P1: SFK/UKS BLBS102-c42 P2: SFK BLBS102-Simpson March 21, 2012 14:27 Trim: 276mm X 219mm 806 Printer Name: Yet to Come Part 8: Food Safety and Food Allegens Major Peanut and Tree Nut Allergens The major allergens identified in peanut are Ara h (glycoprotein, vicilin, MW 63.5 kDa), Ara h (glycoprotein, conglutin, MW 17.5 kDa), Ara h (legumin, MW ∼ 60 kDa), Ara h (legumin, MW 37 kDa), Ara h (profilin, MW 14–15 kDa), Ara h (conglutin, MW 14.5 kDa), Ara h (conglutin, MW 15.8 kDa) and Ara h (pathogenesis-related protein, MW 16.9 kDa; Wen et al 2007, Rajamohamed and Boye 2010) Ara h and Ara h are classified as major allergens and are recognised by the sera of >90% of peanut-allergic patients Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h and Ara h are less frequently recognised by the sera of peanut allergic individuals and are classified as minor allergens (Wen et al 2007, Rajamohamed and Boye 2010) Allergenic proteins in tree nuts vary depending on the type of nuts In a voluntary survey report on tree nut allergy conducted by Sicherer et al (2001), 46% of tree-nut-allergic individuals reacted to multiple tree nuts, and 54% reacted to single tree nuts with the highest reactions being reported toward walnut (34%), cashew (20%) and almond (15%) and lower allergic responses to pecan (9%), pistachio (7%), hazelnut, Brazil nut, macadamia nut, pine nut and hickory (less than 5% each) Table 42.3 provides a list of some of the major proteins identified in tree nuts and their properties (Rajamohamed and Boye 2010) Processing-Induced Changes in Peanut and Tree Nut Allergenic Proteins Processing induces changes in peanut and tree nut proteins, which can modify their allergenic properties Many research studies have found, for example, that roasting increases the immunogenic properties of peanut compared to frying and boiling Using sera of peanut-allergic patients, Beyer et al (2001) found lower IgE-binding intensities of Ara h 1, Ara h and Ara h in fried and boiled peanuts compared to roasted peanuts Similarly, Maleki et al (2000) found significant increases in the allergenic properties of roasted peanut compared to raw peanut On the contrary, Koppelman et al (1999) reported no change in the allergenicity of Ara h 1on heat treatment Hansen et al (2003) also reported that dry roasting of hazelnut reduced its allergenicity compared to raw hazel nut Differences in the effect of the thermal treatment on the molecular structure of the proteins and their solubility may explain the variations in the responses reported FISH AND SHELLFISH ALLERGENS Fish and shellfish represent one of the most common sources of food allergens in the adult population Fish and shellfish species known to cause allergic reactions include but are not limited to cod, flounder, grouper, haddock, halibut, hake, herring, mackerel, pike, sole, snapper, trout, crabs, lobsters, prawns, shrimps, crayfish, octopus, squid, clams, mussels, oysters, scallops and snails The major allergen in fish is parvalbumin (Gad c 1), a 12 kDa protein (O’Neil et al 1993) Tropomyosin, with a MW of ∼36 kDa, is the major allergen found in shrimp, lobster, crab and molluscs such as squid, oyster, snail, mussels, clam and scallops (Daul et al 1993a, 1993b) Both parvalbumin Table 42.3 Major Tree-Nut Allergens and Their Characteristics Tree Nuts Allergen Molecular Weight (kDa) Protein Family Allergen Type Identified Epitopes Allergen Stability Cashew Ana o Ana o Ana o Jug r Jug r Jug r Jug r Cor a Cor a Cor a Cor a Cor a 11 Ber e 50 33 and 53 12 14 44–47 NR 18 14 35–40 47 Vicilin (7S) Legumin (11S) Albumin (2S) Albumin (2S) Vicilin (7S) LTP Legumin (11S) PR-10 Profilin LTP Legumin (11S) Vicilin (7S) Albumin (2S) Major Major Major Major Major NR NR Major Major Major Major NR Major 11 22 16 NR NR NR NR NR NR NR NR NR Ber e NR NR 22 and 35 45 20–22 and 38–42 12 Legumin (11S) Vicilin (7S) Legumin (11S) Minor Major Major NR NR NR Thermostable Thermostable Thermostable NR NR NR NR Thermolabile NR NR NR NR Thermostable and resistant to proteolysis NR NR NR Albumin (2S) Major NR NR Walnut Hazelnut Brazil nut Almond NR Source: Adapted from Rajamohamed and Boye 2010 MW, molecular weight; NR, - not reported; PR, pathogenesis-related protein family; LTP, lipid transfer protein References 51, 54 53, 54 52, 54 55, 56 57 58 58, 76 59, 61 59 63 63 63 64, 65 67 70 68, 69 70 P1: SFK/UKS BLBS102-c42 