Quantification of major 2S allergen protein of yellow mustard using anti-Sin a 1 epitope antibody

Food Control 44 (2014) 233e241

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Quantification of major 2S allergen protein of yellow mustard using anti-Sin a 1 epitope antibody Harsha K. Marambe, Tara C. McIntosh, Bifang Cheng, Janitha P.D. Wanasundara* Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N 0X2, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 December 2013 Received in revised form 27 March 2014 Accepted 28 March 2014 Available online 18 April 2014

The major allergenic protein in yellow mustard (YM, Sinapis alba L.) is the 2S napin Sin a 1. The level of Sin a 1 in YM seeds was quantified using anti-epitope antibody (AE-Ab) generated against the epitope sequence QGPHVISRIYQTAT as the capture antibody in a non-competitive enzyme linked immunosorbent assay (NCI-ELISA), and Sin a 1 containing napin purified from YM (var Andante) as the reference standard. The AE-Ab showed high specificity towards Sin a 1 epitope-containing napin long chain and showed no cross reactivity with other proteins of YM or other Brassicaceae proteins of similar genetic homology. The assay quantified Sin a 1 with a limit of detection and quantitation (LOD and LOQ) of 3.08 ppm and 3.23 ppm, respectively with acceptable recoveries and precision. The Sin a 1 content in YM varieties produced in 2006, 2010, 2011 was in the range of 2.65e4.68 g (AC Base); 3.81e5.98 g (AC Pennant) and 3.11e4.92 g (Andante) per 100 g of seeds. Sin a 1 composed more than 50% of napin protein fraction of YM seed. A trend of increased Sin a 1 level with the increased contents of total seed protein and napin was observed from this data. Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

Keywords: 2S Allergen Mustard Sin a 1 Anti-epitope antibody ELISA

1. Introduction Mustard is consumed worldwide as a condiment and as a food ingredient. In several regions of Asia, mustard seed is a source of edible oil. In the global mustard production, Canada is the largest exporter (75e80%) and the second largest producer while the province of Saskatchewan produces 80% Canadian mustard (www. specialcrops.mb.ca). Among the different crucifer species that are collectively called as mustard, yellow mustard (YM) (Sinapis alba), brown and oriental mustard (Brassica juncea) are grown in Canada. Of these, S. alba seeds are larger, contain less oil (27% vs. 36%) and milder in pungency than B. juncea. YM seed has mucilage containing cells in the seed coat epidermis, higher protein content (30e33%) and less oil content compared to the other crucifer seeds. Therefore, YM finds applications beyond its use as a condiment or source of oil including use in the texture enhancement of food products. These applications include dry milled flour (fine powder from dehulled seeds) for salad dressings, mayonnaise, barbecue sauces, pickles and processed meat, wet milled mustard for mustard paste (e.g. with hot dogs) and whole ground seeds for use in spice mixes, as a seasoning, emulsifying and bulking agent in

* Corresponding author. Tel.: þ1 306 385 9455; fax: þ1 306 395 9482. E-mail address: [email protected] (J.P.D. Wanasundara). http://dx.doi.org/10.1016/j.foodcont.2014.03.053 0956-7135/Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.

meat and other food products (www.specialcrops.mb.ca). Food industry also uses enzyme inactivated (deheated; primarily to inactivate myrosinase) YM flour for the functionalities provided by mustard protein and polysaccharides (Cui & Eskin, 1998). Allergenicity of YM has been reported since 1980 and four different allergenic proteins have been identified and characterized in YM namely, Sin a 1 (14 kDa, a 2S albumin/napin), Sin a 2 (51 kDa, a 11S globulin/cruciferin), Sin a 3 (12.3 kDa, a non-specific lipid transfer protein/nsLTP) and Sin a 4 (14.2 kDa, a profilin) (Menéndez-Arias, Dominguez, Moneo, & Rodríguez, 1990; Palomares et al., 2005; Sirvent et al., 2009). Mustard is in the list of priority food allergens in Canada since 2012 (www.hc.sc.gc.ca) and in European Union it is listed among the 14 allergens to be declared on labels (The Commission of the European Communities, 2007). Mustard allergy accounts for 1e7% of all food allergies in the European regions (European Food Safety Authority, 2007). The prevalence of mustard allergy in Canada and other regions of the world is less known, but the reported severity of its allergy is very high. Ingestion of a minute amount of mustard is reported to cause anaphylaxis (Vidal, Diaz, Saez, Rodriguez, & Iglesias, 1991). According to a recent report of the VITAL expert panel, eggs and mustard were identified as the allergenic foods with lowest eliciting doses. The reported dose of mustard proteins to cause an allergenic reaction is 0.05 mg (Taylor et al., 2014). Patients with mustard allergy have shown a highly significant correlation with

