Food Control 54 (2015) 300e307

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Detection of peanut (Arachis hypogaea) allergens in processed foods by
immunoassay: Influence of selected target protein and ELISA format
nchez a, M. Calvo a,
M. Montserrat a, D. Sanz b, T. Juan c, A. Herrero d, L. Sa
María D. Pe

Tecnología de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain
ZEULAB S.L., Polígono PLAZA, Bari, 25 Duplicado, 50197 Zaragoza, Spain 
n y Tecnología Agroalimentaria de Arago 
n, Avda. Montan
~ ana 930, 50059 Zaragoza, Spain
Centro de Investigacio
~ o km 14, 50180 Utebo, Zaragoza, Spain
Chocolates Lacasa, Autovía de Logron

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 October 2014
Received in revised form
26 January 2015
Accepted 28 January 2015
Available online 17 February 2015

Direct competitive and sandwich ELISA formats developed to determine Ara h1 and Ara h2 proteins were
applied in the detection of peanut in model biscuits prepared with a commercial peanut butter as
ingredient. The sandwich format for Ara h2 protein could detect the addition of 2.5% peanut butter,
whereas the same format for Ara h1 could not detect 5% added peanut. Direct competitive formats for
Ara h1 and Ara h2 proteins could detect the addition of 1% and 0.05% peanut butter, respectively.
Therefore, competitive format for Ara h2 was selected to be evaluated by four laboratories, obtaining
adequate results in term of repeatability and reproducibility. Results obtained indicate that processing
decreased the level of extracted proteins and underestimated the amount of Ara h1 and Ara h2 proteins,
the effect being more severe for Ara h1. The selection of the target protein and the ELISA format applied
greatly influence the detection of peanut in processed foods.
© 2015 Elsevier Ltd. All rights reserved.

Ara h1
Ara h2
Peanut detection
Processed foods
ELISA assay

1. Introduction
Food allergy has emerged as a serious public health problem
over recent years and its prevalence is rising, especially in industrialized countries. The reason appears to be related to changes in
dietary habits as well as to the use of complex technological processes and ingredients in food industry (Nwaru et al., 2014; Sicherer
& Sampson, 2010).
The estimated prevalence of peanut allergy in developed
countries is between 0.6% and 1.0%. Peanut allergy deserves
particular attention because very small amounts of peanut proteins
can induce severe allergic reactions, it persists throughout life and
it accounts for most of food-induced anaphylactic reactions (AlMuhsen, Clarke, & Kagan, 2003; Wen, Borejjsza-Wysocki, De
Cory, & Durst, 2007).

* Corresponding author. Tel.: þ34 876 554240; fax: þ34 976 761590. 
E-mail address: (M.D. Pe
0956-7135/© 2015 Elsevier Ltd. All rights reserved.

Until now, thirteen peanut proteins with allergenic capacity
have been identified, and designated from Ara h1 to Ara h13 (Bublin 
iz, Montealegre, Marina, & García-Ruiz,
& Breiteneder, 2014; Sa
2013). Ara h1 and Ara h2 proteins are considered as the major allergens of peanut, more than 65% of peanut allergic individuals
have specific IgE to Ara h1 and more than 71% to Ara h2 (Scurlock &
Burks, 2004). They are both major proteins in peanut, as they account for 12e16% and 5.9e9.3% of the total seed protein content,
respectively (Koppelman et al., 2001).
Ara h1 is a seed store glycoprotein that belongs to the vicilin
family. It has a molecular mass of 63.5 kDa in its monomeric form
and an isoelectric point of 5.2. It exists as a trimer formed by three
identical monomers stabilized mainly by hydrophobic interactions.
Ara h2 is a glycoprotein of the conglutinin family with a molecular
mass of 17.5 kDa and an isoelectric point of 4.6 (Wen et al., 2007).
Both proteins have been found to maintain the IgE binding capacity
after being exposed to thermal treatments or in vitro digestion with
pepsin, chymotrypsin and trypsin (Lehmann et al., 2006; Maleki,
Chung, Champagne, & Raufman, 2000; Mondoulet et al., 2005).