P2: SFK BLBS102-Simpson March 21, 2012 14:27 Trim: 276mm X 219mm Printer Name: Yet to Come 42 Food Allergens and tropomyosin are muscle cell proteins To date, eight major IgE-binding epiopes have been identified in shrimp tropomyosin (Lehrer et al 2003) Exposure, including handling, consumption and inhalation of air-borne particles from fish and fish ingredients, can induce allergic reaction in sensitised individuals Seafood-induced allergic reactions are generally similar to responses induced by many of the other allergenic foods A study conducted using 30 shrimp-sensitive and 37 fish-allergic individuals reported allergic symptoms ranging from generalised itching, urticaria to swelling of the lips and tongue (Lehrer et al 2003) Other reported symptoms include difficulty breathing, gastrointestinal distress and anaphylactic shock (O’Neil et al 1993, Daul et al 1993a, 1993b) Additionally, occupational reactions among a variety of seafood workers (e.g fish and prawn workers, seafood processing workers, fishermen, canners, restaurant cooks and other workers in the seafood industry) have been reported Cartier et al (1984) showed that workers in the seafood industry were exposed to occupational allergens through direct contact with seafood products as well as inhalation of bits of seafood or water droplets generated during processing Out of the 303 crab workers investigated, 18% reported rhinitis or conjunctivitis, about 24% some sort of skin rash and over a third reported of asthma Very little work has been done on the effect of processing on the allergenicity of seafood As with other allergens, highly refined products from seafoods that not contain residual proteins (e.g refined fish oils, gelatine and isinglass) not pose a risk to allergic consumers However, processing techniques that leave seafood protein fragments in the finished product may pose serious allergenic risk to sensitised individuals As consumers and the food industry become increasingly aware of the health benefits of fish, consumption and utilisation of fish and fish products is likely to increase, which could increase the allergen risk for fish- and shellfish-sensitised individuals ALLERGENS IN CEREALS Certain cereal grains contain proteins that induce immunemediated responses in individuals who are predisposed to CD or who have specific cereal allergy As the mechanisms involved in celiac disease are distinctly different from those involved in IgE-mediated cereal allergy, they will be discussed separately Celiac Disease CD also sometimes known as gluten-sensitive enteropathy or gluten intolerance is an abnormal immunological response to gluten/gliadin, which frequently results in a diseased state characterised by damage of the lining of the gut (villous atrophy) In these individuals, the T lymphocytes in the small intestines respond abnormally to gluten, causing inflammation and damage to the absorptive epithelium of the small intestine, resulting in malabsorption and disorders such as diarrhoea, bloating, weight 807 loss, anaemia, weakness and muscle cramps In children, CD leads to growth retardation and underweight Symptoms linger for some days even after the offending food is avoided due to the fact that the damaged intestine requires time to heal Mortality rate has not been reported but patients are likely to develop malignant lymphomas (Ferguson 1997) CD occurs more commonly in Caucasians than in Blacks, Asians and Hispanics according to present knowledge Whether this is due to under-diagnosis or true biosocial/genetic difference is not clear Reported prevalence rates are 1:200–400 in Europe, 1:133 in America, 1:100–300 in a UK study and 1:120 in a Belfast study (Rostami et al 1999, Gomez et al 2001, Fasano and Catassi 2001, Fasano et al 2003, Garc´ıa Novo et al 2007) CD appears to be genetic with 10% prevalence rates reported among first-degree relatives of CD patients and 70–100% concordance rates amongst twins Higher prevalence rates are reported in women likely due to higher rates of diagnoses The primary offending foods for celiacs are wheat, barley and rye The major proteins present in these cereals are albumins, globulins, gliadin (prolamin) and glutenin (glutelin) and the offending protein for celiacs is the gluten fraction in these cereals, which are the prolamins and glutelins, particularly the prolamins (i.e hordein (barley), secalin (rye) and gliadin (wheat)) Several repeating peptide sequences (e.g QQPFP, QQQP, QQPY, QPYP, PSQQ) in the primary structure of these proteins have been blamed (Osman et al 2001, Kahlenberg et al 2006, Darewicz et al 2008) Although the mechanism involved in the pathogenesis of the disease is unclear, tissue transglutaminase is believed to play a key role in the deamidation of glutamine converting it to glutamic acid, which allows the immune cells to bind, provoking continued immune response (Anderson et al 2000, Mazzeo et al 2003) The principal organ targeted is the gut (i.e small intestine); however, damage to other parts of the body such as the skin (dermatitis herpetiformis), the teeth and the liver has been reported (Lohi 2010) Severity of the disease increases significantly with delays in diagnosis (i.e age of diagnosis) and the degree of susceptibility increase with the rate of gluten consumption (quantity) Tolerance thresholds for the general population of CD patients are not known but some workers have reported values ranging between 10 and 100 mg gluten (Collin et al 2004, Hischenhuber et al 2006, Catassi et al 2007) IgE-Mediated Cereal Allergy IgE-mediated cereal allergy is distinctly different from CD This type of allergy is an immediate-type hypersensitivity occurring minutes to hours after consumption of the offending food Symptoms are similar to those described for the other allergens and include oral allergy syndrome (e.g swelling of lips), respiratory difficulties (e.g asthma), skin reaction (e.g eczema, atopic dermatitis) and gastrointestinal distress (e.g nausea, diarrhoea, vomiting and cramps) There is no accurate data available on the prevalence, but as is the case for the other allergens, genetic susceptibility has been suggested (Becker et al 2004) Severity of IgE-mediated cereal allergy depends on the immune state of the patient (degree of sensitisation/tolerance) and the concentration P1: SFK/UKS BLBS102-c42 P2: SFK BLBS102-Simpson 808 March 21, 2012 14:27 Trim: 276mm X 219mm Printer Name: Yet to Come Part 8: Food Safety and Food Allegens of antigen in the food consumed The proteins responsible for this type of allergy can be the albumins, globulin, gliadins or glutenins and will vary depending on the individual and the specific allergenic food Offending foods include buckwheat, rice, corn, millet, wheat, oats, rye and barley (Cantani 2008) Buckwheat and rice allergies are more frequently observed in Asia than in Europe or North America (Taylor and Hefle 2001, Kumar et al 2007) The pathway for sensitisation and cereal allergy elicitation is by ingestion (mouth) and/or inhalation (nose) (e.g baker’s asthma) Wheat allergy is responsible for up to 30% of occupational asthma in the bakery industry Another type of cereal allergy is exercise-induced cereal allergy Symptoms in this case appear only after food allergen consumption is followed by exercise Wheat, as well as shellfish and nuts, is mostly associated with this type of allergy (Romano et al 2001, Beaudouin et al 2006, Porcel et al 2006) Foods to Avoid for Gluten-Sensitive Enteropathy Patients and IgE-Mediated Cereal-Allergic Patients As with the other allergens, there are presently no cures for CD Major foods to avoid include barley, wheat, durum, farina, kamut, rye, semolina, spelt and triticale Oats was previously included in the list of gluten-containing foods; however, several recent studies suggest that quantities of oats of up to 50 g/day are harmless to the majority of gluten-sensitive individuals (Janatuinen et al 1995, 2000, 2002, Lundin et al 2003) A major challenge for the industry is that commercial oats is very frequently contaminated with high amounts of wheat, barley or rye (Thompson 2004, 2005) as these crops are often grown in the same regions and in close proximity Furthermore, a small percentage of CD patients may react to oat proteins The mechanisms at play in this instance and the specific proteins in oats to which these patients react still remain to be clarified As gluten-containing cereals are often used as ingredients in food formulation, other foods to avoid include hydrolyzed vegetable protein, flavouring, malt, maltodextrin, malted barley, malt vinegar and starch from gluten sources, especially if these contain residual amounts of gluten proteins Many countries have adopted the gluten-free