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specific IgE levels to mustard and skin prick test (SPT) for Sin a 1 (r ¼ 0.98, P: 0.001), and a strong positive correlation with Sin a 2 (r ¼ 0.86, P: 0.001). However, no significant correlation between rSin a 3 (r ¼ 0.15, P:0.41) or rSin a 4 (r ¼ 0.07, P:0.68) was found among mustard allergy patients with the SPT results and the level of allergen protein specific IgE levels of serum (Sirvent, Palomares, Cuesta-Herranz, Villalba, & Rodriguez, 2012; Verada et al., 2011). Sin a 1 is a basic protein and believed to exist as a polymorphic dimer consisting of two polypeptide chains of 39 and 88 amino acids linked by disulfide (SeS) bonds; short chain and long chain, respectively. The Sin a 1 protein belongs to the 2S napin group and in the plants of Brassicaceae/Cruciferae family, napin is expressed by multigene families. Therefore, YM may contain other 2S napin isoforms besides Sin a 1 (Josefsson, Lenman, Ericson, & Rask, 1987). Sin a 1 is reported as highly stable to heat and retains immune reactivity after gastrointestinal (GI) digestion. There are at least two non-overlapping Ab binding sites (epitopes) in Sin a 1; one is conformational in nature whereas the other one is found in the large chain and suggested as a linear (continuous) epitope (Menéndez-Arias et al., 1990). The linear epitope that contains His is considered as the antigenic determinant of Sin a 1 immune response (Monsalve et al., 1993). Currently, mustard allergen detection kits are commercially available and they detect the presence of mustard or quantify the total mustard seed proteins in food, based on polymerized chain reaction (PCR) (Mustorp, Engdahl-Axelsson, Svensson, & Holck, 2008) or ELISA techniques (Lee, Hefle, & Taylor, 2008). Although these detection methods may satisfy the labeling requirements of the food industry, they are not specific for the allergenic proteins of mustard and are therefore less relevant in the study of the allergenic potential of Sin a 1 or seed screening in YM breeding programs. The use of synthetic peptides containing the allergen epitope sequence to generate antibodies (anti-peptide or anti-epitope antibodies; AEAb) that specifically recognize the allergenic protein of interest may be efficient in capturing allergenic protein compared to the traditional antibodies raised against isolated protein. The aim of this study was to use Sin a 1 AE-Ab to detect and quantify Sin a 1 protein in YM varieties that are grown in Canada. 2. Material and methods 2.1. Materials Seeds of YM varieties Andante, AC Pennant, and AC Base produced during 2006, 2010, 2011 and seeds of B. juncea (Centennial brown) and Brassica napus (AC Excel) were from Brassica breeding programs of Agriculture and Agri-Food Canada, Saskatoon Research Centre. The seeds were stored at 4  C in air tight containers. The peptide synthesis and antibody preparations were provided by EZBiolab, USA. The bicinchoninic acid (BCA) assay kit was purchased from Pierce Thermo scientific, USA and the Clarity western ECL substrate for western blotting was from Bio-Rad (Canada). All the other chemicals used were of reagent grade and purchased from Sigma (Canada). 2.2. Methods 2.2.1. Development of Sin a 1 anti-epitope (AE-Ab) and anti-napin (AN-Ab) antibodies The Sin a 1 AE-Ab was developed by immunizing rabbits with the synthesized peptide having the amino acid sequence of the allergenic epitope of Sin a 1 (Fig.1, Monsalve et al., 1993). The rabbit polyclonal anti-napin antibody (AN-Ab) was developed against chromatographically purified napin of S. alba (var. Andante). Antibodies were purified by affinity chromatography, dialyzed, and lyophilized. Antibodies were reconstituted (AE-Ab: 0.5 mg BSA

equivalents/mL; AN-Ab: 9.33 mg BSA equivalents/mL) in borate buffered saline (BSB; 167 mM Boric acid, 125 mM NaCl, pH 8.5), and stored at 20  C as aliquots until use. 2.2.2. Preparation of reference standards YM seed (var. Andante) meal preparation, protein extraction and napin isolation were essentially similar to the procedures described by Shim and Wanasundara (2008). The napin purified using cation exchange chromatography (CEX-1) and hydrophobic interaction chromatography (HIC, Phenyl sepharose) was “unfractionated napin” and used for developing AN-Ab. This unfractionated napin peak of CEX-1 (peak B1 described by Shim & Wanasundara, 2008) was further separated by CEX (CEX-2) using 0e50% of 1 M NaCl gradient (5.5e20 column volumes) and the resulting napin peaks were collected. All protein peaks were dialyzed as needed, lyophilized and stored at 20  C until further use. 2.2.3. Preparation of napin extracts Because of the limited number of seeds and the selective solubility of napin (Wanasundara, Abeysekara, McIntosh, & Falk, 2012), special considerations were used in developing procedures for protein extract preparation. About 5e10 YM seeds were weighed into Polypropylene vials (5 mL) each containing a stainless steel ball (diameter: 1 cm) and pulverized in 0.5 mL hexane using a bench top homogenizer for 4 min (speed level 7) to extract oil. The steel balls were removed from the vials, rinsed with another 0.5 mL of hexane, and the meal slurries were centrifuged at 3074  g for 10 min. The oil containing hexane supernatant was removed with a Pasteur pipette and the oil extraction was repeated two more times with 1 mL of hexane per extraction. The defatted meal was air dried in the same tube under a fume hood overnight. A 0.15 M CaCl2 suspension (pH 3.0) of this defatted YM meal was made with 1 mL of water, 6 mL of 1 M H2SO4 and 0.5 mL of 0.066 g/mL CaCl2$2H2O, homogenized for 10 min (speed level 7) and centrifuged as above. The clear supernatant containing napin was collected and the residual meal was extracted two more times sequentially with water (1.5 mL) and water (1 mL) þ 0.066 g/mL CaCl2$2H2O (0.5 mL), the supernatants were recovered after centrifugation. The pooled napin extracts were filtered using 0.45 mm syringe filters, divided into aliquots and stored at 20  C. 2.2.4. Protein content determination The total N content of ground mustard seeds, meal and purified napin were determined by the combustion method using EDTA as the standard (AOAC, 1997) and converted to total N-based protein content using a conversion factor of 6.25. The protein content of seed extracts was determined using BCA assay (Smith et al., 1985) with the BCA assay kit and bovine serum albumin (BSA) as the standard. The seed extracts at pH 3 were filtered using AMICON centrifugal filters (3 kDa) prior to BCA assay to eliminate any interfering compounds. 2.2.5. Electrophoresis The polypeptide profiles of the samples were obtained by SDSPAGE under reducing (R, heated with b-mercaptoethanol; b-ME)