the enzymatic reaction was stopped by adding 50 mL of 2 M H2SO4 per well. pH 9. 2012. 2013. The care and use of animals were performed following the Spanish Policy for Animal Protection RD 1201/05. 2.2. 2005. 2013.4 (PBS) for 2 h at 37  C and washed with PBS containing 0. 301 2. It is worthwhile to remark that the determination of peanut proteins in foods can be impaired by their interaction with compounds of the complex food matrix and denaturation during processing.2. 2012). Calvo. The implementation of a management plan in the food industry. / Food Control 54 (2015) 300e307 The way to prevent peanut allergy is the strict avoidance of peanut consumption. Spain) and Maxisorp microtitration plates from Nunc (Roskilde. among others (Vierk. wells were blocked with 300 mL of 2% (w/v) ovalbumin in 8 mM Na2HPO4. Enzymelinked immunosorbent assay (ELISA) is the technique most widely used by food industries and official food control agencies for monitoring adventitious contamination of food products by allergenic ingredients because of its sensitivity and specificity (Monaci & Visconti. protein recognition by antibodies is reduced (Chassaigne. Tetramethylbenzidine (TMB) substrate (Reference ZE/TMB125) was obtained from ZEULAB (Zaragoza. 1994). Falci. After washing with PBST. pH 8. Horseradish peroxidase (HRP. 2002). & Pe In this work. plates were coated with 120 mL per well of Ara h1 or Ara h2 proteins (5 mg/mL) in 50 mM sodium carbonate buffer. Brohe Nørgaard. 0. Castillo. Kiening et al. suspended in Tris buffer and filtered. 2007). contamination with hidden allergens can occur due to inefficient cleaning procedures of the production equipment or the use of contaminated raw ingredients. pH 7. Preparation and conjugation of antibodies to Ara h1 and Ara h2 Antisera to Ara h1 and Ara h2 were obtained by immunization of rabbits as previously described (Wehbi et al.3. 2004). Spain). calibration curves for the sandwich assay were obtained using the relationship between the value of absorbance and the logarithm of the concentration of standard solutions. in 50 mM sodium carbonate buffer. However. Poms et al. & van Hengel. 2010). 2005). wells were incubated with 100 mL of TMB substrate for 20 min at room temperature. even though it did not involve the minimum number of laboratories requested by a full interlaboratory study as defined in the ISO 5725 standard (ISO. Therefore. Khuda et al. Sa rez. based on the determination of Ara h1 or Ara h2 proteins (sandwich and direct competitive assay for each protein) have been developed. determined by SDS-PAGE was higher than 95%. Khuda et al. 3 mM KCl. These studies include the design of one ELISA format (sandwich or competitive) and are based on the determination of one selected target (a mixture of peanut proteins or a specific peanut protein) s et al. protein extraction greatly decreases and e. plates were coated with 120 mL per well of anti-Ara h1 or anti-Ara h2 antibodies (5 mg/mL). IL. 2007.5 mM KH2PO4 buffer. 2013. and the absorbance determined at 450 nm using a microplate reader (Labsystem Multiskan.. 2013) and fractions enriched in Ara h2 protein onto a Sephadex G-50 column (80  1 cm). 250e503 units/mg) and goat anti-rabbit IgG antibodies labeled with peroxidase were purchased from Sigma Chemical (Poole. & by Western blotting analysis (Franco. Montserrat et al. 1999. (2013). Calvo. Finally. four ELISA assays for the detection of peanut. 2003. Wolyniak.2. The performance of the four assays was evaluated using biscuits containing defined concentrations of a commercial peanut butter as ingredient.1. Consequently. Several recent studies have shown that results obtained by different ELISA tests give significantly varying results in quantitative assays when they are used to detect peanut in processed foods (Khuda et al. (Holzhauser & Vieths. wells were washed and blocked with ovalbumin as indicated above. Materials Raw peanuts and peanut butter from the Spanish variety was provided by Chocolates Lacasa (Utebo. USA).M. The concentration of Ara h1 and Ara h2 in the test samples was determined by interpolating absorbance data in the corresponding calibration curves. plates were incubated for 30 min at 37  C with . UK). pH 9. Specific antibodies to Ara h1 or Ara h2 were purified by affinity chromatography using immunosorbents of the corresponding proteins as described by Montserrat et al. Proteins precipitated between 40 and 80% ammonium sulfate saturation was collected by centrifugation. 1974). Maks. Antibodies were conjugated with HRP using the periodate method (Nakane & Kawaoi. Isolation of Ara h1 and Ara h2 Peanut proteins were extracted by stirring 20 g of ground raw peanut with 100 mL of 50 mM TriseHCl buffer. Peanut butter was prepared by roasting whole peanuts in a flame oven at 225  C for 27 min and afterwards.14 M NaCl. which meets the European Union Directive 86/609 on the protection of animals used for experimental and other scientific purposes. This variability may be explained by the fact that ELISA tests can use different antigens as targets. Specificity of antisera against Ara h1 or Ara h2 proteins were assessed rez.0 were added to the wells and incubated for 30 min at 37  C. Montserrat. & Hefle. After overnight incubation at 4  C.1. Mayayo. Materials and methods 2. Finland). 