Codex Alimentarius Standards (Joint FAO/WHO Food Standards Programme and Codex Alimentarius Commission 2008), which sets a maximum limit of 20 ppm for gluten-free foods that are naturally free from gluten and 100 ppm for gluten-free foods that have been rendered gluten-free through processing For IgE-mediated cereal allergy, foods to avoid will depend on the particular allergy Major challenges for cereal-allergic individuals are cross-reactivity and cross-contamination of foods Food ingredients that are particularly problematic if not properly labelled are starches, spices, seasonings, sauces, flavourings, colourings, some vinegars, hydrolysed plant protein, syrups (e.g brown rice syrup), beverages (e.g beer, ale, etc.) Other potential hidden sources of cereals or cereal ingredients are cosmetics, pillows, toy stuffings and certain medications when products derived from these are used as ingredients SESAME AND MUSTARD ALLERGENS Sesame Allergy Sesame (Sesamum indicum) is a herbaceous plant of the Pedaliaceae family originating from India, which is now grown in many countries It is also known as Benne, Gingelly, Til or Teel, Simsim and Ajonjoli and is now a common ingredient used extensively in everyday foods because of its high nutritional value (Perkins 2000) Sesame proteins are rich in methionine (Dalal et al 2002, Wolff et al 2003) Common sesame products include biscuits, crackers, breadsticks, rice cakes, etc., as well as prepacked delicatessen and processed foods such as noodles, dips, soups, sausages, samosas, processed meats, vegeburgers, chutneys, etc., (Perkins 2000, Allergyexpert 2010) Sesame seeds, which may be used whole or crushed, are extremely potent allergens, causing severe allergic reactions in susceptible individuals The first case of sesame allergy was reported in 1950 (Gangur et al 2005) More recently, a study of Australian children showed that allergic reactions to sesame ranked fourth behind reactions to egg, milk, and peanuts, and sesame was also found to be the third most common allergyinducing food in Israeli children (Gangur et al 2005) Another recent study showed that sesame allergy in Israeli children was more common than peanut allergy (More 2009) Sesame allergy seems to affect people of all ages, which imply that this food allergy is not commonly outgrown The symptoms of sesame allergy can include urticaria, angioedema, asthma, atopic dermatitis, oral allergy syndrome, allergic rhinitis and anaphylaxis One of the major sesame seed allergens is the kDa, 2S albumin (Pastorello et al 2001) Beyer et al (2002) also identified 10 IgE-binding proteins in sesame, four of which had MWs of 7, 34, 45 and 78 kDa Wolff et al (2003) have also reported a 14 kDa sesame allergenic protein belonging to the 2S albumin family Sesame products are used in a wide variety of food products and may represent hidden allergens in foods Fatal anaphylactic reactions have occurred as a result of consuming sesame (Gangur et al 2005) Unrefined sesame oil may be used in food products, which may trigger allergic reactions in susceptible individuals The oil resists rancidity and is popular with Oriental chefs In the bakery industry, workers have reported allergic reactions, which include asthma, to sesame products In addition to its use in the food industry, sesame and sesame products are used in the pharmaceutical and cosmetic industries Sesame products used in cosmetics and ointments may cause allergic dermatitis, an inflammatory condition of the skin, in sensitised individuals Contact dermatitis as a result of direct exposure to cosmetics or pharmaceutical products containing sesame allergens has been reported Specific instances of sesame allergy resulting in skin rashes and inflammation after baking with sesame seeds and skin rashes from cosmetics that contain sesame oil have also been reported (Stoppler and Marks 2005) As sesame allergens are similar in biochemical structure to peanut allergens, people with sesame allergy may be at risk of having allergic reactions as a result of eating peanuts and viceversa (Gangur et al 2005) Cross-reactivity may also exist with rye, kiwi, poppy seed and various tree nuts (such as hazelnut, P1: SFK/UKS BLBS102-c42 P2: SFK BLBS102-Simpson March 21, 2012 14:27 Trim: 276mm X 219mm Printer Name: Yet to Come 42 Food Allergens black walnut, cashew, macadamia and pistachio), but