Fig. 1. Primary sequence of allergenic napin isoform Sin a 1 (ALL1_SINAL) of Sinapis alba. Epitope sequence (17AA peptide) identified by Monsalve et al. (1993) is highlighted and used for synthesizing antigen peptide for Sin a 1 AE-Ab generation.

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and/or non-reducing (NR, heated without b-ME) conditions essentially similar to Wanasundara et al. (2012). 2.2.6. Western blotting The specificity of Sin a 1 AE-Ab and AN-AB was determined using protein extracts of defatted meals of dehulled S. alba (Andante), B. juncea (Centennial Brown) and B. napus (AC Excel) by western blotting with the antibodies. First, the meals were mixed with sample buffer (0.2 mg/mL; 62.5 mM TriseHCl þ 2% SDS þ 25% glycerol þ 0.01% bromophenol blue þ 5% b-ME) and loaded (30 mL/well) into 12% or 4e20% Mini protean TGX precast gels (BioRad). Electrophoresis was carried out using 10  running buffer (25 mM Tris þ 192 mM glycine þ 0.1% SDS) at 200 mA for 40 min. One gel was stained with Coomassie blue to detect migration of polypeptide bands and the other unstained gel was used for western blotting. The separated polypeptide bands of each unstained gel were transferred to separate PVDF membranes using cold transfer buffer (20% methanol v/v, 0.303% Tris base w/w, 1.441% glycine w/w) at 100 V constant voltage for 70 min. The PVDF membranes with transferred proteins were washed three times with water for 5 min each on a plate shaker and incubated with blocking buffer (4% skim milk powder in phosphate buffered saline containing Tween 20; PBST; 0.115% Na2HPO4 w/w, 0.02% NaHPO4 w/w, 0.16% NaCl w/ w þ 0.2% Tween 20 v/v) separately for overnight with gentle agitation at 4  C. After this incubation period, the blocking buffer was replaced with 5 mL of fresh blocking buffer containing antibodies (AE-Ab: 25.7 ng of BSA equivalents/mL; AN-Ab 9.3 ng of BSA equivalents/mL) and incubated on a plate shaker for 1 h at ambient temperature. Then the PVDF membranes were washed three times (10 min each) with PBST (with agitation) and incubated for another hour with 10 mL of fresh blocking buffer containing the secondary antibody (goat anti rabbit IgGehorseradish peroxidase (HRP) conjugate, dilution factor: 2  103 for AE-Ab and 2.5  103 for AN-Ab). The washed membranes (four times for 10 min each with PBST with agitation) were added with 2 mL of clarity western ECL substrate (1:1 mixture, V/V). The blots were detected by exposing the membranes to an X-ray film for 10 s to 1 min in a dark room. 2.2.7. Non-competitive indirect enzyme linked immunosorbent assay (NCI-ELISA) using Sin a 1 AE-Ab The Sin a 1 content of the YM seed napin extract was determined under reducing and denaturing (R þ D) and non-reducing and non-denaturing (NR þ ND) conditions. The napin extracts (100 mL) were mixed with 400 mL of BSB containing 0.33% (v/v) bME and heated at 99  C for 10 min in a thermal mixer (Brinkmann) at 900 rpm for the R þ D condition and BSB without b-ME or heat was used for NR þ ND condition. The wells of the microtitre plates were coated to have separate wells for blanks (no antigen), reference standard (0e27 mg of BSA equivalents/mL in BSB) and napin extract (100 mL/well, diluted appropriately) and incubated for 1 h with shaking at 37  C. After incubation, wells were washed three times with BSB containing 0.05% (v/v) Tween 20 (BSB-T20), and uncoated areas were blocked with BSA by incubating with 300 mL BSA solution/well (10 mg of BSA/mL of BSB) for 1 h on a micro plate shaker. Then the AE-Ab stock solution (0.9 mg/mL of BSB) diluted with BSB (1:25,000) was added to the wells (100 mL/well) and incubated for 1 h to capture antigen. Any AE-Ab not captured by Sin a 1 molecules of the extract or reference standard was removed by washing the wells three times with BSB-T20. For the secondary antibody, a 100 mL of goat anti rabbit IgGeHRP conjugate (secondary Ab) diluted in BSB (1:2000 v/v) was added to each well and incubated for 1 h with shaking. Excess HRP conjugate that was unbound to the Sin a 1 captured AE-Ab was removed by three successive washings with BSB-T20. The bound HRP conjugate to AE-Ab was detected by adding 100 mL of chromogenic substrate

