Article pubs.acs.org/JAFC
Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay Changqi Liu, Guneet S. Chhabra, and Shridhar K. Sathe* Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, Florida 32306, United States ABSTRACT: A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5−36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10−50000 ppm) food matrices were 93.1−125.6% and 35.7−112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples. KEYWORDS: pistachio, mAb, antibody, ELISA
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analysis of food allergen recalls for the fiscal years 2007−2012, representing the effective FALCPA years, indicates that bakery (31.5%), candy (10.0%), and snack (12.1%) foods, together, account for 53.6% of the total 732 recalls.12 Among the 933 recalls (counting each recall multiple times if the recall involved multiple allergens) covered in the analysis, milk (296), wheat (171), soy (153), tree nuts (119), and egg (108) were the top five allergens, representing 90.78% of the total recalls.12 Recent realtime PCR analysis of 229 commercial food products that did not declare pistachio or tree nut presence detected pistachio presence in 29 samples.13 With the likely increasing use of pistachio in food products, it is important to develop reliable pistachio detection assays. Several commercial polyclonal antibody (pAb)-based ELISA kits are available for pistachio detection including those from Astori Tecnica; Bio-Check (UK), Ltd.; Romer Laboratories, Inc.; Immunolab GmbH; and Genon Laboratories, Ltd. Typically, pAb-based assays exhibit measurable cross-reactivity.14−20 Antibody-based assays may encounter signal reduction/elimination for thermally processed foods/ingredients. This signal loss may result from epitope denaturation, destruction, or both. Such signal loss attributable to destruction of a clinically relevant and immunodominant conformational epitope, targeted by the assay detection antibody, is of interest. If, however, the tested food contains a heat stable human allergy relevant protein/epitope, targeting the heat stable protein/epitope is important for designing the detection method, regardless of the tested food/ ingredient/matrix and pAb/mAb use. Recently, a murine mAbbased direct sandwich ELISA kit for pistachio detection has been
INTRODUCTION Pistacia vera, a plant in the Anacardiaceae family, produces pistachio nut seeds with desirable sensory attributes and nutritional value1 that enjoy good consumer acceptability. Over the past three decades, pistachio production has increased >20 times. In 2013−2014, pistachio was the third largest produced tree nut in the United States with 235,000 metric tons (MT), following almond (1,732,800 MT) and walnut (482,000 MT).2 In 2013, the U.S. pistachio production (196,930 MT valued at U.S. $646,744,902) was second, behind that of Iran.3 Pistachio seeds are commonly consumed as a snack food or are used as an ingredient in ice cream and confections such as baklava, chocolate, halva, Turkish delight, and marzipan. Although safely enjoyed by most, upon exposure, sensitive individuals may experience adverse reactions to pistachios. The U.S. prevalence of tree nut allergy was estimated to be 0.6% of the population in 2008, of which 9% of the cases were attributed to pistachio sensitivity.4 The prevalence of pistachio allergy in Iran was reported to be 0.65% for people living in pistachio cultivation regions and 0.3% for those living outside those regions.5 Pistachio-induced anaphylaxis6 and fatality7 have been documented. Pistachio oral allergy syndrome (OAS) has also been reported.8 Although clinical trial efforts are continuing to show how to desensitize food allergy patients, currently avoidance of the offending allergens remains the best course of action for sensitive consumers.9 According to 2014 FDA data, food allergens have become the single largest cause of food recalls, representing ∼47% of the total recalls.10 Available 2015 data (April 15−June 10 recalls) indicate that of the listed 34 recalls, 11 (32.35%) were food allergen related.10 Although allergen declaration is required according to the FDA Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004,11 undeclared allergens may still be present in food products due to cross-contamination, mislabeling, or both. A comprehensive © 2015 American Chemical Society