clinical studies are lacking While the number of reports of sesame allergy has steadily increased, it is still not clear whether this increase is in the number of reactions or an increased rate of detection and reporting of these allergic reactions (Stoppler and Marks 2005) Mustard Seed Allergy The mustard plant belongs to the family Brassicaceae with cabbage, cauliflower, broccoli, Brussel sprout, turnip and radish (Monreal et al 1992) Mustard is often consumed as a condiment prepared from the mustard seed The powder is made from a mixture of two species, Sinapis alba L (yellow mustard) and Brassica juncea L (oriental mustard) The varieties Brassica nigra and Brassica juncea are used for food products In addition to mustard powder, mustard is also usually found in salad dressing, mayonnaise, soups and sauces (Ensminger et al 1983) Mustard allergy is now considered to be a very common food allergy, accounting for about 1.1% of food allergies in children (Morisset et al 2003b) and ranks fourth in children’s food allergies after eggs, peanuts and cow’s milk (Rance et al 2000) The major allergen of yellow mustard is Sin a 1, which has been found to be resistant to heating and proteolysis (Dominguez et al 1990, Gonzalez de la Pena et al 1996, Rance et al 2000) Bra j is the major allergen in oriental mustard with a structure similar to that of Sin a (Gonzalez de la Pena et al 1991, Caballero et al 1994) Mustard allergy can cause a wide array of symptoms Rance et al (2000) reported that allergic reactions to mustard start early in life and are probably linked to early consumption of baby foods Mustard-allergic patients will react to any food that comes from the mustard plant, including jars of mustard, mustard powder, mustard leaves, seeds and flowers, sprouted mustard seeds, mustard oil and foods that contain these (Bock 2008) Common symptoms of mustard allergy include difficulty in breathing, shortness of breath and other breathing complications, a rash or hives, itchy skin or general skin irritation In some severe cases, it can lead to anaphylaxis, and if left untreated, anaphylaxis can lead to anaphylactic shock and even death Some of the common symptoms of anaphylaxis include constriction of airways in the throat and lungs, anaphylactic shock, severe drop in blood pressure, heightened pulse and heavy heartbeat, dizziness, nausea and abdominal pain, confusion and disorientation, and loss of consciousness Panconesi et al (1980) reported the first case of mustard-induced anaphylaxis after the subject had consumed pizza Incidents of cross-reactions have been rarely reported However, single cases have been described of cross-allergy to cauliflower, broccoli, cabbage and Brazil nuts, which may be linked to sequence homology of some proteins (Moneret-Vautrin 2006) One of the major challenges of mustard allergy is that many foods contain mustard even when it would seem unlikely (e.g lunchmeat and hot dogs) Thus, careful reading of labels on processed foods is important for mustard-allergic patients 809 MINOR FOOD ALLERGENS As previously indicated, over 170 foods are known to provoke allergic reactions in humans (Taylor 2000) In addition to the nine major priority allergens, other minor food allergens and/or emerging allergens include lupin, pea, chickpea, lentil, fruits (e.g apple, apricot, avocado, banana, cherry, grape, kiwi, mango, melon, peach, pear, pineapple and strawberry) and vegetables (celery, carrot, eggplant, lettuce, potato, pumpkin and tomato) The reader is referred to the following references for further reading (Pereira et al 2002, Fern´andez-Rivas 2003, Fern´andezRivas et al 2008, Harish Babu et al 2008, Towell 2009, Skypala 2009, Jappe and Vieths 2010) MANAGEMENT OF FOOD ALLERGY Many of the priority food allergens such as milk, eggs, nuts and soya bean are commonly used in food processing (e.g processed beef, sausages, salad dressings, breads, cakes, soups and sauces) and pharmaceutical products (e.g casein hydrolysates as “drug” carriers) due to their desirable properties (Monaci et al 2006) Extensive use of these foods as ingredients in various products increases the chances of their presence as hidden allergens, particularly when they are undeclared or present as a result of cross-contact As there are currently no cures for food allergy, the best management tool is the reading of food labels and