235

(1 mg/mL of 3,30 ,5,50 -tetramethylbenzidine/TMB in methanol) per well and incubating for 20 min with mixing. At the end of the incubation period, the color development in wells was arrested by adding 50 mL 2 M H2SO4/well. The optical density (OD) of the contents in each well was measured at 450 nm using a micro plate reader. In the NCI-ELISA assay both standards and samples were assayed in quadruplicate. The concentration of Sin a 1 in napin extracts were interpolated from the linear portion of the standard curve. Except for the first incubation step, all the other incubation steps were performed at ambient temperature. 2.2.8. Determination of limit of quantitation (LOQ) and limit of detection (LOD) Sensitivity of the Sin a1 ELISA assay to quantify Sin a 1 was evaluated by assessing LOD and LOQ. The LOD was determined as the lowest amount of Sin a 1 in a sample extract that can be distinguished from a true blank sample at a specified probability level (Eq. (1)). The LOQ was determined as the lowest amount of Sin a 1 in an extract sample that can be reasonably quantified at a specified level of precision (Eq. (2)). The average OD and standard deviation (SD) of 74 blank samples (with no protein) were obtained in six separate ELISA runs. The LOD and LOQ of the AE-Ab based NCI-ELISA assay were calculated as below.

LOD ¼ Mean OD þ ð3  SDÞ

(1)

LOQ ¼ Mean OD þ ð10  SDÞ

(2)

where, OD is optical density of the blank and SD is the standard deviation. 2.2.9. Determination of inter and intra assay coefficient of variation (CV) The precision of ELISA assay was determined by evaluating the inter-assay and intra-assay CV that indicates the variability of results of replicate samples between and within runs, respectively. Precision was assessed by analyzing each reference standard concentration (0, 1.08, 2.16, 3.24, 4.32, 5.4, 10.8 and 27 mg of BSA equivalents/mL) of 4 replicates on 3 different dates. The % CV was determined for each set of replicates within the same and between different dates of analysis. The % CV was determined as the standard deviation divided by mean and multiplied by 100. 2.2.10. Determination of spike recovery of Sin a 1 The percent recovery of Sin a 1 in napin extracts was evaluated by spiking defatted B. napus (AC Excel) meal with three levels (Low: 30 mg/g of meal; Medium: 80 mg/g of meal; High: 153 mg/g of meal) of chromatographically purified napin. The spiked B. napus meals were extracted similarly to the sample preparation for YM seeds as described above and the extracts were assayed for Sin a 1 content using AE-Ab based NCI-ELISA. 2.2.11. Statistical analysis All the standards and samples used in ELISA assays were analyzed in four replicates. The other analyses were carried out in three replicates. The values were reported as mean  standard deviation. Statistical data analysis was performed as a generalized linear model (one way analysis of variance-ANOVA) with multiple comparisons of means [Tukey’s pair wise comparisons; probability (p) at 0.05] using SAS 9.1 software (SAS Institute Inc., U. S.). 3. Results and discussion Among the allergenic proteins of YM, Sin a 1 is the most important allergen in terms of allergenic potential (Monsalve,

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Villalba, & Rodriguez, 2001; Sirvent, Palomares, Cuesta-Herranz, Villalba, & Rodríguez, 2012). Quantitation of Sin a 1 is needed to determine the level of this allergen in the seed and the products to estimate the level of allergen exposure from the mustardcontaining food and also to assess Sin a 1 levels in mustard germplasm for breeding programs. Sin a 1-specific antibodies and a suitable Sin a 1 reference standard are the major requirements for Sin a 1 quantitation. The commercial mustard protein detection ELISA kits recognize cruciferin, napin and other proteins of YM flour together without differentiation because these kits are for detecting total mustard protein. 3.1. Reference standards Chromatographically purified napin of S. alba (var Andante) was used as the Sin a 1 reference standard. The first cation exchange chromatography step (CEX-1) provided reasonable separation of napin (w14 kDa, Fig. 2a) and cruciferin from the extract (Fig. 2b and c). The polypeptide profile of the meal residue remained after extraction (Fig. 2a) indicates partial extraction of cruciferin (peptide bands >20 kDa) but reasonably good extraction of napin under

the conditions used. In the CEX-1 separation (mobile phase pH 8.5), positively charged napin (mobile phase pH < IEP) was strongly bound to the column material and eluted when a salt gradient was applied (Fig. 2b). Surprisingly, a fraction of napin was co-eluted with cruciferin in the unbound proteins (Fig. 2c) and was subsequently recovered in the SEC step (Fig. 2d and e). Further purification of these two napin collections (CEX-1 and SEC) by HIC (Shim & Wanasundara, 2008) yielded 8.88 mg solids/g of Andante defatted meal. The napin obtained from CEX-1 and SEC steps had total Nbased protein content of 92% and 82%, respectively. According to Fig. 2b, the CEX-1 step resulted in a cluster of small napin peaks indicating a mixed nature of ionization of the proteins present. Napin is encoded by multigene families therefore isoforms with a high degree of sequence similarity may be present. In addition to inducing allergic reactions, napins have been shown to have other biological activities such as antifungal activity and calmodulin antagonist capability (Barciszweski, Szymanski, & Haertle, 2000) owing to the slight differences in certain amino acids, which may result in differences in ionizability of the molecules to perform different functions. Although further separation of the CEX-1 napin peak using a different salt gradient (0e50%) was

Fig. 2. SDS PAGE profiles and chromatograms of S. alba (Andante) napin purification: (a) SDS PAGE profile of defatted meal, residue left from protein extraction, and the protein peak obtained from desalting and de-pigmentation step, (b) separation of cruciferin and napin by cation exchange chromatography (CEX-1), (c) SDS PAGE profile of CEX-1 separated cruciferin and napin, (d) Separation of napin in the cruciferin peak of CEX-1 by size exclusion chromatography (SEC), (e) SDS PAGE profile of cruciferin and napin separated by SEC, and (f) further separation of napin fractions by CEX (CEX-2) under different gradient system. NR: Non-reducing; R: Reducing conditions.

