Analytical Methods for Allergens

1. Analytical Methods for Allergens Sue Hefle, Ph.D. Associate Professor and Co-Director Food Allergy Research and Resource Program (FARRP) University of Nebraska shefle1@unl.edu www.farrp.unl.edu 402-472-4430 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. 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 • Neogen • R-Biopharm • ELISA Technologies • Tepnel • Pro-Lab Diagnostics • Two peanut lateral flow devices are currently in a JRC interlab trial • Neogen • Tepnel • Only one matrix, though - cookies Food Allergy Research and Resource Program  2005

6. “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 • http://www.hc-sc.gc.ca/food-aliment/cs-ipc/fr-ra/e_allergen_compendium.html Food Allergy Research and Resource Program  2005

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

8. “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 • Reference materials • 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. “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. 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

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

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

17. Cross-Contact Associated with Milk-Allergic Consumer Complaints(Hefle and Lambrecht, J. Food Prot., Sept. 2004) 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