began coming out with test kits
for residues of allergens several years ago. “We
have reasonably decent test methods for major
allergenic foods that companies want to test
for,” he said. “New tests are being developed
for other, minor allergens, e.g., mustard, but
they have a much more limited market.”
     Two other testing approaches, Taylor said, are
polymerase chain reaction (PCR) and ATP. PCR
detects DNA rather than protein, so it is considered
less relevant for detection of allergens, which are
protein residues. It also is not easily quantified.
ATP methods are widely used and effective for
the assessment of sanitation, since they detect
residual soil very easily, but ATP levels may not
correlate with allergen levels. Nevertheless,
Taylor said, inexpensive ATP tests can be used
as a first-pass system, but if ATP is negative,
then specific allergen testing should be done.

Challenges Ahead
Taylor said that several challenges lie ahead. One
is harmonization. Different test kit companies
might use different criteria for judging the success
of their methods, and the user might not know
which test works best for their purposes.
    Another challenge, Taylor said, is obtaining
consensus on threshold levels, setting a baseline
for analytical sensitivity for these tests. FDA has

                                                             09.08 • www.ift.org   71   pg

                                                Testing for Allergens continued...
                                                talked about setting threshold levels for        for Major Food Allergens and for Gluten in
                                                allergen tests but hasn’t yet. FDA’s Threshold   Food” (www.cfsan.fda.gov/~dms/alrgn2.
                                                Working Group in 2006 issued a report            html) and published an article of the same
                                                titled “Approaches to Establish Thresholds       name in the May 2008 issue of Journal of
                                                                                                 Food Protection. The report and article
                                                                                                 provide an excellent discussion of the topic.
IFTers Present Allergen Research Papers                                                              A third challenge, Taylor said, is the
                                                                                                 fact that we don’t yet have good methods

                                                                                                 for some allergens. For example, it is very
     apers presented during the 2008 IFT        nology that has been shown to inactivate         difficult to develop a soybean test, since
     Annual Meeting & Food Expo, June           peanut allergens—to reduce allergens in          different forms of soy proteins, such as soy
     28–July 1, included presentations on       cow’s milk and concluded that it has the         protein isolate and soy protein concentrate,
research being conducted to develop and/        potential to reduce the allergenicity of milk.   differ immunologically, and all forms of
or improve assays for various allergens.             • Mustard. Researchers at the               soy are being used by the food industry.
    • Almonds. Researchers at the National      University of Nebraska, Lincoln, have            Another problematic allergenic food is fish.
Center for Food Safety and Technology,          developed and validated a specific and            There are many different species, and the
Summit-Argo, Ill., studied ways to improve      sensitive ELISA for the detection and            allergenic proteins differ with the species.
the extraction of almond proteins from          quantification of mustard residues in foods.          We’re also missing tests for some minor
processed foods to improve ELISA                     Researchers at Agriculture and              tree nuts: pistachio, Brazil, macadamia,
accuracy. They determined that combining        Agri-Food Canada, Saskatoon, SK,                 and pine nuts, he added. His laboratory has
temperature with sonication increased           Canada, have developed a sandwich-               developed ELISAs for almond, hazelnut,
the total amount of protein extracted.          ELISA method to quantify the allergenic          walnut, peanut, cashew, and pecan, but