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attempted under different column volumes ranging from 5.5 to 20, only two distinct fractions (Napin I and Napin II) were possible at 6.5 column volumes (Fig. 2f; CEX-2). Of the napins purified from CEX-1 (unfractionated) and CEX-2 (Napin I and Napin II) and SEC, the most suitable reference standard for ELISA assay was selected based on their ability to bind with AE-Ab and AN-Ab antibodies. 3.2. Specificity of AE-Ab and AN-Ab The AE-Ab showed specific binding with YM napin with or without SeS bonds (Fig 3a). No cross reactivity was observed between AE-Ab and non-napin proteins of YM or the related napin proteins of other Brassicas such as B. juncea and B. napus (Fig. 3a). When SeS bonds were reduced, the AE-Ab affinity was towards the large chain of YM napin confirming that the YM epitope (QGPHLQHVISRIYQTAT) used in developing AE-Ab is a linear epitope located in the large chain of Sin a 1. With conformational epitopes, AE-Ab binding ability may not be retained upon SeS bond reduction and heat denaturation. In contrast to AE-Ab, the AN-Ab raised against chromatographically purified (CEX-1; unfractionated) YM

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napin showed a high degree of non-specific binding for the proteins in YM seed extracts under non-reducing conditions (Fig. 3b). In addition to recognizing napin, AN-Ab showed affinity to bind polypeptide bands derived from cruciferin, and cross-reactivity with napin and cruciferin in related species, B. juncea and B. napus (Fig. 3b). However, AN-Ab did not bind cruciferin and showed a faint signal in the region of napin large chain when SeS bonds were broken (Fig. 3b). The cruciferin and napin of S. alba has no significant amino acid sequence homology, however, an 11% sequence identity can be observed (results are not presented) indicating the possibility of existing conformational (discontinuous) epitope sharing common immune-reactive regions in cruciferin which could be recognized by the AN-Ab. Complete loss of cruciferin-antibody binding upon SeS bonds disruption further confirms this possibility (Fig. 3b). Reduction of SeS bonds exposes the linear epitope located in the large chain of napin, therefore only napin large chain was recognized by the AN-Ab under reducing conditions. Although cross reactivity between allergens with low sequence identity is rare, IgE cross reactivity among non-homologous peanut allergens was

Fig. 3. SDS-PAGE profile and Western blot for B. napus, B. juncea and S. alba meal proteins show (a) binding of Sin a 1 AE-Ab with S. alba napin large chain only, and (b) cross reactivity of S. alba AN-Ab with B. napus, and B. juncea. MWM: molecular weight markers, NR: Non-reducing, R: Reducing conditions. 12% T homogenous gels, 2.5 mg protein/well were used. AE-Ab 0.01 mg/mL, secondary antibody dilution 5.0  103 (goat anti rabbit IgGeHRP conjugate), For AN-Ab 4e20% gels, 2.5 mg protein/well, AN-Ab 0.009 mg/ml, secondary antibody dilution 2.0  103 (goat anti rabbit IgGeHRP conjugate), X ray film exposure was 30 s.

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reported recently by Merima et al. (2013). Occurrence of similar short peptide sequences on the surface of folded proteins has been suggested as the reason for this cross reactivity (Merima et al., 2013). Of the conformational and linear epitopes, a linear epitope contains a stretch of contiguous amino acids in the antigen whereas a conformational epitope contains amino acid residues distant in the primary sequence but proximate in the folded protein (Robotham, Teuber, Sathe, & Roux, 2002). The secondary or tertiary structure of the allergen brings together different segments of the polypeptide chain to form the conformational epitope that binds with IgE. Linear epitopes persist after the protein is denatured, yet a conformational epitope persists only in a properly folded protein and loses the ability to bind with its relevant antibody when SeS bonds stabilizing higher order structures are no longer present upon reducing and denaturing (Ladner, 2007). Linear epitopes are highly prevalent in major food allergens such as peanut (Ara h 1, Ara h 2 and Ara h 3), milk, soy, shrimp and cashew and only encountered by the immune system upon partial denaturation and digestion (Bannon, 2004). Results of the present study are in agreement with Menéndez-Arias et al. (1990) reporting on epitope mapping of Sin a 1 and the proposed location of the immunodominant linear epitope on the large chain. Present results also suggest lack of specificity of AN-Ab against napin and its non-reliability to quantify Sin a 1. The AE-Ab, which showed specific binding to YM

