Manaaki Tangata Taiao Hoki protecting people and their environment through science Specialist Science Solutions FOOD ALLERGEN TESTING Peter Cressey ESR Allergen Seminar Waipuna Hotel and Conference Centre 21 March 2007

© ESR 2007 Presentation Content What are we testing for? What techniques are available? What are the strengths and weaknesses of various methods? Common pitfalls and problems in allergen testing When to use testing

© ESR 2007 What are we testing for? True food allergies are an immunological response to specific food proteins Test methods can target proteins or the genetic material that produces the proteins (DNA) The test method needs to be as specific as possible for the food material of concern The test method need to be able to detect the material of concern in as wide as possible a range of circumstances (e.g. processed informs of the food including heat treated)

© ESR 2007 What Techniques are Available? Protein-based methods (usually antibody based) that determine proteins characteristic of the allergenic source material (but not necessarily the allergenic proteins) DNA-based methods that determine a characteristic sequence of base pairs (not necessarily related to the allergenic proteins) Most allergen testing utilises protein-based methods (Enzyme-Linked ImmunoSorbent Assay – ELISA) detecting a specific sequence of amino acids (epitope)

© ESR 2007 Rapid Antibody-Based Methods So called ‘dipstick’ or ‘lateral flow’ methods Extracted proteins are captured on coloured particles with antibodies attached Particles are allowed to migrate through a support medium, containing a zone of antibodies A positive result is visualised as a coloured line on a dipstick or test strip

© ESR 2007

© ESR 2007 Rapid Allergen Methods Fast (<10 minutes) Sensitive (detection limits ~5 ppm) Qualitative Suitable for screening of foods and environmental swabs Currently available for peanut, gliadin (gluten), milk

© ESR 2007 Sandwich ELISA

© ESR 2007 Sandwich ELISA Extracts added to reaction well coated with bound antibodies Second set of antibodies are then added with enzyme linked to the antibody Coloured chemical added, the enzyme converts the chemical to a different form with a different colour

© ESR 2007

Sandwich ELISA Test methods are exacting and usually take 1-2 hours to complete Sensitive (sub-ppm to ~5 ppm, depending on kit) Quantitative* Available for most major allergenic source materials, except fish

© ESR 2007 Pitfalls with ELISA Detection level too high (some kits designed for adulteration detection – percentage level, not ppm) Kit not validated for substrate or state of allergen (heat-treated, non-heat- treated) Not following kit insert instructions Hydrolysed or fermented proteins Non-homogeneity in test material Too low detection limit – what do you do with the result?

© ESR 2007 Limit of Detection too High Until recently one of the few soy kits available had a detection limit of 5000 ppm (0.5%) soy protein Soy allergy sufferers have been reproted to react to as little as 88 mg soy protein or 18 g of a food containing 5000 ppm of soy protein This kit was designed for grain adulteration not allergen detection

© ESR 2007 Kit not Validated for Substrate or State of Allergen With some kits cooked egg protein only showed 5-20% of the response of raw egg protein. However, most egg allergic individuals are allergic to both raw and cooked egg Almond protein was recovered from a biscuit matrix quantitatively, however recoveries from a fruit pulp were less than 20% of expected values Tropomyosin, the target protein for crustacean kits varies significantly in content between prawn species and between prawns an dotehr crustacea

© ESR 2007 Not Following Kit Insert Elisa kit methods are highly empirical and changing times, temperatures and concentration may not have the expected results A US regulatory agency caused an unjustified Class I recall through a decision to multiply and analytical result by a factor of 25 We have no local examples of this, BECAUSE WE DON’T DO IT

© ESR 2007 Hydrolysed or Fermented Proteins The sequence of amino acids utilised by the test may be different to the sequence causing the allergic reaction Hydrolysis and fermentation break up protein structure and may destroy either the test or allergic sequence Consequently, the food may test positive while it is not allergenic (probably okay) or it may test negative when it is still allergenic (not okay)

© ESR 2007 Non-Homogeneity of Test Sample The test is only as good as the sample tested! Allergen contamination of a food has the potential to be distributed in a non-uniform manner (‘hot spots’) Sampling needs to take this into account

© ESR 2007 Limit of Detection Too Low Some kits are extremely sensitive (e.g. limit of detection 0.5 ppm) and will elicit positive test results at even lower concentration (e.g. 0.1 ppm) The most sensitive individiuals reported in the literature react to about 0.1 mg of protein from the allergenic source At a detection level of 0.1 ppm, the most sensitive allergy sufferers would need to consume 1 kg of the offending food to experience an adverse reaction However, positive test results cannot be ignored, no matter how low

© ESR 2007 Qualitative or Quantiative? Many kits can adequately detect the qualitative presence of allergenic source material Quantitative measurement requires a target protein that is: - Always present at about the same concentration - Unaffected by food processing - Able to be extracted quantitatively from a range of foods Many kits appear to be quantitative when, in fact, they are only qualitative

© ESR 2007 Test kit validation – what is ESR doing? Sensitivity – what is the actual limit of detection? Specificity – does the kit cross react with other than the target proteins? Precision – how repeatable are results? Accuracy – does the kit result represent the actual amount of material present? Confounders – does processing affect the performance of the kit (e.g. heating of ingredients)?

© ESR 2007 Test kit validation – typical results (Tepnel peanut kit) Sensitivity – LOD approximately 1.5 ppm of peanut Specificity – no detectable cross reaction with range of foods (soy, almond, cashew, lentils, sesame, cornmeal, linseed) Precision – CoV of approximately 20% Accuracy – average spike recovery 106% Heat affect – 90% recovery after heating for 2 hours at 100 C Validated for biscuit matrix, other matrices would need to be validated on a case by case basis

© ESR 2007 Performance of Currently Available Test Kits While kits all appear similar, there seems to be considerable variation in the degree of development and validation carried out Peanut kits from major manufacturers are generally the best and have been through AOAC method assessment process Some kits are perfectly satisfactory for screening, but not really suitable for quantitative testing Some kits (e.g. crustacea) would only be of limited value for screening, unless the test situation was well defined (i.e. known allergen source in known food)

© ESR 2007 Conclusions A range of antibody-based assays are available for most common food allergens Assays require no or minimal laboratory resources (unless quantitation is required) Limitations of assay need to be acknowledged Assays should be validated for particular situations

© ESR 2007 When Should Allergen Testing be used? Investigation of consumer complaints (critical incidents) Validation of sanitation methods Incoming raw product validation (Finished product testing?)