Received: Revised: Accepted: Published: 9139
June 21, 2015 September 17, 2015 September 28, 2015 September 28, 2015 DOI: 10.1021/acs.jafc.5b03066 J. Agric. Food Chem. 2015, 63, 9139−9149
Article
Journal of Agricultural and Food Chemistry
For the pistachio dough, 50 g of pistachio full-fat flour, 60 g of Publix confectionery sugar, and 1 g of McCormick cardamom were ground in an Osterizer blender (speed setting “grind”) for 1 min followed by the addition of 0.5 g of McCormick green food coloring and 8 g of water. The mixture was manually kneaded into dough. To prepare the rolls, 10 g of cashew dough was rolled into a flat sheet (∼3 mm thick) and cut into a rectangular shape, whereas pistachio dough (0.1, 0.6, and 1.2 g) was formed into log shapes (2−10 mm diameter) and placed in the center of the cashew sheets. The cashew sheets were tightly rolled around the pistachio logs to form the kaju rolls. Pistachio contents in rolls prepared according to recipe 1 (Nestlé recipe) were 0, 0.6, 3.4, and 6.3% (w/w) and in rolls prepared according to recipe 2 (laboratory recipe) were 0, 0.4, 2.4, and 4.5% (w/w). Corn Flakes. Forty-five grams of Publix sweet corn kernels were ground in an Osterizer blender (speed setting “grind”) for 1 min with 50 mL of distilled water and baked at 120 °C in a KitchenAid Architect II oven (KitchenAid, Benton Harbor, MI, USA) until dry. The moisture content of the sweet corn was 78% as determined according to AOAC Official Method 925.40.23 The final yield of the corn flakes was approximately 10 g. Pistachio full-fat flour was incurred at 10, 50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to baking. Sponge Cakes. Seventeen grams of egg was beaten by a KitchenAid hand mixer (speed setting “1”) for 4 min. Thirty-four grams of Publix sugar was added, and the mixture was beaten for another 4−5 min until light and fluffy. Twenty-three grams of Publix all-purpose flour, 0.7 g of Argo baking powder, and 0.2 g of Publix salt were sifted into the mixture, and 0.4 g of McCormick vanilla extract was added. In a sauce pan, 20 g of Publix whole milk and 4.7 g of Publix butter were heated on low heat until the butter was melted. The milk and butter were combined with the batter and baked in greased cake pans at 163 °C in a KitchenAid Architect II oven for 30 min. Pistachio full-fat flour was incurred at 10, 50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to baking. Sugar Cookies. Twenty-three grams of Publix butter was creamed with 30 g of Publix sugar using a KitchenAid hand mixer (speed setting “1”) and were then mixed with 6 g of egg and 0.3 g of McCormick vanilla extract. Subsequently, 40 g of Publix all-purpose flour, 0.5 g of Arm & Hammer baking soda, and 0.2 g of Argo baking powder were sifted into the batter and blended to form the dough. The dough was rolled to 0.69 cm thickness and baked at 190 °C in a KitchenAid Architect II oven for 12 min. Pistachio full-fat flour was incurred at 10, 50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to baking. Flour Preparation. All high moisture content ingredients and foods were dried as described earlier.17,18 Specifically, heat-sensitive foods (e.g., ice cream, cheese) were freeze-dried (VirTis BenchTop K freezedryer, SP Scientific, Warminster, PA, USA), whereas fresh produce (e.g., fruits and vegetables) and high-sugar dried fruit (e.g., raisins, dates) were oven-dried for 24 h at 50−60 °C. All seeds, food ingredients, and processed foods, in their dried from, were ground in an Osterizer blender (speed setting “grind”; Galaxy model 869-18R, Jaden Consumer Solutions, Boca Raton, FL, USA) to obtain uniform flours. As needed, the flours were defatted for 8 h using a Soxhlet apparatus (Thermo Fisher Scientific Inc., Waltham, MA, USA) with petroleum ether (boiling point range of 38.2−54.3 °C) as the solvent (flour-to-solvent ratio of 1:10 w/v). After overnight drying in a fume hood at room temperature, the powders were passed through a 40 mesh sieve and then stored in screw-capped plastic bottles at −20 °C until further use. Protein Extract Preparation. Protein extracts