napin, was used as the primary Ab in developing an ELISA method to quantify Sin a 1. 3.3. NCI-ELISA standard curve Napin obtained from CEX-1gave higher OD values under both NR þ ND and R þ D conditions than napin from SEC when assayed by NCI-ELISA as the standard (results not shown). Reduction of SeS bonds resulted in higher OD values for napin of CEX-1 than intact protein under non-denaturing conditions (Fig. 4a). This is an indication that in native Sin a 1 the linear epitope (used for raising AEAb) is partially exposed to the protein surface and the extent of exposure increases with heat denaturation and SeS disruption, consequently, binding more AE-Ab molecules leading to high OD. Increased accessibility of antibody to the epitope is necessary for accurate quantification; therefore, R þ D conditions were favorable for both protein standards and seed extracts in recognizing Sin a 1 using AE-Ab. Napin I and II fractions (CEX-2) and unfractionated napin had fairly similar AE-Ab binding ability (Fig. 4b). Therefore, unfractionated napin from CEX-1, which may be a mixture of the Sin a 1 isoform and other napin isoforms, was considered as a suitable standard for Sin a 1 quantitation. The NCI-ELISA method was capable of quantifying Sin a 1 with a LOD and LOQ of 3.08 ppm and 3.23 ppm, respectively. The AE-Ab based NCI-ELISA used in the present study meets the recommended requirement of a 1e10 ppm sensitivity range for allergen analysis methods (Poms, Klein, & Anklam, 2004). Both inter- and intra assay CV values were below 15% (Table 1) indicating the reproducibility of the assay. 3.4. Spike recovery The spike recovery study using B. napus (AC Excel) meal and CEX-1 purified napin of S. alba validated the procedure for Sin a 1 quantitation (Table 1). The recovery % was significantly lower (56.96%; P < 0.05) at low spike levels whereas significantly higher (P < 0.05) recoveries (99.38e104.48%) were obtained at high spike levels. Abbott et al. (2010) recommend 80e120% spike recovery levels as ideal in validating the performance characteristics of quantitative ELISA methods for food allergen determination and allow 50e150% spike recovery considering the matrix effects and differences between the incurred samples and spiked sample. Therefore, the extraction of napin at pH 3 and quantitation by AEAb based NCI-ELISA as used in this study provided ideal recoveries

Table 1 Intra- and inter-assay coefficient of variation (CV) of AE-Ab based NCI ELISA assay for Sin a 1 quantification. Napin protein levels

Assay level (mg protein/mL) 0.00 1.08 2.16 3.24 4.32 5.40 10.8 27.0

Fig. 4. Standard curves for NCI-ELISA show (a) increased binding of AE-Ab with SeS bonds reduced S. alba (Andante) napin (CEX-1), (b) AE-Ab binding pattern of unfractionated napin and napin fractions (napin I and napin II) obtained from CEX-2.

Coefficient of variation, % Intra-assay

Inter-assay

1.97e8.64 6.83e11.57 9.16e13.89 5.51e12.16 6.76e11.05 2.39e7.68 0.36e0.69 0.35e1.27

6.67 14.14 13.16 9.44 12.45 8.05 1.70 1.45

a

Spike level (mg/g meal)b

Recovery, %

Coefficient of variation, %

30 (Low) 80 (Medium) 153 (High)

56.96  2.64 99.38  2.53 104.48  2.14

4.63 2.54 2.05

a b

Protein as BSA equivalents. Meal is defatted Brassica napus (canola).

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Values are means  standard deviations. Mean values of each content (total protein, napin and Sin a 1) category in the same continuous row followed by the same letter are not significantly different (p < 0.05). X Determined by total nitrogen analysis (% N  6.25). Y Determined by BCA assay using BSA as the standard (pH 3 extracts were filtered by AMICON 3K centrifugal filters to remove interfering compounds prior to BCA assay). Z Determined by AE-Ab based NCI ELISA.

60.17  2.42Y 90.39  2.71Y 65.65  3.77Z 57.31  3.31Y 58.97  0.33X 54.89  0.80X 3.48  0.27Y 4.03  0.10Y 3.72  0.23Y 2.65  0.14X 3.81  0.08X 3.11  0.22X 4.68  0.22Z 5.98  0.60Z 4.92  0.48Z 8.22  0.46Y 9.25  0.47Z 8.15  0.41Z 32.35  1.04Y 31.37  0.98Y 34.93  0.58Y AC Base AC Pennant Andante

2011

4.43  0.29X 4.27  0.34X 4.77  0.22X 27.10  0.25X 26.23  1.38X 24.44  0.75X 35.81  0.34Z 34.61  0.22Z 35.90  0.43Y

6.88  0.28Y 6.49  0.89Y 6.18  0.48Y

2010 Year

2006 2011 2010 2006 2010 2006

2010 Year Year

2011

2006

Percent (%) of napin content (g/100 g) Percent (%) of seed weight (g/100 g)

Sin a 1 contentZ Napin content (g/100 g seeds)Y Total protein content (g/100 g seeds)X Variety

Table 2 Levels of total seed protein, extractable protein at pH 3 (napin) and Sin a 1 content in three commercial YM varieties.