2. All procedures were approved by the Ethic Committee for Animal Experiments from the University of Zaragoza (Project Licence PI 48/ 10). Pe S anchez. Anklam. Afterwards. wells were incubated with 100 mL of anti-Ara h1 or anti-Ara h2 antibodies HRP-conjugated diluted 1/6000 and 1/10.. pH 9. Denmark). & Klonz.1 M sodium borate buffer.2. 2010).2. The bicinchoninic acid (BCA) assay kit was from Pierce (Rockford. Calibration curves for the sandwich assay of Ara h1 was obtained by plotting absorbance versus the concentration of standard solutions.2.. this part of the study is called interlaboratory study. the enforcement of labeling rules and its control by authorities are important strategies for protecting against allergic reactions...6 overnight at 4  C. 100 mL of Ara h1 and Ara h2 standards or samples diluted in 0. The purity of isolated proteins. For Ara h2. Fu & Maks. Pome Stephan & Vieths.6. After washing with PBST. Taylor. Several studies have been performed to develop ELISA techniques to detect peanut in foods. Sandwich and direct competitive ELISA assays for Ara h1 and Ara h2 For the sandwich ELISA. Methods 2.5 cm) as previously described (Montserrat et al.000. by grinding in a stone mill to obtain an emulsion with dark color. Helsinki. van Hengel. For clarity and explanation. Then. Then... 2005). The ELISA format and the target protein that gave the best sensitivity was selected to determine peanut content in model biscuit samples in blind duplicate by four laboratories.. For the direct competitive ELISA. The extract was applied onto a Sephacryl S-200 column (90  2 cm). reliable methods to detect peanut are required to ensure compliance with the labeling legislation and to assist food manufacturers in order to improve consumer protection. respectively in the same buffer for 30 min at 37  C. 2. 2012. 1.5% Tween 20 (PBST). Fractions enriched in Ara h1 were applied onto a QSepharose column (15  1. antibodies for antigen recognition and assay formats (Fu & nchez.

Oster.25% samples with the blank sample into plastic tubes to give a total weight of 3. The coordinator provided two sets of 8 pre-weighed test samples. The ingredients were kneaded for 30 min using a bread and dough maker (Deluxe: Bread and Dough Maker. In this study. Results 3. biscuit samples without added peanut gave a concentration value below the cut-off calculated for each format assay.000 and 1/40.0 and incubated in a shaking water bath at 30  C for 15 min. USA) equipped with a blade type “pigtail”.01 g. Each set of samples was extracted once in different days and analyzed in triplicate in the ELISA assay.0 and 50 mL of HRP-labeled anti-Ara h1 or anti-Ara h2 antibodies diluted 1/30. 2006) (Table 1). biscuits were introduced into an oven and cooked at 160  C for 12 min.3. 2. Preparation of model biscuits Biscuits were prepared at the pilot plant of the University of Zaragoza following standard manufacturing processes.0%.01. & Oliver. respectively.1 M borate buffer. and the supernatants stored in aliquots at 20  C until use.25. The detection limit (LOD) of the immunoassays tests was determined as the mean concentration of Ara h1 and Ara h2 corresponding to the absorbance of eight replicates of the blank standard plus 3. 40 g of homogenized material was placed in a baking mould (10 cm diameter) and pressed to obtain round cookies of 1 cm height. The relationship found was linear within the range of concentrations between 20 ng/mL and 2 mg/mL for direct competitive assays and for the sandwich format of Ara h2. Then.985 (Fig. Specificity of antisera to Ara h1 and Ara h2 The specificity of antisera against Ara h1 and Ara h2 proteins were assessed by Western blotting (Fig. Fig. after washing wells were incubated with TMB substrate and enzymatic reaction stopped with H2SO4 before measuring absorbance at 450 nm.3 times the value of its stan et al. 300 g wheat flour. 150 g sugar and peanut butter to obtain final concentrations of 0. pH 9. Evaluation of direct competitive ELISA for Ara h2 The evaluation study was performed following the procedure previously described (Abbott et al. Results showed that antibodies to Ara h1 only reacts with Ara h1 and antibodies to Ara h2 only bind to Ara h2. In both cases.302 M. 1). Extraction of test samples was performed as indicated above. Previously. 2. Four laboratories with ELISA experience participated in this study to evaluate the direct competitive ELISA for Ara h2 protein to detect peanut in model biscuits. 1. respectively in the same buffer. 2. AOAC. The samples to be sent to the participants were prepared as follows. An amount of 3. Supernatants were directly assayed in the ELISA plates. 0. All assays gave regression coefficients r2  0. A plot of logit (r) of standards against the log10 of the concentration. Absorbance data of calibration standards and blind samples of each set were sent to the coordinator. 0. Direct competitive assays for Ara h1 and Ara h2 proteins could detect biscuits samples containing 1.5.. Biscuits with peanut butter concentrations of 0. whereas the same format for Ara h1 could not detect samples containing 5. This value was estimated as the average concentration of the blank biscuit plus 3. 