1. Analytical Methods for Allergens
Sue Hefle, Ph.D.
Associate Professor and Co-Director
Food Allergy Research and
Resource Program (FARRP)
University of Nebraska
Food Allergy Research and Resource Program  2005

2. Basics of Analytical Methods for Allergens
Most used is ELISA-based (includes lateral flow)
Most successful kits use polyclonal antibodies, but occasional kit uses monoclonal antibodies directed against a single protein
Usually, antibodies are directed against a crude extract of allergenic food
Not necessary to measure allergen; industry really just cares if “any” peanut is there, not if Ara h 1 is there!
Challenge: different standards used in different kits; also different antibodies
Food Allergy Research and Resource Program  2005

3. Basics of Analytical Methods for Allergens
Detection limits range from 0.1 – 2.5 ppm for quantitative methods
Using a method that has a very low detection limit:
Every kit has the ability to have a low detection limit
Clinical relevance vs. chasing molecules around a food plant (“paralysis by analysis”)
Adversely affects the quality of life for food-allergic individuals because of the industry reaction in the form of increased use of “may contain”-type labeling
Current detection limits have worked very well for 7 years
Food Allergy Research and Resource Program  2005

4. Status of Allergen Testing in the U.S.
Many companies are testing for allergen residues
ELISA or lateral flow is the preferred method
Some do in-house testing, others use contractor labs
Most companies are not testing finished product
Are testing to validate sanitation methods; environmental swabbing
Some testing of finished product done when product is under full control
PCR(DNA) tests available – FARRP does not recommend for allergenic residue detection
Not practical for in-plant use; expensive equipment and isolated lab required
Does not prove presence or absence of protein/allergen
ATP tests – do not correlate completely with allergen ELISA results
Food Allergy Research and Resource Program  2005

5. Food Allergy Research and Resource Program  2005
Peanut Detection Kits
Three peanut ELISA kits have been “performance tested” by FDA through AOAC-RI
Neogen
R-Biopharm
Tepnel
Five peanut ELISA kits have been studied in one JRC interlab trial
ELISA Technologies
Pro-Lab Diagnostics
Two peanut lateral flow devices are currently in a JRC interlab trial
Only one matrix, though - cookies
Food Allergy Research and Resource Program  2005

6. Food Allergy Research and Resource Program  2005
“Validation” of Kits
FDA-AOAC have said they plan more validation studies with other test kits; this has been the case for more than 2 years with no apparent progress
U.S. food industry and other regulatory agencies (i.e. Canada) have moved way ahead of FDA/AOAC
U.S. industry has been testing for 7 years, since first peanut test came out, and has increased the amount of testing each year
Health Canada/CFIA Compendium of Food Allergen Methodologies; commercial and in-house methods
Food Allergy Research and Resource Program  2005

7. Food Allergy Research and Resource Program  2005
“Validation” of Kits
More JRC trials likely
Other groups planning interlab trials, some with “model” foods
Food Allergy Research and Resource Program  2005

8. Food Allergy Research and Resource Program  2005
“Validation” of Kits
Kit companies do much more extensive validation than will ever be done by any regulatory agency or academic center
Have liability issues, reputation issues
Food Allergy Research and Resource Program  2005

9. Reference Materials and “Model” Foods
Not many available; REALLY needed
NIST is one source, but NIST standards were not made for allergen testing and often do not represent the type of allergenic materials used in the food industry
Standard used in AOAC-RI-FDA study = peanut butter; varieties not known with certainty (manufacturer would not divulge to FDA); different peanut varieties have different responses in kits
Other sources of materials that could be used as reference materials
JRC, FARRP
Effect of processing on extraction/kit performance
Most kits are not validated using “model” foods
International call for use of “model” foods – “spiking” provides some useful information but “manufacturing” gives best information about how a kit will work
Food Allergy Research and Resource Program  2005

10. Food Allergy Research and Resource Program  2005
“Model” Foods
“Model” foods must be made on a pilot plant or industrial-sized scale
Ex. Simply making mini-cookies in a home-sized oven does not mimic industrial practices
Results not practical or useful for the food industry
Invaluable for assessing how a kit is going to work with a specific commodity and how efficient extraction method is
Becoming more important to use these types of standards in assessing a kit’s performance for certain commodities/processing
“Spiking” is really passé in this area - only good for initial assessment of possible matrix interferences
Food Allergy Research and Resource Program  2005