The total N-based seed protein content in YM varieties grown in Saskatoon ranged from 24.4 to 35.9 g/100 g seeds (Table 2) and for each variety a significant difference (p < 0.05) with the production year was evident. The lowest total protein content (w24e27%) was recorded for year 2010 compared to those of 2006 (w34e35%) and 2011 (w31e34%). The harvest survey reports of the Canadian Grain Commission (www.grainscanada.ca) also confirm that YM seeds produced in 2006 had higher protein content and lower oil content than those of 2010 and 2011. The influence of environmental factors on growth, location and growing year on the crude protein content of YM mustard seeds has been previously reported (Bell, Rakow, & Downey, 2000). Extracts of YM meals prepared at pH 3.0 with CaCl2 contained napin only (Fig. 5a) and an absence of napin in the residual meal polypeptide profile (Fig. 5b) further confirming that these conditions were selective for napin solubilization. Therefore, the pH 3 extractable protein represents total napin in YM seeds. Currently, inefficient extraction of allergens from the food matrix is a major issue in reliable quantification of food allergens using ELISA assays. The present study shows complete recovery of napin can be achieved when an extraction medium at pH 3 containing CaCl2 (0.15 M) is used. The total napin content of YM seeds ranged from 4.8 to 9.2 g/ 100 g seeds (Table 2) and showed a significant variation (p < 0.05) with the production year. A similar changing pattern was observed for the total protein content (Table 2, Supplementary data Fig. S1a). The highest total protein as well as the napin levels were found in the seeds produced in 2006. The napin level of each variety as a fraction of total protein content of the seeds was in the ranges of 16e19% in 2010, 22e26% in 2006 and 17e21% in 2011. According to Bell et al. (2000) cruciferin, napin and oleosin contributes 50%, 20% and 10%, respectively to the total seed protein in mustard and the values of the present study show that the napin content of YM protein may vary from 16 to 26%. The Sin a 1 level of seeds of AC Base, AC Pennant and Andante ranged from 2.6 to 4.7%, 3.8 to 5.9% and 3.1 to 4.9%, respectively within the three crop years (Table 2). On the basis of seed weight, seeds produced in 2010 had the lowest Sin a 1 level and comparatively low contents of total protein and napin (Table 2, Supplementary data Fig. S1a & b). In a given year differences between varieties for the contents of total protein, napin and Sin a 1 did not show a high variability. Sin a 1 accounted for >50% of the napin content of all varieties (Table 2) and was in the range of 50e 60%, 55e90% and 55e65% for AC Base, AC Pennant and Andante, respectively with an exceptionally high value for AC Pennant from 2010 year. The present study shows that the napin protein fraction of YM also contains proteins devoid of the Sin a 1 epitope sequence therefore all YM napin may not contribute to the 2S allergenicity. In the previous reporting of Sin a 1 levels using a polyclonal antibody based sandwich ELISA (pAbS-ELISA) and seed extracts prepared at pH 8.5, values between 0.18 and 0.29 g/100 g meal were recorded (Shim & Wanasundara, 2008). The Present study shows that napin is not totally solubilized at pH 8.5, which may lead to inaccuracies in the estimation of total Sin a 1 levels in YM seeds using that approach. We also found that when the extracts of pH 8.5 are diluted for ELISA assay, napin as a lesser protein species in the extract dilutes down to an undetectable level. In contrast, the pH 3 extracts prepared with CaCl2 predominantly contain napin and facilitate much better estimation of Sin a1. Moreover, the high

Year

3.5. Total protein, napin and Sin a 1 levels in commercial YM varieties

2011

when the sample has high levels of Sin a 1. The recovery levels were still acceptable at low concentrations of the antigen.

49.94  3.08X 55.13  1.41X 60.29  1.97Y

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Fig. 5. SDS PAGE profiles of (a) pH 3 seed extract, and (b) remaining seed residue of different YM varieties showing selective extractability of napin. MWM: molecular weight markers, 8e25% T gradient gels, polypeptide separation under non-reducing conditions.

specificity of AE-Ab towards the Sin a 1 epitope suggests more reliability on the results based on AE-Ab based ELISA compared to pAb based ELISAs. Use of epitope based peptide for antibody generation overcomes the purification of antigen protein, which is quite tedious and limited by the strength of identification tools available. For example, identity confirmation of purified Sin a 1 (as antigen) using mass spectroscopy is not specific to Sin a 1 because of the very limited S. alba napin sequences in the protein databases. 4. Conclusion AE-Ab based NCI-ELISA is useful in quantitation of Sin a 1 in YM seeds and will have the potential to determine actual allergen level present in the seed and products. The level of Sin a 1 varies with YM variety and the environmental factors may be significant. The values of Sin a 1 for Canadian grown YM varieties were 50e90% of pH 3 soluble proteins or 2.6e5.9% of seed weight and showed a positive relationship with the total content of seed protein and napin. Acknowledgment Authors thank Agriculture Agri-Food Canada Risk mitigation Initiative project (RBPI #2105) and Agriculture Development Fund (ADF Project 20100144) of Saskatchewan Ministry of Agriculture for financial support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.foodcont.2014.03.053. References Abbott, M., Heyward, S., Ross, W., Godefroy, S. B., Ulberth, F., Van Hengel, A., et al. (2010). Validation procedures for quantitative food allergen ELISA methods: community guidance and best practices. Journal of AOAC International, 93, 442e450. AOAC. (1997). AOAC official methods of analysis (16th ed.). Washington, DC: Association of Official Analytical Chemists. Bannon, G. A. (2004). What makes a food protein an allergen. Current Allergy and Asthma Reports, 4, 43e46.

