A – Raw Food B – Processed Food A – Raw Food B – Processed Food 1 . Apple Apple

Background: Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner, all commercial food antigens are prepared from raw food. Furthermore, all IgE and IgG antibodies against dietary proteins offered by many clinical laboratories are measured against raw food antigens. Methods: We developed an enzyme-linked immunosorbent assay for the measurement of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Sera with low or high reactivity to modified food antigens were subjected to myelin basic protein, oxidized low density lipoprotein, and advanced glycation end products (AGE) such as AGE-human serum albumin and AGE-hemoglobin. Results: Compared to raw food antigens, IgE antibodies showed a 3–8-fold increase against processed food antigens in 31% of the patients. Similarly, IgG, IgA and IgM antibodies against modified food antigens overall were found at much higher levels than antibody reactions against raw food antigens. Almost every tested serum with high levels of antibodies against modified food antigens showed very high levels of antibodies against myelin basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-hemoglobin. Conclusion: We conclude that the determination of food allergy, intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Antibodies against modified food antigens, by reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and neuroautoimmunity. Background Adverse reactions to foods in which the pathogenesis involves an immunological response to food components are appropriately called food-hypersensitivity reactions. This term is considered to be synonymous with "food allergy." This adverse immune reaction to food proteins affects many children and adults [1]. In a study using double-blind placebo-controlled food challenge, 39% of participants showed hypersensitivity to food antigens [2]. Based on clinical presentation and antibody response, immune-mediated adverse reactions to foods can be divided into immediate and delayed hypersensitivity reactions. Immediate reactions to food antigens are IgE-mediPublished: 12 May 2009 Nutrition & Metabolism 2009, 6:22 doi:10.1186/1743-7075-6-22 Received: 26 March 2009 Accepted: 12 May 2009 This article is available from: http://www.nutritionandmetabolism.com/content/6/1/22 © 2009 Vojdani; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page 1 of 17 (page number not for citation purposes) Nutrition & Metabolism 2009, 6:22 http://www.nutritionandmetabolism.com/content/6/1/22 ated and dependent on activation of mast cells in specific tissues, including the skin, respiratory tract, gastrointestinal, mucosal, and cardiovascular system [3-5]. The delayed immune reaction to food antigens are mediated by IgG, IgA and IgM. Unlike the immediate effects of IgE-mediated allergy, the IgG, IgM and IgA-mediated food allergy and intolerance reactions can take several days to appear. Therefore, levels of IgG, IgM and IgA antibodies in the blood against different food antigens have been used for demonstration of delayed food allergy and intolerance reactions [6,7]. Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner [8], all commercial food antigens are prepared from raw food. However, for demonstration of both immediate and delayed hypersensitivity to food, antibodies are measured against antigens prepared from raw food [9-11]. Processed foods and their ingredients are subjected to a variety of conditions, which may cause alterations in immunodominant epitopes, potentially affecting allergenic properties. This processing may destroy existing epitopes on a protein or may cause new ones to be formed (neoallergen formation) as a result of change in protein conformation. Neoallergen formation has been known for at least three decades [12]; it may be part of the reason why some individuals can tolerate a raw food or raw food ingredient but will react to the same food when it is processed. Studies have found neoallergens from pecans [13], wheat flour [14], roasted peanuts [15], lentil [16], almond, cashew nut and walnut [17], soybean [18,19], shrimp, scallop, tuna, egg, apple, plum, milk and potato [2,11,20-22]. The different types of food processing includes thermal as well as non-thermal treatments, and each type of process may have a different effect on epitopes. In evaluating allergen stability, then, the different effects of individual treatments must be considered carefully. Thermal processing may be done by dry heat (e.g. oven roasting, oil roasting, infra-red heating, ohmic heating) or wet heat (e.g. boiling, microwave cooking, pressure cooking, autoclaving, extrusion, blanching, steaming). Non-thermal treatments include irradiation, soaking, germination, milling, fermentation, high-pressure processing, dehulling and dehusking, and grinding. Processing may affect food in a manner that may induce the masking or unmasking of allergenic epitopes, thereby enhancing or reducing allergen recognition and potentially altering the allergenicity of the offending food [21]. In relation to common processing methods, including mechanical, enzymatic, heating, drying, peeling, pulping, blanching, mashing, pasteurization and multiple-treatment effects on the allergenicity of processed food antigens, all the published articles dealt with immediate hypersensitivity reaction which is IgE mediated. None of these articles dealt with delayed immune reaction to processed food antigens [1-22]. This sampling of articles also illustrates that in a majority of cases some of the technological processing treatments not only maintained their antigenicity and allergenicity but also induced the modification and introduction of neoantigens. Therefore, this study was designed to assess both IgEand non-IgE-mediated hypersensitivity against both raw and processed food antigens. The measurements of IgE, IgG, IgA and IgM antibodies in blood against processed food antigens results in an enhancement in the detection of delayed food sensitivities. This would not be possible by merely measuring these same antibodies against antigens prepared from raw or unprocessed foods alone. Methods Sera from 40 food-allergic individuals and 40 healthy subjects were obtained from DPC Inc., Los Angeles, CA, and Innovative Research Inc., Novi, MI. Food antigens were prepared from food products purchased from large supermarket chains to reflect American purchasing and accessibility of food products. Each advertised ingredient of the prepared, cooked and processed foods was carefully catalogued at the lab. Some examples of the raw and processed foods used for the extraction of antigens are shown in Table 1. Hemoglobin (Hb), human serum albumin (HSA) and myelin basic protein (MBP) were purchased from Sigma Aldrich, St. Louis, Missouri, USA. Analytical method for identification and characterization of food antigens Each raw or processed food was ground at 4°C using a food processor and extraction buffers and reagents, such as Coco buffer (0.55% NaHCO3, 1% NaCl), 0.1 M phosphate buffer saline pH 7.4, 70% ethanol, and cold acetone. Each food was mixed in four different solvents and kept on the stirrer for 2 h at room temperature. After centrifugation at 2000 g for 15 minutes the liquid phase from each buffer and solvent was removed and dialysed against 0.01 M PBS using dialysis bags with a cutoff of 6,000. The pellet from the acetone was dissolved in 0.1 M PBS buffer and dialysed. Dialysis was repeated for three times in order to make sure that all small molecules are removed. After dialysis extracted antigens from the above conditions were combined, and protein concentrations were measured using a kit provided by Bio-Rad (Hercules, CA). For quality control in separating the antigens from the foods, sodium dodecylsulfate (SDS) gel electrophoresis was performed. The electrophoretic methods included Page 2 of 17 (page number not for citation purposes) Nutrition & Metabolism 2009, 6:22 http://www.nutritionandmetabolism.com/content/6/1/22 SDS acrylamide gel (gradient of 7–15%), and Western blot [23,24]. Detection of IgE, IgG, IgA and IgM antibodies by EnzymeLinked ImmunoSorbent Assay (ELISA) Dietary proteins and peptides were dissolved in phosphate buffered saline (PBS) at a concentration of 1.0 mg/ ml then diluted 1:100 in 0.1 M carbonate-bicarbonate buffer, pH 9.5. 