    • Buckwheat. Researchers at the             protein in yellow mustard using polyclonal       only the almond and hazelnut tests are
University of Nebraska, Lincoln, have gen-      antibodies. They found that heat treatment       available in commercial kit form. His
erated highly specific polyclonal antibodies     reduced the allergenic protein content           laboratory is the only one that offers
against buckwheat for use in sandwich           and possibly the allergenic potential.           tests for the other tree nut allergens.
ELISAs for detection of undeclared buck-             • Soy. Researchers at Alabama A&M               Another key question, he said, is
wheat residues in food. Buckwheat is used       University, Normal, and the U.S. Dept. of        which commercial test kit is best for a
in Western countries as a substitute for        Agriculture’s Southern Regional Research         food company’s needs. For example, for
wheat flour for gluten-sensitive subjects,       Center, New Orleans, are studying the            testing milk, the kits available from three
but recent studies indicate that some           use of pulsed UV light to reduce the             major suppliers differ in what they test for.
buckwheat proteins are potent allergens.        allergenicity of soybeans, since the             Neogen Corp. tests for both casein and whey;
    • Corn. Researchers at the University       allergens are thermostable and traditional       r-Biopharm has separate tests for the key
of Nebraska, Lincoln, stated that reports       thermal treatments are ineffective.              milk fractions casein and the whey protein
of allergy to corn are rare but a number of          Researchers at Oklahoma State               beta-lactoglobulin; and ELISA Systems tests
people experience significant reactions to       University, Stillwater, studied use of           for casein but not whey. Depending on the
corn lipid transfer protein. The researchers    real-time PCR DNA detection to confirm            application, one or the other might be best.
concluded that this protein is localized        the presence of soy in a food product.               Whether a test kit for some of these
in the outer pericarp of corn grain and         Detection of allergens in soy is particularly    allergens will be developed is really a
suggest that removing the seed coat and         difficult because soy products are so highly      financial consideration, Taylor said. First, is
pericarp may lower the risk of allergy.         processed that ELISA-based detection             it worth the investment to have a graduate
    • Fish. Researchers at Florida State Uni-   of the protein allergen can give incorrect       student devote several years to develop
versity, Tallahassee, used non-competitive      results. The researchers studied whole           an immunoassay with a limited market?
indirect ELISA to examine the immuno-           soybeans, soybean protein concentrates,          And second, even if a test kit for a minor
reactivity of soluble proteins extracted        soybean protein isolates, soybean flour, and      allergen is developed, it might not be in
from 51 common species of cooked food           soybean protein–fiber–lecithin products.          high-enough demand to make it worthwhile
finfish, 3 types of shellfish, and 13 species           Researchers at the University of            for a company to commercialize it.
of meat and poultry to anti-parvalbumin         Illinois, Urbana, developed and validated
antibodies. They found that MAb 3E1 is          an immunochemical method for detection           Harmonization Activities
specific to fish parvalbumin, which would         and quantification of the major allergenic        Samuel Godefroy (samuel_godefroy@hc-sc.
be a useful probe for the detection of the      proteins in a variety of commercial soy          gc.ca), Director of the Bureau of Chemical
major fish allergen in processed food.           ingredients and products, including soy          Safety at HealthCanada’s Food Directorate
    • Milk. Researchers at Alabama A&M          protein isolate, soy protein concentrate,        and Cochair of AOAC International’s
University, Normal, studied the use of          soy flour, soy grits, soymilk, tofu, soy          Presidential Task Force on Food Allergens,
pulsed ultraviolet light—a nonthermal tech-     cheese, meat analogs, and soy pudding.           said that his task force—consisting of
                                                                                                 representatives of regulatory agencies in the

