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 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
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 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
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
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
Validation of Kits More JRC trials likely Other groups planning interlab trials, some with model foods
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
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
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 kits performance for certain commodities/processing Spiking is really pass in this area - only good for initial assessment of possible matrix interferences
Food Allergy Research and Resource Program 2005
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
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
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
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 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
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 FARRPs opinion ex. dark chocolate and milk chocolate on same line 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
HRO (Highly Refined Oils) What does HRO mean? In FARRPs 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 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