2). Calibration curves for direct competitive assays were obtained using the logit log model (Nix & Wild. 1. SDS-PAGE (a) and Western-blotting against rabbit antiserum to Ara h1 (b) and Ara h2 (c) of raw peanut extract. The assumption dard deviation (Lexmaulova of this value ensures that interference caused by the matrix effect in each assay is minimized.1.0 and 2.2. Biscuit samples were extracted in three different days and assayed by triplicate.25.6.1% were prepared by mixing appropriate quantities of the ground 0.2. Finally.4. where B is the absorbance of each standard and B0 the absorbance of the blank standard was calculated.1 M sodium borate buffer. 2010. 0. (w/w). Extracts were clarified by centrifugation at 3000g for 15 min.01 g was weighted into 50 mL plastic tubes. Calibration curves were obtained for each ELISA assay using the logit log model.0.. 2000). reagents. The fraction bound (r ¼ B/B0).2. 2. that were chosen for the validation and the interlaboratory study. .000. They were made by mixing 6 hen eggs (55e65 g). and curvilinear between 20 ng/ mL and 800 mg/mL for the sandwich format of Ara h1 protein. Montserrat et al.5.5 and 5. pH 9.5% peanut butter were ground and 3.3 times the standard deviation (Miller. Torrance. 2012).05% of peanut addition. Then. Extraction procedure Food samples purchased from local retailers and model biscuits were ground into fine powder with a mincer. / Food Control 54 (2015) 300e307 50 mL of protein standards or samples diluted in 0.01 g of ground samples were extracted in 30 mL of 0. and ZEULAB provided the ELISA kits containing plates. randomly coded.5. where logit (r) ¼ ln [(1  r)/r] was obtained.00 ± 0. a cut-off value was established to consider a sample as positive for peanut addition for each ELISA test. 3.2. 120 g butter. Biscuits containing 0. no reaction was observed with any other protein from crude peanut extract demonstrating that antisera obtained were specific for each protein.0% peanut. standards and instructions.00 þ 0. 3. 3. Determination of repeatability and reproducibility data were calculated according to ISO 5725. The study was coordinated by the group of the University of Zaragoza. The concentration of Ara h1 and Ara h2 in tests samples was determined from its fraction bound.5% peanut. 1. 2013) (Table 1). Determination of peanut in model biscuits Results obtained in the analysis of model biscuits which contained different amounts of peanut butter using sandwich and direct competitive assays to determine Ara h1 and Ara h2 proteins are shown in Fig. 0. 3. Development of sandwich and direct competitive ELISA for Ara h1 and Ara h2 Immunoassay formats for Ara h1 and Ara h2 were optimized to choose the assay conditions which gave the highest sensitivity.00 ± 0. which is the ratio between absorbance of the sample and absorbance of the blank standard (B0).05 and 0. 0. The sandwich format based on Ara h2 protein could detect the addition of 2.0% and 0.

Montserrat et al. b) and direct competitive (c. Concentration of immunoreactive Ara h1 (a.0-5.01) 0. d) ELISA. 3.24 ± ± ± ± 0.07 (0. Lines indicate the cut-off value above which biscuits are considered positive for peanut butter addition. estimated using the direct competitive assays was 1000 ± 20 and 2750 ± 13 mg/kg.0-8. The protein concentration in the peanut butter extract was of 8. Samples of raw peanut from the same variety were also analyzed and a protein content of Table 1 Limit of detection (LOD) of the ELISA tests for Ara h1 and Ara h2 and cut-off establish for the ELISA tests to determine a biscuit sample as positive for peanut addition.02 ± ± ± ± Cut-off (mg/kg) 0.3 SD of the blank biscuit.M.06 h1 h2 h1 h2 (0. Values are the mean þ SD of three sample extractions assayed by triplicate expressed in mg/kg.0-20. c) and Ara h2 (b.01) 0. estimated by the bicinchoninic acid assay. d) in model biscuits added with different amounts of peanut butter.07) 0. Calibration points correspond to the protein concentration of standards used in each ELISA tests.13 0.11 (0. On the other hand. d) concentration in standard solutions of pure proteins. / Food Control 54 (2015) 300e307 303 Fig. Sandwich (a. .0 0-0. c) and Ara h2 (b. Biscuit samples which contained a lower percentage of peanut than those indicated above gave false-negative results in the corresponding assays and those which contained higher percentages gave a concentration of Ara h1 and Ara h2 that increased gradually.04 (0.12) Calibration points (mg/kg) 0-0.64 (0.30 0.08) 0.5-20.0 0-0.03) 0.19 0.0-8.20 0.42 0.2-2. d) ELISA formats for determination of Ara h1 (a.10 (0.0 Fig.16 (0. b) and direct competitive (c.5-20.1 ± 0. Mean value þ SD are given in brackets. Test format Target protein LOD (mg/kg) Sandwich Sandwich Competitive Competitive Ara Ara Ara Ara 0.2-1. 2.0-5.05) 0. and of Ara h1 and Ara h2 by ELISA was determined in peanut butter and in raw dough of biscuits.02) 0. and were calculated as the mean value þ 3. respectively.10 0.0 0-0. Calibration curves obtained for sandwich (a.05 (0.2-2.0-5.4% (w/w) and the concentration of Ara h1 and Ara h2 proteins.2-1. the concentration of soluble proteins.