were prepared as per the ELISA kit instructions. Briefly, sample flours (100 mg each for pistachio and other food ingredients, 1 g each for commercially prepared foods, pistachio-spiked samples, and samples incurred with ≥5000 ppm of pistachio) were extracted in extraction buffer (provided with the ELISA kit; flour-to-solvent ratio of 1:10 w/v) at 60 °C for 10 min with vigorous manual vortex mixing every 2 min. For corn flakes, sponge cakes, and sugar cookies incurred with ≤100 ppm of pistachio, the entire batch of each sample was blended in an Osterizer blender (speed setting “grind”) with borate saline buffer (BSB, 0.1 M boric acid, 0.025 M sodium borate, 0.075 M sodium chloride, pH 8.45; sample-to-solvent ratio of 1:10 w/v) at room temperature for 1 min and then magnetically stirred at room temperature for 1 h. The extracts were subsequently centrifuged at 2000g (Centrific 225 centrifuge, Thermo Fisher Scientific
introduced into the market by BioFront Technologies (Tallahassee, FL, USA). The kit claims include targeting a thermally stable antigen, assay specificity, 0.3 ppm sensitivity, and good recovery (>85%) from spiked samples. We were interested in assessing the kit specificity, sensitivity, and robustness. Included in these investigations were (a) laboratory-prepared ingredients and foods, (b) commercially processed and sold foods with declared and undeclared pistachio presence, (c) pistachio seed/flour and pistachio-containing foods/matrices exposed to select food-processing methods in the laboratory, and (d) commercial and laboratory-prepared foods spiked with known amounts of pistachio proteins.
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MATERIALS AND METHODS
Materials. Dehulled raw pistachio seeds, food ingredients, and commercially processed foods were purchased from local grocery stores and restaurants and were processed as needed as earlier described in detail by Tiwari et al.18 The MonoTrace pistachio ELISA kits were purchased from BioFront Technologies (Tallahassee, FL, USA). Sources of chemicals, reagents, supplies, and instruments have been reported earlier.20 Methods. Pistachio Seed Processing. Dehulled whole pistachio raw seeds were processed as follows.21 1. Pressure cooking in a SterileMax benchtop autoclave (Barnstead International, Inc., Dubuque, IA, USA) at 121 °C, 15 psi, for 15 and 30 min. Autoclaved samples were air-dried at room temperature (∼25 °C) in a fume hood until constant weight. 2. Blanching in boiling water (94 °C) for 5 and 10 min. The ratio of nut seeds to water was 1:10 w/v. Samples were patted dry on paper towels and further air-dried at room temperature in a fume hood until constant weight. 3. Frying in Crisco vegetable oil at 191 °C for 1 min. Excess oil was allowed to drain completely on paper towels prior to further handling. 4. Microwave heating in a Panasonic microwave oven (Sears, Roebuck and Co., Hoffman Estates, IL, USA) at 50% (500 W) and 100% power (1000 W) for 3 min. 5. Dry roasting at 140 °C for 30 min and at 168 °C for 12 min. Samples were placed in aluminum pans and subjected to roasting in a TempCon oven (American Scientific Products, McGaw Park, IL, USA) previously heated to the desired temperatures and monitored using a thermometer. Kaju Roll. Kaju roll is a sweet, popular in India, made by rolling a cashew dough sheet around a pistachio dough log. Cashew and pistachio doughs were each prepared separately using two different recipes: (1) Nestlé recipe22 and (2) laboratory recipe. Doughs were prepared as described below. 1. Nestle ́ Recipe. For the cashew dough, 60 g of full-fat cashew nut seed flour, 40 g of Nestlé sweetened condensed milk, 1 g of Ziyad ghee (Indian clarified butter), and 1 g of Dabur rose water were manually mixed in a pan with a spatula and heated at medium-low heat [stove temperature = 130 °C, dough temperature = 77 °C, monitored by a Traceable infrared thermometer (VWR International, LLC, Radnor, PA, USA)] for 10 min to form the dough. Subsequently, 3 g of Nestlé nonfat dry milk powder was added to the dough and the dough was allowed to cool to room temperature. For the pistachio dough, 60 g of full-fat pistachio seed flour, 40 g of Nestlé sweetened condensed milk, 1 g of Ziyad ghee, 1 g of McCormick cardamom, and 0.5 g of McCormick green food coloring (FD&C Yellow 5 and Blue 1) were manually mixed in a pan with a spatula and heated at medium-low heat for 10 min to form the dough. Subsequently, 3 g of Nestlé nonfat dry milk was added to the dough and the dough was allowed to cool to room temperature. 