avoidance of foods containing allergens or allergen-derived ingredients As a result, many countries presently require the use of the common names of priority allergens when they are used as ingredients in foods A major development in the last decade has been the use of precautionary allergen labelling to warn allergic consumers of the likely presence of allergens Unfortunately, consumers with food allergy have become less avoidant to products with advisory labels, such as “may contain” or “shared equipment” due to their misuse (Hefle et al 2007) Pieretti et al (2009) studied the use of advisory labels in the United States and found that 17% of 20,241 manufactured foods contained advisory labels Thus, the unregulated use of advisory labels has become a source of confusion and frustration, which could eventually pose challenges for allergic consumers (Pieretti et al 2009) Recent emergence of the hygiene hypothesis (Yazdanbakhsh et al 2002) brings a new approach for the management of allergic diseases Several epidemiological studies have suggested that allergic diseases are more common in industrialised nations and urban areas compared to developing countries and rural areas Less frequent microbial exposures in the developed world has been regarded as an important predisposing factor for having higher allergic population in these regions To compensate the inadequate exposure of microbial load, probiotics (live bacteria, usually Lactobacillus and Bifidobacterium) are now being added in some infant formula along with oligosaccharides (prebiotics; Matricardi et al 2003) The goal is to achieve the development of regulatory T cell or balanced Th1/Th2 activity, which could eventually prevent allergy development (Cross et al 2001, Prioult et al 2004) Further research will be required to confirm the purported effects P1: SFK/UKS BLBS102-c42 P2: SFK BLBS102-Simpson 810 March 21, 2012 14:27 Trim: 276mm X 219mm Printer Name: Yet to Come Part 8: Food Safety and Food Allegens Another area of interest is specific oral tolerance induction (SOTI) Taking milk as an example, about 15% of children with milk allergy maintain the susceptibility permanently in their life Strict avoidance of cow’s milk and cow’s milk derivatives remains the gold standard for allergen management for such CMA patients For these patients, however, the likelihood of exposing the offending allergen unintentionally always remains and thus total avoidance cannot be guaranteed Similar concerns exist for patients with persistent peanut and tree nut allergies SOTI is a promising approach particularly for patients with persistent food allergy Staden et al (2007) reported that SOTI treatment (using a daily dose of CMP, starting from 0.002 mg CMP) remarkably increased the threshold dose for allergic reaction in CMA patients As a result, the patient could be protected against potentially fatal allergic reactions when small amounts of the hidden allergen are accidentally ingested Since this approach uses the specific offending allergen to induce tolerance, development of mild to moderate side effects (such as itching in the mouth, nausea and wheals, etc.) is very common Close supervision by an experienced professional is, therefore, necessary for SOTI treatment Similar promising studies have been reported for peanut (Blumchen et al 2008, Clark et al 2009, Jones et al 2009) For the food industry, food allergen testing has become an increasingly important tool for the management and control of allergens during food production and processing The rest of the chapter provides a brief summary of some of the methods currently available for allergen detection METHODS FOR DETECTING ALLERGENS Table 42.4 provides examples of some of the commonly used methods for food allergen detection Food allergens may be detected either by the presence of the target allergen itself or by using a marker in the target food The method of choice will depend on factors such as food matrix interference, nature and quantity of the target allergen, the desired level of detection, specificity and time and resources required for running the assay The threshold dose for sensitised individuals to manifest allergic reactions is quite low and variable among individuals (BindslevJensen et al 2002) However, it is commonly agreed that food allergen detection methods should be sensitive enough to detect 