42 0.61 1. Evaluation of direct competitive ELISA for Ara h2 The direct competitive ELISA test to determine Ara h2 protein was evaluated by four laboratories for the detection of peanut in the model biscuits.38 2. legumes (chick pea..16 43.30 0.00 2.05% of peanut butter.22 0.25a 0. using the direct competitive ELISA format. Fu & Maks.93 62. Schmitt. The cut-off value for the interlaboratorial study was determined as 3. respectively. seasoning blends. homology at surface residues requires a higher degree of amino acid identity s et al.20 0. Ara h2 was detected in samples with a percentage equal or higher than 0. Lab 2 ± ± ± ± ± ± ± ± a 0. 3. 2010).19 0.52 0. cashew nut.09 0.48 9. the two major peanut allergens.91 20.5.24 0. Concentration of Ara h2 in two set of blind biscuit samples prepared with peanut butter were determined. Protein concentration of extracts assayed ranged from 0 to 32 mg/kg.76 5.35 1.11 15. creams and cereal bars.37 5. soya.3 times the reproducibility (SR) of the blank biscuit  et al.98 1. obtaining a value of 0. We selected this processed material to prepare biscuits because it is commonly used in the elaboration of nougats.09 0. (Lexmaulova The four laboratories obtained concentrations of Ara h2 in the blank biscuit samples below the cut-off established for interlaboratory study to consider a sample as positive.25 0.45 Lab 1 ± ± ± ± ± ± ± ± a 0.69 14.11 1.13a 0. 2004). 2000). / Food Control 54 (2015) 300e307 16.29 0.69 4.46 5. Our results are in good agreement with those previously reported on the effect of thermal processing of peanut on protein solubility and detectability by ELISA techniques (Chassaigne et al. chocolate. Zeece.60 4.40 1.36a 0.02 9.81 mg/kg.50 1. green pea and lentil)..11 0.29 0. At higher percentages.27 3.244 ± 68 and 5873 ± 87 mg/kg respectively.33 0.53 8.42 0.13 0. indicating that no false-positive samples were found. Hurlburt.53 0.10 0. obtaining regression coefficients higher than 0. 4.65 6. Fu and Maks (2013) studied the effect of heat treatment of peanut flour on the Table 2 Results obtained by the four participating laboratories for the determination of Ara h2 (mg/kg) in model biscuits added with different percentages of peanut butter.12 0. frostings.09 0.53 0.11 0.00 2.46 0. & Maleki.38 0. Cross-reactivity study The specificity of anti-Ara h1 and Ara h2 antibodies was also examined by testing its cross-reactivity with other food ingredients such as. Cheng.70 2.86 6.60 0.01% of peanut addition. The concentration of Ara h2 in test samples was calculated as indicated above.31 0.81 1.41 1. confectionery products.29a 0. Concentration values of Ara h1 and Ara h2 determined in these ingredients were below the cut-off established for each ELISA assay to consider a sample as positive for peanut protein. All ingredients gave a small decrease (in competitive format) or increase (in sandwich format) of the absorbance value compared to the blank standard indicating a certain degree of interference (results not shown). pistachio. 2003).53 51.87 Lab 4 ± ± ± ± ± ± ± ± a 0. the concentration of Ara h1 and Ara h2 proteins under mild and strong roasting of peanuts. concentration of Ara h2 increased for all laboratories.32 Food samples with concentration values below the cut-off established for the interlaboratory study.82 6.43 Lab 3 ± ± ± ± ± ± ± ± a 0.88 0.50 a 0.93 2. Sarath.304 M. Nesbit. The optimum conditions led to the development of sandwich and direct competitive ELISA tests with sensitivities comparable to s those previously obtained for Ara h1 and Ara h2 proteins (Pome et al. we did not observe a higher level (Pome of interference when analyzing legumes compared to other foods. respectively.87 2.58 44.19 0.97 15.25 0. the concentration of Ara h2 was below the cut-off with the exception of one laboratory. The existence of cross-reactivity between Ara h1 and other vicilin storage proteins of legumes such as soya. Peanut butter (%) Assay 1 Assay 2 Lab 1 0 0.33 49. 3.02 7. Model biscuits containing several different percentages of peanut butter as ingredient were analyzed using developed ELISA assays.2 ± 0.73 1. Thus.60 2.21 0. Thus.74 2. milk.16 1.69 27.04 2.18 and 111.4.38 0.33 21. the concentration of Ara h1 and Ara h2 was found to be about 1% and 45% of that in the raw dough before the baking treatment. Discussion The search for the selection of an immunoassay format and a target protein to detect peanut in processed foods led us to develop direct competitive and sandwich ELISA formats to determine Ara h1 and Ara h2 proteins.65 1. Results and performance characteristics (repeatability and reproducibility data) of the interlaboratory study are summarized in Table 3.05 0.47 