100 μl of each antigen was added to duplicate wells of a polystyrene flat-bottom ELISA plate. Plates were incubated overnight at 4°C and then washed three times with 20 mM Tris-buffered saline (TBS) containing 0.05% Tween 20. After washing, the plates were coated with 200 μl of 1.5% bovine serum albumin (BSA) and 1.5% gelatin in TBS and then incubated for 2 hours at room temperature and then overnight at 4°C. When used in combination, BSA+gelatin results in a very low nonspecific binding OD <0.1. After the overnight incubation, the BSA+gelatin was removed. Plates were washed three times with 20 mM TBS containing 0.05% Tween 20, dried and stored at 4°C. Quality control was performed by the addition of serum with low, medium and high titers of antibodies against different food antigens. In addition, plates were studied for the detection of non-specific reaction by the addition of all reagents except serum. After the performance of quality control, the plates were kept at 4°C until used. For IgE determination the sera were diluted 1:2 in serum diluent buffer (20 mM TBS containing 1% BSA, 0.05% Tween 20, and 0.01% sodium azide, while for IgG, IgA and IgM determination the sera were diluted 1:100 in the same buffer; 100 μl of diluted sera were then added to duplicate wells of microtiter plates and incubated for an appropriate length of time: 2 hours for IgG, IgA and IgM, and overnight for IgE at 4°C. A standard curve was constructed using serum with a titer of 1:128 for IgE against hydrolyzed milk (protein concentration 1 mg/mL). A different serum with a titer of 1:12,800 against wheat proteins (concentration 1 mg/mL) was used to construct standard curves for IgG, IgA and IgM antibodies using antigens prepared from cereal. Plates were washed 5 Table 1: Examples of raw and processed foods used for antigenic extraction and measurement of IgE, IgG, IgM and IgA antibodies in blood A – Raw Food B – Processed Food A – Raw Food B – Processed Food 1. Apple Apple Cider 26. Wheat (Semolina) Pasta 2. Pork Bacon 27. Peanut Peanut, Roasted 3. Barley, Hops, Yeast Beer 28. Pecan Pecan, Roasted 4. Chicken Wing, Wheat, Soy Buffalo Wing 29. Cucumber, Vinegar Pickles 5. Wheat, Barley, Yeast, Soy, Sugar Doughnut 30. Wheat, Milk, Cheese, Olives, Tomato, Spinach, Mushroom, Broccoli Pizza 6. Wheat, Egg, Corn, Milk, Sugar Cake 31. Corn, Butter Popcorn 7. Wheat, Oat, Corn, Rice, Barley, Milk, Sugar Cereal 32. Egg, Potato, Cucumber, Corn, Mustard Seed, Vinegar, Lemon Potato Salad 8. Chicken, Wheat, Rice, Veg. Oil Chicken Chow Mein 33. Pumpkin, Wheat, Egg, Corn, Milk, Soy Pumpkin Pie 9. Chicken, Wheat, Veg. Oil Chicken, Fried 34. Rice, Wheat, Peanut, Soy Rice Cake 10. Chicken, Wheat, Orange, Veg. Oil Chicken, Orange 35. Salmon Salmon, Baked 11. Beans, Chili Powder, Tomato, Onion, Corn, Potato, Garlic Chili (Vegetarian) 36. Salmon Salmon, Fried 12. Coffee Coffee, Roasted 37. Beef, Pork, Soy, Wheat Sausage 13. Cranberry, Corn Cranberry Sauce 38. Shrimp Shrimp, Cooked 14. Egg, Raw Egg, Cooked 39. Soybean Soy Agglutinin 15. Egg, Assorted Vegs., Wheat, Soy Egg Roll 40. Beef Filet Mignon Steak, Filet Mignon 16. Corn Starch, Sugar Dextrose, Yellow #5, Yellow #6, Red #3, Red #40, Blue #1 Food Coloring 41. New York Strip Steak, New York 17. Potato, Veg. Oil, Wheat, Milk French Fries 42. Potato, Veg. Oil Tater Tots 18. Beef, Onion, Seasoning Hamburger 43. Soybean, Casein Tofu 19. Beef, Pork, Turkey, Corn, Wheat Hotdog 44. Tuna Tuna, Canned 20. Milk, Corn, Cocoa, Gum, Strawberries, Sugar Ice Cream 45. Tomato, Carrot, Celery, Beet, Parsley, Lettuce, Spinach Vegetable Juice 21. Tomato, Corn, Onion, Garlic, Vinegar Ketchup 46. Wheat Wheat Germ Agglutinin 22. Lentil Lentil, Boiled 47. Milk, Corn, Coconut, Palm Oil, Veg. Oil Whipped Cream 