pg   72                09.08 • www.ift.org
Table 1. Food allergen test methods undergoing validation for acceptance as AOAC official methods.

Manufacturer         Food allergen            Trade name                      Technique              Manufacturer     Food allergen               Trade name                       Technique

Neogen            Almond                 Alert® and Veratox® for            Sandwich ELISA           Tepnel         Almond                   BioKits Rapid 3-D™                  Lateral-flow device
                                            Almond Allergen                                                                                  Almond Test Kit
                  Egg (whites)           Alert for Egg Allergen             Sandwich ELISA                                                   BioKits Almond Assay Kit            Sandwich ELISA
                                         Veratox for Egg Allergen           Sandwich ELISA                          β-Lactoglobulin/whey     BioKits β-Lactoglobulin)            Inhibition/
                  Gliadin/Gluten         Alert for Gliadin                  Sandwich ELISA                                                      Assay Kit                           competitive ELISA
                                         Veratox for Gliadin                Sandwich ELISA                          Milk (cow)               BioKits Allergen DNA                PCR
                                                                                                                                                Selection Modules for
                  Hazelnut               Veratox for Hazelnut Allergen      Sandwich ELISA                                                      Detection of Milk
                  Milk (total)           Alert for Total Milk Allergen      Sandwich ELISA                          Casein                   BioKits Rapid 3-D Casein Test Kit   Lateral-flow device
                                         Veratox for Total Milk Allergen    Sandwich ELISA                                                   BioKits Casein Assay Kit            ELISA-IC
                                         Reveal® for Total Milk Allergen    Lateral-flow device                      Egg                      BioKits Rapid 3-D Test Kit          LFD
                  Peanut                 Alert for Peanut Allergen          Sandwich ELISA                                                   BioKits Egg Assay Kit               Sandwich ELISA
                                         Veratox for Peanut Allergen*       Sandwich ELISA                          Gluten                   BioKits Rapid 3-D™ Test Kit         Lateral-flow device
                                         Reveal for Peanut Allergen         Lateral-flow device                                               BioKits Gluten Assay                Sandwich ELISA
                  Soy (flour)             Alert for Soy Flour                Sandwich ELISA                          Hazelnut                 BioKits Rapid 3-D Test Kit          Lateral-flow device
                                         Veratox for Soy Flour              Sandwich ELISA                                                   BioKits Hazelnut Assay              Sandwich ELISA
                                                                                                                    Peanut                   BioKits Rapid 3-D Peanut Test Kit   Lateral-flow device
R-Biopharm        Casein (milk)           Ridascreen® Fast Casein           Sandwich ELISA                                                   BioKits Peanut Assay Kit*           Sandwich ELISA
                  Lupin                   SureFood® Allergen Lupin          PCR (real-time)                                                  BioKits Allergen DNA                PCR
                  Gliadin                 Ridascreen Gliadin*               Sandwich ELISA                                                      selection modules for
                                                                                                                                                detection of Peanut*
                                          Ridascreen Fast Gliadin           Sandwich ELISA
                                                                                                                    Sesame                   BioKits Sesame Protein Test Kit     Sandwich ELISA
                                          Ridascreen Gliadin competitive    Competitive ELISA
                                                                                                                    Shellfish                 BioKits Rapid 3-D™ Shellfish         Lateral-flow device
                                          Rida® Quick Gliadin               Lateral-flow device                                                  Test Kit
                                          SureFood Allergen Gluten          PCR (real-time)                                                  BioKits Shellfish Assay Kit          Sandwich ELISA
                  Peanut                  Ridascreen Peanut                 Sandwich ELISA                          Soya Lectin              BioKits Allergen DNA selection      PCR
                                          Ridascreen Fast Peanut*           Sandwich ELISA                                                      modules for detection of Soya
                                          RidaQuick Peanut                  Lateral-flow device
                                          SureFood Allergen Peanut          PCR (real-time)          Tecra          Whole egg                Aller-tect™ Whole Egg Protein       Lateral-flow device
                  Hazelnut                Ridascreen Fast Hazelnut                                                  Egg                      Egg VIA™                            ELISA
                                          RidaQuick Hazelnut                                                        Whole peanut             Aller-tect Whole Peanut Protein     Lateral-flow device
                                          SureFood Allergen Hazelnut        PCR (real-time)                         Peanut                   Peanut VIA                          ELISA
                  Almond                  Ridascreen Fast Mandel / Almond   Sandwich ELISA                          Whole milk               Aller-tect Whole Milk Powder        Lateral-flow device
                                          SureFood Allergen Almond          PCR (real-time)
                                                                                                                    Casein                   Casein VIA                          ELISA
                  Egg                     Ridascreen Fast Ei/Egg Protein    Sandwich ELISA
                                                                                                                    β-Lactoglobulin          β-Lactoglobulin VIA                 ELISA
                  Soya                    SureFood Allergen Soya            PCR (real-time)
                                                                                                                    Gluten flour              Aller-tect Gluten Flour Protein     Lateral-flow device
                  Celery                  SureFood Allergen Celery          PCR (real-time)
                                                                                                                    Soy flour                 Aller-tect Soy Flour Protein        Lateral-flow device
                  Walnut                  SureFood Allergen Walnut          PCR (real-time)
                                                                                                                    Whole almond             Aller-tect Whole Almond Protein     Lateral-flow device
                  Sesame                  SureFood Allergen Sesame          PCR (real-time)
                                                                                                                    Buckwheat flour           Aller-tect Buckwheat Protein        Lateral-flow device
                  Mustard                 SureFood Allergen Mustard         PCR (real-time)
                  Fish                    SureFood Allergen Fish            PCR (real-time)
                                                                                                     Morinaga       Egg (ovalbumin)        Egg Protein ELISA Kit                 Sandwich ELISA
                  β-Lactoglobulin (milk) Ridascreen β-Lactoglobulin         Sandwich ELISA
                                                                                                                    Milk (casein)          Milk Protein ELISA Kit (Casein)       Sandwich ELISA
                  Crustaceans (prawns,    SureFood Allergen Crustaceans     PCR (real-time)
                     crawfish,lobster,                                                                               Milk (β-Lactoglobulin) Milk Protein ELISA Kit                Sandwich ELISA
                     crabs)                                                                                                                   (β-Lactoglobulin)
                  Molluscs (snails,       SureFood Allergen Molluscs        PCR (real-time)                         Peanut                 Peanut Protein ELISA Kit              Sandwich ELISA
                     mussels, cephalopod)                                                                           Wheat (gliadin)        Wheat Protein ELISA Kit               Sandwich ELISA