6. (2007) found that roasting of peanuts under mild or strong conditions decreased extraction efficiency of proteins by 75% and 82%.07% and values of reproducibility RSD (RSDR) between 30.10 7. green pea and beans have been reported (Beardslee.50 0.57 0.16 0.07a 0.31 3.22 0. walnut and hazelnut). Certain degree of interference was observed between Ara h1 and Ara h2 with basic food ingredients when they were analyzed using competitive ELISA tests. Values of repeatability RSD (RSDr) ranged between 15.0% peanut butter. When these proteins were determined in biscuits added with 1..01 0. tree nuts (almond.09 0.02a 0.59 0.21 1.28 0. Sicherer. fillings. However. & Markwell.06 5. For all laboratories. respectively. and ingredients used in the elaboration of biscuits (wheat.48 0. Extracts of all ingredients and peanuts were prepared following the extraction protocol and tested undiluted. it is assumed that interference could be produced by nonspecific interaction between components of the food matrix and antibodies. Results obtained indicated that the processing of peanut to obtain butter caused a decrease in the level of extracted proteins of about 50% and a loss of immunoreactive proteins of about 95% and 53% for Ara h1 and Ara h2.18 0. Chassaigne et al.21 . The mean concentration of Ara h2 obtained for each set of samples by each laboratory is shown in Table 2.56 21.03 1.03 0.21a 0.28 1.20 1.11 20. At 0. 2007. bakery mixes.10 6.95 1.24 1.75 ± ± ± ± ± ± ± ± 0.75 5.83 and 44. 2003.75 Lab 2 ± ± ± ± ± ± ± ± a 0.15 Lab 3 ± ± ± ± ± ± ± ± a 0. Montserrat et al. determined by ELISA kits.18 0.0 and 5. These proteins have some 30e45% of amino acids in common with peanuts and a similar folding.57 0. 2013). were obtained.34 0. & Burks.42 1. In this study. In the same study. 2013.55 Lab 4 ± ± ± ± ± ± ± ± a 0.38 0.74 3..13%.27 0.45 3. Schmitt.4% (w/w) and concentrations of Ara h1 and Ara h2 of 20. Maleki.03 7. calibration curves were obtained for every ELISA plate using the logit log model. & Burks.47 1.69 0. were reported to be about 15% and 8% of that of the raw peanut extract for Ara h1 and 59% and 47% for Ara h2.72 3.62 9.18 0. Sampson.48 1. Using the standards of Ara h2 indicated in Table 1. 2000.60 0.76 2. Cheng.976. egg and sugar).

56 53.28 14.44 48.. which result in a lower level of extracted Ara h1 and/or in a lower recognition of this protein by their specific antibodies.397 5. Peng et al. Our results and those obtained by other authors (Chassaigne et al.49 0.62 13.18 4. 2010) support the previously reported good thermal stability of Ara h2 (OwusuApenten. as it relates to immunoreactivity and solubility. the accessibility of adsorbed antibodies may differ from the antibodies in solution. & Lambrecht.38 30. treatment such as sterilized pa Our results also show that sandwich and direct competitive assays based on the determination of Ara h2 protein are able to detect lower percentages of added peanut compared to their counterparts for Ara h1.10 0. Pome used are composed in all these studies of Ara h1. They observed that the recovery of Ara h1 progressively decreased when lower amounts of peanut were added to those foods. we used food samples.4% at 260  C for kits targeted whole peanut proteins and Ara h1. which causes a higher loss of epitopes recognized by antibodies and a higher reduction of its solubility. Our study confirms that thermal processing of peanuts decreases solubility of peanut proteins as well as immunoreactivity of Ara h1 and Ara h2 proteins.05 0. Performance characteristics Total number of laboratories Total number of replicates Mean value Repeatability SD Reproducibility SD Repeatability RSD Reproducibility RSD Repeatability limit Reproducibility limit Abbreviation P n X Sr SR RSDr RSDR r R Peanut butter (%) 0.714 5.253 0..00 2. the two ELISA kits underestimated the level of proteins in the samples.221 0. food products analyzed were spiked with pure Ara h1 (Peng et al. The use of spiked foods is useful to deter(Pome mine the effect of food matrix but they do not provide information about the effect of processing on assay performance.2%.7% and 75.42 0. 2013) or with a raw peanut extract s et al. allows evaluating the actual effect of processing on the detection efficiency of an immunoassay (Khuda et al. is necessary to evaluate the performance of ELISA methods to detect allergens in processed foods.964 38.755 37.91 58. 2009).247 39.473 0. Khuda et al. about 62. 