23. Egg, Soybean Oil, Lemon, Vinegar Mayonnaise 48. Whitefish Whitefish, Baked 24. Mustard Seed, Turmeric, Paprika, Vinegar Mustard 49. Whitefish Whitefish, Fried 25. Peanut, Veg. Oil Peanut Butter 50. Grape, Yeast Wine Page 3 of 17 (page number not for citation purposes) Nutrition & Metabolism 2009, 6:22 http://www.nutritionandmetabolism.com/content/6/1/22 times, and then 100 μl of alkaline phosphatase labeled goat anti-human IgE, IgG, IgM or IgA F(ab')2 fragments (KPI, Gaithersburg, MD) at an optimal dilution of 1:400 – 1:2000 in 1% BSA-TBS were added to each well; plates were incubated for an additional 2 h at room temperature. After washing five times with TBS-Tween buffer, the enzyme reaction was started by adding 100 μl of paranitrophenylphosphate (PNPP) in 0.1 mL diethanolamine buffer containing 1 mM MgCl2 and sodium azide pH 9.8. The reaction was stopped 45 min later with 50 μl of 1 N NaOH. The optical density (OD) was read at 405 nm by means of a microtiter reader. To detect non-specific binding, several control wells containing all reagents except human serum were added and used in each assay. The OD for all control wells coated with different antigens first and then with BSA+gelatin was less than 0.1 or 2 EU. The ELISA units (EU) of IgE, IgG, IgA or IgM antibodies against specific food antigens was determined by plotting the antibody's concentration on the log scale against the OD on the linear scale for seven calibrators with a known titer of 4, 8, 16, 32, 64, 96 and 128. The EU was determined using a computer program with linear regression capability and the generation of a curve that best fits the data and the following formula: For example, using this formula, optical densities of 0.17, 0.3, 0.5, 0.8, 1.4, 2.0 and 2.7 resulted in EUs of 4, 8, 16, 32, 64, 96 and 128 respectively. Preparation of Advance Glycation End product (AGE) proteins AGE-hemoglobin (AGE-Hb) and AGE-human serum albumin (AGE-HSA) were prepared as described previously [25] and later modified [26]. Briefly, 0.5 g HSA or Hb was dissolved with 3.0 g D-glucose in 10 mL of 0.1 M phosphate buffer saline pH 7.4 containing 0.05% sodium azide. For controls Hb and HSA were dissolved in PBS without D-glucose. Each solution was deoxygenated with nitrogen, sterilized by ultrafiltration (0.2 μm filter) and incubated for 45 days at 37°C. The samples were then dialyzed against 0.1 M PBS and were separated on a gradient of 7–15% of acrylamide. In comparison to samples without glucose, shift in Hb and HSA band in samples containing D-glucose was used for confirmation of AGE-Hb and AGE-HSA formation. Measurement of IgG antibodies against AGE-Hb and AGEHSA Hb, HSA, AGE-Hb and AGE-HSA were diluted to 100 μg/ mL, and similar to the above ELISA, 100 μl of each antigen was added to duplicate wells of microtiter plated followed by incubation, washing, coating with BSA-gelatin, and repeated washing. Eight different sera with severe reaction against modified food antigens and 8 healthy control sera with very mild reaction against processed food antigens were applied to duplicate wells coated with Hb, HSA, AGE-Hb or AGE-HSA. After incubation, washing, addition of anti-human IgG secondary antibody, repeated washing and substrate, color development was measured. Sera from individuals with a titer of 1:12,800 against boiled egg antigens were used to construct a standard curve and the calculation of antibody units against AGE-Hb or AGEHSA. For the determination of specific IgG antibodies against AGE-Hb and AGE-HSA ELISA values obtained against Hb or HSA alone were subtracted from the ELISA values of AGE-Hb or AGE-HSA. Measurement of IgG antibodies against myelin basic protein (MBP) MBP IgG antibody was measured according to the previously described method [27]. Measurement of IgG antibodies against oxidized low density lipoprotein (ox-LDL) ox-LDL IgG antibody was measured by a kit manufactured by Biomedica Gruppe and distributed in the US by ALPCO Diagnostics, Salem, New Hampshire. Results Detection of IgE antibodies against raw and processed food antigens Sera from 40 individuals with known type-1 allergic reaction to foods and sera from 40 healthy controls were screened for food-specific IgE antibodies by the Immulite DPC test system. 