Elisa Systems     Almond                 Almond Residue Assay             ELISA                      Charm Sciences Crustacean (shrimp)      AllerGiene® Food Residue            ATP
                  Beta Lactoglobulin     Beta Lactoglobulin Residue Assay ELISA                                     Egg (whole egg powder)   AllerGiene Food Residue             ATP
                  Crustacean             Crustacean Residue Assay         ELISA                                     Milk (non-fat            AllerGiene Food Residue             ATP
                     (Tropomyosin)                                                                                     milk powder)
                  Buckwheat              Buckwheat Protein Assay            ELISA                                   Peanut (butter)          AllerGiene Food Residue             ATP
                  Casein                 Casein Residue Assay               ELISA                                   Soybeans                 AllerGiene Food Residue             ATP
                  Egg (whites)           Egg residue Assay                  ELISA                                   Wheat (whole-wheat       AllerGiene Food Residue             ATP
                  Hazelnut               Hazelnut Residue Assay             ELISA                                      flour)
                  Peanut                 Peanut Residue Assay               ELISA
                  Mustard                Mustard Seed Residue Assay         ELISA
                  Sesame                 Sesame Seed Residue Assay          ELISA
                  Soy                    Soy Assay                          Assay detects multiple
                                                                            Soy proteins and is
                                                                            based on comparison
                                                                            to Soy Flour Protein
                  Gluten                 Gluten Residue Assay               ELISA

*AOAC Performance Tested Method

                                                                                                                                                   09.08 • www.ift.org                            73   pg

          Testing for Allergens continued...
                    U.S., Canada, Japan, Australia, and       food allergen test kits have been          the process of validation for accep-
                    New Zealand, test kit manufacturers,      granted this status: BioKits Peanut        tance as AOAC official methods.
                    and others with food industry             Assay Kit from Tepnel BioSystems                Another international effort on
                    expertise—expects to complete             Ltd. detects peanuts in breakfast          harmonization is being conducted by
                    development this month of a               cereal, cookies/biscuits, ice cream,       the MoniQA Network of Excellence
                    harmonized validation protocol for        milk chocolate, plain chocolate,           (www.moniqa.org), a European
                    ELISA-based methods to determine          and milled nuts; Veratox® for Peanut       Union–funded network of more than
                                                                                                         30 partners whose goal is to make
          Another key question is which commercial test                                                  food safer by harmonizing methods
                                            kit is best for a food company’s needs.                      for food analyses. MoniQA (for
                                                                                                         “monitoring and quality assurance
                                                                                                         in the food supply chain”) was
                    food allergen markers. The                Allergen from Neogen Corp. detects         organized last year and will hold
                    document will provide guidance to         peanuts in breakfast cereal, cookies,      its 1st International Conference,
                    manufacturers of test kits on how to      ice cream, and milk chocolate.             “Increasing Trust in Rapid Analysis
                    design and validate their studies and     Ridascreen®Fast Peanut from r-Bio-         for Food Safety and Quality,” on
                    submit the results for acceptance         pharm AG detects peanuts in such           October 8–10, 2008, in Rome, Italy.
                    as an AOAC official method.                products as breakfast cereal, cook-        Details are available at www.
                        The AOAC Research Institute           ies, ice cream, and milk chocolate.        moniqa.org/rome2008. FT
                    provides an independent third-party       Ridascreen Gliadin from r-Biopharm
                    review of test kit performance claims     AG detects gliadin, secalins, and                   Neil H. Mermelstein, a Fellow of
                                                                                                                  IFT, is Editor Emeritus of
                    and grants Performance Tested             hordeins in wheat, buckwheat, rice,                 Food Technology
                    Methods status to test kits that fulfill   corn, oats, syrup, and sausage. Table               • nhmermelstein@comcast.net
                    their claims. Currently, the following    1 is a list of test methods currently in

                                        1/2 Horiz Bleed

pg   74           09.08 • www.ift.org

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