2013. in which the allergenic food is added as ingredient and afterwards.39 0. both formats performed well to detect egg added to pasteurized sausages and baked bread whereas only the competitive format could detect egg in foods subjected to severe heat ^te .7% decrease in the amount of proteins extracted.691 4 8 0. in which a commercial peanut butter was added at the ingredient stage and afterwards subjected to processing.856 1. The limitations of immunoassays should be considered when .0% at 232  C and 98. Recently. In that study.864 24. Taylor. 2002) and suggest that Ara h2 would be a better target than Ara h1 when immunoassays are going to be used for the detection of peanut in processed foods. 2003)... These authors obtained that recovery was drastically reduced after baking at 190  C for 30 min.713 solubility of proteins and compared the performance of two commercial ELISA test kits targeting whole peanut proteins or Ara h1 for quantitation of residual peanut.572 1. It should be also considered that the way that specific antibodies are presented to its target protein is different depending on the ELISA format. whereas in the others.45 to 105. capture antibodies are coated on the wells whereas in the competitive format antibodies are in solution and thus. Although incurred samples are considered difficult and costly to obtain.788 49. / Food Control 54 (2015) 300e307 305 Table 3 Results of the interlaboratory study. the potential effects of processing on the quantitation of proteins by ELISA have become recognized. as indicated above. 2003). Our study and others demonstrates that ELISA tests could not give accurate results when they are used to determine allergenic proteins present in thermal processed foods due to changes in solubility and immunoreactivity of the target proteins (Fu & Maks. In the last few years.169 0.907 39. Montserrat et al. These findings can be attributed to a more severe denaturation and/or aggregation of Ara h1 compared to Ara h2 induced by the baking process.. respectively. Schmitt et al.05 44. being less than 18% at all added levels. Noordle.698 4 8 40.199 16..638 44. (2013) developed a monoclonal-antibody sandwich ELISA for Ara h1 that could detect milk samples spiked with pure Ara h1 at levels between 60 and 240 ng/mL. processed in a manner mimicking as closely as possible the actual conditions under which the sample matrix would normally be manufactured.708 1. obtaining recoveries ranging from 95.2% of ground peanut.721 0. Our results are in accordance with those reported by de Luis et al.018 2.506 15. The performance of the assays developed in our work to detect peanut addition is difficult to compare with other studies s et al. Performance criteria (repeatability and reproducibility data).57 0. In the sandwich format.07 111. This fact indicates that compounds of the matrix impaired recognition of Ara h1 by its specific antibodies.416 4 8 1.83 36.50 4 8 0. 2007. 2012). obtaining recoveries in biscuits of 86% and 6% at spiked levels of 16% and 0..13 0. (2008) using competitive and sandwich ELISA assays based on the determination of ovomucoid to detect egg in model foods. some regulatory bodies may be unwilling to consider approval of validation data without the inclusion of data generated with incurred samples prepared with material for the allergen being targeted (AOAC.07 41.32 0.618 1.787 4 8 1. Niemann. the degree of immunoreactivity loss being greater for the kit targeted to Ara h1 than for the kit targeted to whole peanut proteins. Therefore.39 0.924 17. This fact could be attributed to a higher degree of denaturation and/or aggregation of Ara h1 compared to Ara h2. (2003) developed a sandwich ELISA for Ara h1 to Pome monitor peanut allergen in foods that could detect peanut in cookies and pancake mix spiked with 0. They found that dry heating at 232 and 260  C for 10 min caused an approximately 49. Likewise.00 0. s et al.99 0.946 24.5% and 99. 2012.18%. 2012). 2013. The use of incurred samples. Differences in the recognition of antigen by competitive and sandwich ELISAs could be due to the former requires only one site of interaction with the antibodies whereas the later requires two binding sites. (2012) performed a study to establish the effect of food processing on peanut detection by five commercial ELISA kits using cookie dough prepared with defatted light-roasted peanut flour before baking.50 1.401 5.26 4.9% and 85.01 0.220 4 8 6. Khuda et al.25 0.M. Results obtained in the analysis of model biscuits which contained different amounts of peanut butter indicate that direct competitive formats have a higher sensitivity to detect added peanut butter than the sandwich formats. an understanding of the effects of processing on allergen structure in a specific matrix.540 4 8 3.19 45.449 4 8 12.. the effect being more marked for Ara h1. respectively.55 2.47 2. respectively.45 1. Although the standards (Peng et al.