26 out of the 40 samples from the known allergic individuals and 3 out of the 40 samples from healthy control subjects that demonstrated IgE antibodies > 15 IU against five or more food antigens were selected for simultaneous measurement of IgE antibodies against raw and modified food antigens. These 29 samples with food-specific IgE antibodies > 15 IU were subjected to ELISA plates coated with raw and modified food antigens. 20 out of the 29 specimens showed very similar IgE antibody levels (± 20%) against different raw and modified food antigens. The other 9 samples (31%) showed 3– 8-fold increase in IgE antibodies against modified food antigens as compared to the raw food antigens. For example, Sample #1 with OD of 0.32 or 10 EU of IgE against raw meat, OD of 0.41 or 14 EU against soy, and OD of 0.59 or 22 EU against wheat exhibited OD of 1.9 or 86 EU of IgE antibodies against antigens extracted from sausage. Sample #9 with OD of 0.6 had a level of 23 EU of IgE against raw egg, but had an OD of 3.2 or IgE level of 139 EU against boiled egg antigens (Figure 1). The data presented in Figure 1 clearly shows significant elevation in IgE, IgG, IgA or IgM ELISA units Values of calibrators Abs = × orbance of test specimen Absorbance of calibrators Page 4 of 17 (page number not for citation purposes) Nutrition & Metabolism 2009, 6:22 http://www.nutritionandmetabolism.com/content/6/1/22 IgE antibodies against processed food in 9 serum samples from food-allergic individuals. Detection of IgG, IgA and IgM antibodies against raw and modified food antigens All 80 sera specimens (40 with known IgE reaction to foods and 40 healthy controls) were subjected to ELISA plates coated with raw and modified food antigens for measurement of IgG-, IgAand IgM-specific antibodies. Compared to sera from healthy controls, specimens with IgE reaction to food antibodies demonstrated elevations in IgG, IgA and IgM antibodies against both raw and modified food antigens. Overall this immune reaction against different modified food antigens was much higher than reaction against raw food antigens. Due to the high volume of data generated in this study, for clarity's sake only one patient's results are presented. Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9A show the concentration of IgG antibodies in serum expressed by ELISA units against raw versus processed foods. Note in this example the high levels expressed against a majority of the processed or modified versions of the foods in comparison to their raw or crude forms. The results of 8 patients with high IgG reactivity against many modified food antigens were compared to 8 controls with low reactivity against these same modified antigens, as is summarized in Table 2. The total number of reactions >30 EU against modified food antigens in the 8 patients with high reactivity was 91 out of 360 determinations, while in the control group only 13 out of the same 360 determinations were >30 EU. The difference in reactivity against the same modified food antigens in these two groups was highly significant (p < 0.000001). Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9B show the concentration of IgA antibodies in serum expressed by ELISA units against raw versus processed foods. Note in this example the high levels of IgA antibodies expressed against a majority of the procSerum levels of IgE Antibodies against Raw (white square) vs Processed (black square) Food Antigens expressed by ELISA unit Fig re 1 Serum levels of IgE Antibodies against Raw (white square) vs Processed (black square) Food Antigens expressed by ELISA unit. Measurement of IgE antibodies against different raw or crude ingredients versus the processed or cooked version of the foods in the sera of nine individuals with food allergy expressed in ELISA units. 14 10 22 86