11. Factors Affecting Test Performance
Extraction method – sufficient, recovery good?
Some foods are challenging (ex. tannins/polyphenols in dark chocolate bind protein, high fat level “hides” allergen in other types of ingredients)
Hydrolysis – cannot analyze hydrolyzed or fermented ingredients
Methods meant to detect intact proteins, not peptides
Processing
Food Allergy Research and Resource Program  2005

12. Factors Affecting Test Performance
Processing
Most kits for most allergens have good reactivity with processed forms of allergenic food
Use of polyclonal antibodies and crude extracts, and making antibodies against processed forms are recipes for successful kits
Monoclonals ok if against a heat-resistant epitope
Some of the egg residue kits have some issues in this regard
Industry has been able to adjust and adapt – many survey raw material or use a kit that has antibodies against raw AND processed egg
Food Allergy Research and Resource Program  2005

13. Factors Affecting Test Performance
Matrix effects
My lab has used all of the ELISA-based test kits available on the market in our own validations and tests
Matrix effects not usually a problem for the vast majority, and kit companies have added extraction additives to their extraction buffers to assist in this regard, esp. with well-known matrix issues like dark chocolate
“Model” foods of great use in assessing this; spiking useful also
Cross-reactivity
Even though most methods use polyclonal antibodies directed against crude extracts, do not see cross-reactivity issues for the most part
Food Allergy Research and Resource Program  2005

14. Food Allergy Research and Resource Program  2005
Testing Issues
Hydrolyzed proteins
Most ELISAs are rendered useless when trying to analyze for a hydrolyzed protein
But, a negative result does not mean that there is no allergenic residue left -must ascertain residual allergenicity via IgE methods (Western, RAST)
Another related area is analysis of fermented ingredients (gums, Lactobacillus, etc.)
Companies do not tell contract labs the nature of their samples, and when it comes out negative, they report that to their customers!
Food Allergy Research and Resource Program  2005
Food Allergy Research & Resource Program

15. Food Testing – Consumer Reactions
My laboratory performs testing for food-allergic consumers, their physicians, or their lawyers (gratis) when they report a reaction to a food
Food Allergy and Anaphylaxis Network, others
In ten years of doing this, we have only seen “large” amounts of undeclared allergenic food cause reactions
Food Allergy Research and Resource Program  2005
Food Allergy Research & Resource Program

16. Practical Issues for the Industry
Cannot currently do “immediate” monitoring
Technology does not exist – lateral flow devices will help, but not available for many allergens yet
Therefore, sanitation verification most practical, not “test and release”
Do not have tests for some allergens
Ex. fish
Cannot test for hydrolyzed or fermented allergen sources
Some types of cross-contact are not homogenous or 100% cleaning is not possible due to nature of product
Cannot take enough samples to practically test to be 100% sure
In some of these cases, precautionary labeling justified, in FARRP’s opinion – ex. dark chocolate and milk chocolate on same line
Food Allergy Research and Resource Program  2005
Food Allergy Research & Resource Program

17. Food Allergy Research and Resource Program  2005
Cross-Contact Associated with Milk-Allergic Consumer Complaints (Hefle and Lambrecht, J. Food Prot., Sept. 2004)
Product
Casein (ppm)
Whole Wheat Roll
5,500
Tofu Cheesecake
16,100
Soy coffee drink
43,500
Dark Chocolate (kosher)
12,700*
Bakery icing (kosher)
44,500
Energy bar (“dairy-free”)
27,300
Cupcake (“dairy-free”)
13,700
Food Allergy Research and Resource Program  2005

18. HRO (Highly Refined Oils)
What does HRO mean?
In FARRP’s opinion, HRO = neutralized (alkali-refined), bleached, and deodorized (NBD), or refined, bleached and deodorized (RBD)
Opinion based on scientific review of oral challenges with oils in the literature
Available quantitative methods
Methods used in literature include ELISA, etc.
None of these have been validated in interlab trials or other types of validation for protein-in-oil determination to date
Question as to whether small amount of protein in HRO is completely extracted in aqueous buffer (usual method used), or whether some of the more hydrophobic proteins stay in the oil fraction, and therefore not extracted/determined
My lab uses an amino acid determination based on Edman degradation, but also aqueous extraction (limitations), but report results as “relative” and not a complete picture of the possible protein content of HRO oil
Food Allergy Research and Resource Program  2005

19. HRO (Highly Refined Oils)
Protein levels of HRO reported in literature
Caveats – aqueous buffers, epitope recognition by antibody, etc., relating “total” nitrogen to intact proteins, limitations of dye binding protein methods, etc.
Protein levels reported in the literature
Usually a few mg/kg = few ppms
Food Allergy Research and Resource Program  2005