Muraro. Y. Journal of Analytical Toxicology. S. 189e229). Owusu-Apenten.. E. This ELISA test could detect percentages of peanut butter addition higher than 0. Allergy.4 and 127. L. Holzhauser. R. nchez. Journal of Allergy and Clinical Immunology. B. Peng. Amy. et al.. (2008). Virginia. M. N. K.. Journal of Agricultural and Food Chemistry. Journal of Dairy Science. L. Godefroy. A. C. K. Matsuda et al. G.. Analysis of food al (Ed. Kuang. van Reijsen.. Torrance. Nakane. M.. (1999). 443e450.. Biochemical Journal.. et al. (2006) evaluated the analytical performance of two ELISA kits to detect peanut in an interlaboratory study and found RSDR values of 14% and 9% for cookies added with peanut proteins at a level of 10 mg/g  et al.. Food Chemistry. G.. H. AOAC International.. A. Hickstein. (2000). Soybean glycinin G1 acidic chain shares IgE epitopes with peanut allergen Ara h3. W. ELISA assays based on the determination of Ara h2 protein were able to detect lower percentages of peanut than their counterparts for Ara h1.. & Visconti. S.. Journal of Agricultural and Food Chemistry. T.. et al. H. Kiening. M.. (1994). It has been shown that relatively low values of RSDR from 30. Journal of AOAC International. D... M. S. & Oliver. A. M.. M. J. & Pe Evaluation of indirect competitive and double antibody sandwich ELISA tests to determine b-lactoglobulin and ovomucoid in model processed foods. M. A. M. H. Anklam. Indirect competitive ELISA for determination of traces of peanut (Arachis hypogaea L. Cardona. Food protein analysis: Quantitative effects on processing (1st ed. Helm. & Markwell.. 2897e2905. Appendix M: validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. Gosling (Ed. J. F. 1600e1608. 3321e3327... the effect being more marked in the case of Ara h1. & Pe rez. van Hengel. (2013). F.. 30. Becker. (2006). T.. Bublin. Suhr.. (2010). Matsuda.. M. Lexmaulova a. milk. 60. Amsterdam: Elsevier. L. L. M. bred in different parts of the world. D. S rez. Calvo. Peroxidase-labeled antibody a new method of conjugation. Koppelman. A. & Wild.. Moreover. A. T. Gabroysk . 10. de Luis.. Journal of Agricultural and Food Chemistry. of a scholarship (Ref. I. E.18 to 53. Khuda. Miller. Stumr. Current Allergy and Asthma Reports. (1974). S. S. Vlooswijk. 272e283. Accuracy (trueness and precision) of measurement methods and results. & Maks. I.01%). Maleki. 62e75. Validation of the immunalysis microplate ELISA for the detection of buprenorphine and its metabolite norbuprenorphine in urine. 5. 111.4 and 59. K.. The effects of roasting on the allergenic properties of peanut proteins. 93. A... & Sa nchez. (2000).. RSDR values obtained in our study are in the range of those reported in other studies. 1e12. 463e472. Journal of Allergy and Clinical Immunology. 14. 4480e4489. L. Slate. 19. Immunoassays practical approach (pp.. S.). S.. R. Knippels.. Mata. and Valencia.). Knol. 61. Schweimer. Drumare.. Calvo. (2005) carried out an interlaboratory validation of five commercial ELISA test kits for the determination of peanut in two food matrices (biscuits and dark chocolate) at four levels of peanut contamination.. M. direct competitive and sandwich ELISA formats to determine Ara h1 and Ara h2 proteins were developed and assayed in model biscuits prepared with a commercial peanut butter as ingredient. Y. Song. R. Montserrat et al. 168. 47. W. In J.. M. Niessner.. Journal of Agricultural and Food Chemistry. K. Rysova F.  . Acknowledgments n This work was supported by Grant P1078/09 from the Arago Government and European Social Fund. A. Al-Muhsen.. M. Lexmaulova validate an ELISA method for the quantitative determination of peanut protein in foods. M. Diaz-Amigo. et al. H. T. E. (2010).05e5% peanut addition. Montserrat is recipient n. Al-Taher.. the target protein and the food processing conditions... M.. E. 640e645. 5649e5658. Fu. D. (2013) performed a collaborative study to of food.. Effect of bovine Franco. 21. (2007). Results obtained revealed that detected levels of Ara h1 and Ara h2 were drastically reduced after the roasting of peanuts to obtain the peanut butter used as ingredient and also after the baking of biscuits. Canadian Medical Association Journal. et al. Pe lactoferrin addition to milk in yogurt manufacturing. P. W. ISO 5725e2. J. Lavilla. Beardslee. (2013). obtaining adequate results in term of repeatability and reproducibility. S.. International Journal of Environmental Research and Public Health. Drs.. M. Hayward. E. et al. direct competitive format for Ara h2 were selected to be evaluated by four laboratories. et al. (2001). V. 93. Journal of Agricultural and Food Chemistry. 123. & Raufman.. van Hengel. Trends in Food Science and Technology.. et al. J. Liu. 4547e4553.. J. L. et al. 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