1–100 mg of analyte (allergen) per kg of processed food (Poms et al 2004) Increasing numbers of rapid and user-friendly test kits to detect different food allergens are now commercially available with sensitivities ranging from 0.05 to 10 mg/kg (Table 42.5) Protein-based food allergen detection methods include immunoblotting, rocket immunoelectrophoresis, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), protein microarray and biosensors More recently developed DNA-based methods are available as supplementary and complementary methods to protein-based methods and are particularly useful in species differentiation and detection of genetically modified food (Mustorp et al 2008) This chapter will focus on ELISA-based detection, lateral-flow assays (LFA) and dipstick test, proteomic approaches and DNA-based allergen detection methods ELISA-Based Detection Methods Among the growing number of food allergen detection techniques, ELISA has gained prominence and has become an extensively used technique due to its high sensitivity and specificity, availability of automation and user friendliness Some ELISAs are designed to detect specific allergens (e.g BLG, Ara h1 (a major peanut allergen) and shrimp tropomyosin), whereas other ELISAs detect mixtures of proteins from the allergenic source (e.g total milk, egg, peanut and almond-soluble proteins; Taylor et al 2009) Detection is based on binding of an allergen or a specific marker protein with an antibody specifically generated to recognise and bind to these proteins This binding complex is visualised by a colourimetric assay when the enzymes, which are labelled to the allergen specific antibody, interact with the substrate solution The concentration of the allergen can be quantified by obtaining optical density (OD) values with a microplate reader and plotting these values using a standard curve There are certain limitations of ELISA-based detection methods, however (Yeung 2006) ELISAs are based on aqueous systems and they not work well in detecting insoluble proteins or allergens derived from edible oil-producing foods In addition, ELISA may not detect oleosins, a family of protein involved in the formation of oil bodies in peanut and sesame (Pons et al 2002 and Leduc et al 2006) and soya lecithin containing more than 50 ppm residual protein (Taylor et al 2009) ELISAs also fail to distinguish certain closely related foods For example, walnut antisera react with pecan (Niemann et al 2009) and mustard antisera react to rapeseed (Lee et al 2008) These may be caused by the presence of cross-reactive epitopes or proteins between two foods (Taylor et al 2009) Regardless of the above-mentioned limitations, ELISA is still a very powerful tool and remains the method of choice for food allergen detection ELISA-based food allergen detection methods are developed using either the sandwich ELISA or competitive ELISA format Sandwich ELISA In this assay, as indicated by its self-explanatory name, an allergen of interest is captured between two allergen-specific antibodies The first allergen-specific antibody is immobilised on the solid phase to capture the allergen, and the second allergenspecific antibody is labelled with enzyme to detect the captured allergen Allergens need to have enough binding sites, for example, large molecules such as proteins, to allow binding of the two allergen-specific antibodies (Schubert-Ullrich et al 2009) Competitive ELISA This assay is based on the competitive binding of unknown analyte (allergen) in a sample of interest and a known analyte (allergen) to the allergen-specific antibody The antigen of interest in this case is incubated in the presence of the unlabelled antibody Antibody/antigen complexes are formed and the solution is then ... sensitisation/tolerance) and the concentration P1: SFK/UKS BLBS102-c42 P2: SFK BLBS102-Simpson 80 8 March 21, 2012 14:27 Trim: 276mm X 219mm Printer Name: Yet to Come Part 8: Food Safety and Food Allegens... Alimentarius Standards (Joint FAO/WHO Food Standards Programme and Codex Alimentarius Commission 20 08) , which sets a maximum limit of 20 ppm for gluten-free foods that are naturally free from gluten and. .. Fern´andezRivas et al 20 08, Harish Babu et al 20 08, Towell 2009, Skypala 2009, Jappe and Vieths 2010) MANAGEMENT OF FOOD ALLERGY Many of the priority food allergens such as milk, eggs, nuts and