Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens

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-mediated 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 IgE- and 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.

Delayed food sensitivity is associated with a multitude of disorders, such as multiple sclerosis, autism and rheumatoid arthritis, and affects an estimated 40% of the population. Patients presenting with clustered symtpoms of migraine, mood swings, fatigue, intestinal upset, joint pain, high blood pressure and attention problems are often found to have delayed immune reaction to food antigens [2, 3, 28]. Thus, it is vital to offer the medical community a sensitive method for the detection of food allergy and intolerance testing that is scientifically supported.

One reason traditional food sensitivity testing fails is that it does not reflect a true, real-world, contemporary, non-raw food diet. Widely-used food antigen testing uses only raw food isolates. Since the discovery of fire, fewer and fewer people consume a diet consisting solely of raw foods. As was shown in the introduction, researchers have demonstrated that chemical/molecular changes occur during food preparation, cooking and processing. In addition to altering the makeup of a single food, other changes to the food can occur when this food is combined with another during cooking and processing. Thus, a person who is allergic to ketchup may not have an immune reaction to a raw tomato. If tested using traditional food sensitivity assays, this patient would result negative for tomato, and the patient's problems would be unresolved [10, 11, 13, 14, 16, 18, 19, 21, 22, 29–32]. Despite all of these findings documented in scientific literature and the demonstration of IgE-mediated reaction to many processed food antigens while not reacting to raw foods, almost all commercial laboratories measure IgE and IgG antibodies primarily against raw food antigens.

In this study we sought to prepare extracts from processed foods and compare them to extracts from raw food samples. Since all examples of new allergenicity to food antigens shown here and many others published in scientific journals deal only with IgE mediated or type-I allergic reaction, we decided to confirm these findings with raw and processed food antigens prepared in our laboratory. For example, in Serum #1 the assay detected 10 units of IgE against raw meat, 22 units of IgE against wheat antigens, 14 units of IgE against soy, and 86 units of IgE against wheat isolates prepared from sausage. Or in Serum #7, only 17 units of IgE were detected against raw bacon, but 134 units of IgE were detected against antigens prepared from fried bacon, which is part of a contemporary American diet (Figure 1). These results confirm an earlier study [14] that reported a case of food allergy to a wheat isolate used in sausage and pork pie, but without any allergic reaction to native wheat flour. This allergic reaction to wheat isolates was attributed to the induction of cryptic allergenic or the formation of new allergenic epitopes by technological and chemical processes [14]. Similarly, a case of contact urticaria has recently been attributed to hydrolyzed wheat in cosmetics combined with a generalized urticaria induced with the ingestion of sausages with lentils and a French cassoulet [14]. It was concluded that wheat isolates should be tested when a food allergy to finished food is suspected. In addition to IgE-mediated food sensitivity, delayed food sensitivity has become a growing concern for practitioners in many medical fields. In light of this we decided to extend this investigation to non-IgE-mediated antibodies produced against extracts prepared from modified foods purchased from supermarkets or restaurants (as shown in Table 1). This includes the measurement in duplicate of IgG, IgM and IgA in blood against modified food antigens. Only data from one out of eighty tested sera is presented in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9. This and other data (not shown) clearly indicate that an individual may not show IgG, IgA and IgM antibodies against antigens prepared from raw food, but testing the same patient against antigens prepared from cooked food might result in a severe immune reaction.

To examine possible cross-reactivity and the clinical significance of these antibodies in inflammation and autoimmunities, we selected 8 sera from patients with significant reaction and 8 sera from healthy controls with no or very moderate reaction against modified food antigens and reacted them with AGE-HSA and AGE-Hb. The data presented in Table 2 shows that out of 8 samples with IgG antibodies >30 EU against 8–16 modified food antigens, 6 had IgG antibodies against AGE-HSA and 6 against AGE-Hb. In comparison healthy controls with low levels of antibodies against modified food antigens produced very low levels of antibodies against AGE-HSA and AGE-Hb. These results suggest that AGE formation through the reaction of protein amino groups with sugar may be an important chemical pathway that leads to distinct patterns of modification in foods. These new molecules are highly antigenic, and many important neoantigens found in cooked or stored foods are produced through Maillard reaction [33]. Therefore, the foundation of AGE during food processing through this mechanism of action and IgG, IgA and IgM antibodies produced against them can have a potent impact in tissue inflammation, a process linked to diverse biological settings such as diabetes, metabolic syndrome, renal failure and aging [34, 35].

Elevation of antibodies against modified food antigens and their possible cross-reaction with AGE-HSA, AGE-Hb and ox-LDL suggest formation of high levels of glycated and lipoxidated proteins and peptides, including HbA1c [36, 37].

RAGE, a receptor for AGE that has been known since 1992, induces activation of nuclear factor κB (NFκB) and converts long-lasting proinflammatory signals into cellular dysfunction, resulting in disease [38–40]. The binding of AGE to its receptor RAGE results in RAGE activation and the production of dysfunctioning proteins in tissues and in blood, and is strongly associated with a series of diseases from allergy and Alzheimers to rheuamtoid arthritis and urogenital disorders [34].

During the processing of food many lipids may become oxidized [30]. Auto-oxidized lipids could interact with various proteins and form new antigenic materials. For this reason, we measured ox-LDL antibodies in samples with or without antibodies aqgainst modified food antigens. IgG-ox-LDL antibody correlated with AGE-HSA, AGE-Hb and modified food antigens (Table 2). In addition to oxidation, LDL, like most plasma proteins, is also susceptible to AGE modification. AGE-modified food proteins are immunogenic, a property that has been used to great advantage for their detection in serum and localization in tissues [41–43]. Antibody formations against ox-LDL and AGE-LDL are able to combine with circulating ox-LDL or AGE-modified antigens and form soluble immune complexes that may contribute to inflammation and autoimmunity [44, 45].

Finally, since the earlier studies it has been suggested that modification in food antigen results in formation of AGE, and that AGEs play a signifcant role in neuronal stress, thus exacerbating aging and neurodegenerative processes in the brain [13, 18, 21, 29, 32, 35, 45, 46]. We measured antibodies against MBP in patients with high levels of antibodies against modified food antigens, AGE-HSA, AGE-Hb and ox-LDL. Results showed that the majority of samples with antibodies against modified proteins and lipoproteins also produced antibodies against MBP. These results further support the idea that formation of AGEs by modified food antigens not only results in antibody formation against dietary proteins and peptides but could also result in antibody production against self proteins, possibly contributing to inflammation, autoimmunity, aging, diabetes and neurodegeneration [34].

From the results presented here, we propose that the determination of allergy and sensitivity to food in the population could be improved by measuring IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Further research is needed to examine just how much an elimination diet could contribute to the reduction of these autoantibodies and a concomitant improvement in the conditions of those suffering from autoimmunity, metabolic syndrome, aging and neurodegenerative disorders.