Barciszweski, J., Szymanski, M., & Haertle, T. (2000). Minireview: analysis of rapeseed napin structure and potential role of the storage protein. Journal of Protein Chemistry, 19, 249e254. Bell, J. M., Rakow, G., & Downey, R. K. (2000). Comparisons of amino acid and protein levels in oil-extracted seeds of Brassica and Sinapis species, with observations on environmental effects. Canadian Journal of Animal Science, 80, 169e174. Cui, W., & Eskin, N. A. M. (1998). Processing and properties of mustard products and components. In G. Mazza (Ed.), Functional foods biochemical and processing aspects (pp. 235e264). Lancaster PA Technomic Publishing Co Inc. European Food Safety Authority. (2007). Opinion of the scientific panel on dietetic products, nutrition and allergies on a request from the Commission related to the notification from IFF on mustard seed oil pursuant to article 6, paragraph 11 of directive 2000/13/EC. The European Food Safety Authority Journal, 481, 1e7. Josefsson, L. G., Lenman, M., Ericson, M. L., & Rask, L. (1987). Structure of a gene encoding the 1.7S storage protein, napin, from Brassica napus. The Journal of Biological Chemistry, 262, 12196e12201. Ladner, R. C. (2007). Mapping the epitopes of antibodies. Biotechnology and Genetic Engineering Reviews, 24, 1e30. Lee, P. W., Hefle, S. L., & Taylor, S. L. (2008). Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of mustard in foods. Journal of Food Science, 73, T62eT68. Menéndez-Arias, L., Dominguez, J., Moneo, I., & Rodríguez, R. (1990). Epitope mapping of the major allergen from yellow mustard, Sin a 1. Molecular Immunology, 27, 143e150. Merima, B., Kostadinova, M., Radauer, C., Hafner, C., Szepfalusi, Z., Varga, E. M., et al. (2013). Ig-E cross reactivity between the major peanut allergen Ara h 2 and the non- homologous allergens Ara h 1 and Ara h 3. Journal of Allergy and Clinical Immunology, 132, 118e124. Monsalve, R. I., González de la Peña, M. A., Menéndez-Arias, L., López-Otín, C., Villalba, M., & Rodríguez, R. (1993). Characterization of a new oriental-mustard (Brassica juncea) allergen, Bra j I E: detection of an allergenic epitope. Biochemical Journal, 293, 625e632. Monsalve, R. I., Villalba, M., & Rodríguez, R. (2001). Allergy to mustard seeds: the importance of 2S albumins as food allergens. Internet Symposium on Food Allergens, 3, 57e69. Mustorp, S., Engdahl-Axelsson, C., Svensson, U., & Holck, A. (2008). Detection of celery (Apium graveolens), mustard (Sinapis alba, Brassica juncea, Brassica nigra) and sesame (Sesamum indicum) in food by real-time PCR. European Food Research and Technology, 226, 771e778. Palomares, O., Cuesta-Heranz, J., Vereda, A., Sirvent, S., Villalba, M., & Rodríguez, R. (2005). Isolation and identification of an 11S globulin as a new major allergen in mustard seeds. Annals of Allergy, Asthma and Immunology, 94, 586e592. Poms, R. E., Klein, C. L., & Anklam, E. (2004). Methods for allergen analysis in food: a review. Food Additives and Contaminants, 21, 1e31. Robotham, J. M., Teuber, S. S., Sathe, S. K., & Roux, K. H. (2002). Linear IgE epitope mapping of the English walnut (Juglans regia) major food allergen jug r1. Journal of Allergy and Clinical Immunology, 109, 143e149. Shim, Y. Y., & Wanasundara, J. P. (2008). Quantitative detection of allergenic protein Sin a1 from yellow mustard (Sinapis alba L.) using enzyme linked immunosorbent assay. Journal of Agricultural and Food Chemistry, 56, 1184e1192.

H.K. Marambe et al. / Food Control 44 (2014) 233e241 Sirvent, S., Palomares, O., Cuesta-Herranz, J., Villalba, M., & Rodríguez, R. (2012). Analysis of the structural and immunological stability of 2S albumin, nonspecific lipid transfer protein, and profilin allergens from mustard seeds. Journal of Agricultural and Food Chemistry, 60, 6011e6018. Sirvent, S., Palomares, O., Vereda, A., Villalba, M., Herranz, J. C., & Rodríguez, R. (2009). nsLTP and profilin are allergens in mustard seeds: cloning, sequencing and recombinant production of Sin a 3 and Sin a 4. Clinical & Experimental Allergy, 39, 1929e1936. Smith, P. K., Krohgn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., et al. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150, 76e85. Taylor, S. L., Baumert, J. L., Kruizinga, A. G., Remington, B. C., Crevel, R. W. R., BrookeTaylor, S., et al. (2014). Establishment of reference doses for residues of

241

allergenic foods: report of the VITAL expert panel. Food and Chemical Toxicology, 63, 9e17. The Commission of the European Communities. (2007). Commission Directive 2007/68/EC. Official Journal of the European Union, L310, 11, 28.11.2007. Verada, A., Sirvent, S., Villalba, M., Rodriguez, R., Cuesta-Herranz, J., & Palomares, O. (2011). Improvement of mustard (Sinapis alba) allergy diagnosis and management by linking clinical features and component resolved approaches. Journal of Allergy and Clinical Immunology, 127, 1304e1307. Vidal, C., Diaz, C., Saez, A., Rodriguez, M., & Iglesias, A. (1991). Anaphylaxis to mustard. Postgraduate Medical Journal, 67, 404. Wanasundara, P. K. J. P. D., Abeysekara, S. J., McIntosh, T. C., & Falk, K. C. (2012). Solubility differences of major storage proteins of Brassicaceae oilseeds. Journal of the American Oil Chemists’ Society, 89, 869e881.