2. Laboratory Recipe. For the cashew dough, 50 g of cashew full-fat flour and 60 g of Publix confectionery sugar were ground in an Osterizer blender (speed setting “grind”; Galaxy model 869-18R, Jaden Consumer Solutions, Boca Raton, FL, USA) for 1 min followed by the addition of 1 g of Dabur rose water and 8 g of water. The mixture was manually kneaded into dough. 9140
DOI: 10.1021/acs.jafc.5b03066 J. Agric. Food Chem. 2015, 63, 9139−9149
Article
Journal of Agricultural and Food Chemistry
Table 1. Food Ingredients (100000 ppm) Tested for Assessing Cross-Reactivity of the MonoTrace Pistachio ELISA Kita
a Food matrix from a single batch was weighed in triplicate, and each sample was extracted using buffer provided in the kit. No cross-reactivity was detected (i.e., signal equivalent to 0.99) 1.73 ± 0.32 5.76 ± 1.07 3.64 ± 1.36 7.20 ± 2.40
a Data are expressed as mean ± SD. The 50% maximum signal concentration and linear detection range were determined using protein extracts from three different batches of pistachios and repeated in two different kits. The limits of detection and quantification were determined in three different kits using pistachio standards provided in those kits. bLimit of detection 1 (LOD1) was determined on the basis of the standard deviation of the blank (σ) and the slope of the standard curve (S) according to the formula LOD1 = 3σ/S. cLimit of detection 2 (LOD2) was determined on the basis of the mean absorbance (M) and standard deviation of the blank (σ) according to the formula LOD2 = M + 3σ. dLimit of quantification 1 (LOQ1) was determined on the basis of the standard deviation of the blank (σ) and the slope of the standard curve (S) according to the formula LOQ1 = 10σ/S. eLimit of quantification 2 (LOQ2) was determined based on the mean absorbance (M) and standard deviation of the blank (σ) according to the formula LOQ2 = M + 10σ.
Figure 1. Typical MonoTrace pistachio ELISA kit four-parameter curve (A) and linear standard curve using pistachio protein standards (0, 0.5, 2, 6, 18, and 36 ppm pistachio) provided within the kit (B). The standard curves were repeated three times within a kit.
population would react) for an allergic reaction in 1% of the population estimated for hazelnut and peanut were 0.2 and 0.1 mg of protein, respectively, and the reference dose for cashew (lower 95% confidence interval of ED05) was 2.0 mg of protein.39,40 The reference dose for most allergenic foods ranged from 0.03 mg of protein (ED01 for egg) to 10 mg of protein (lower 95% confidence interval of ED05 for shrimp). Currently, no threshold data are available for pistachio and other tree nuts, and thus the tested kit detection range 0.5−36 ppm must be interpreted with caution. Assay Efficiency. The MonoTrace pistachio ELISA was rapid with an assay time of approximately 1.5 h (extraction, 10 min; incubation with antigen, 30 min; incubation with detection antibody, 30 min; and color development, 10 min). The updated version of the MonoTrace Pistachio ELISA claims improvement in the assay efficiency and short incubations with antigen/food sample extract, detection antibody, and substrate (10 min each). To determine if the antigen extraction was sufficient, pistachio full-fat flours were extracted in four different buffers (EXB, BSB, PBS, and SBC) under two separate conditions (60 °C, 10 min and room temperature, 1 h). Under both conditions, the kitprovided buffer, EXB, yielded the highest soluble protein extractability and antigen recovery (Table 3). Protein extractions by EXB at 60 °C for 10 min and at room temperature for 1 h were equivalent (P > 0.05). Assay Specificity. Individuals allergic to one tree nut often exhibit IgE reactivity to other edible nut seed proteins.41 Crossreactive tree nuts are usually found in the same botanical family. Strong cross-reactivities have been documented in nut seed proteins of (a) pecan and walnut species of the Juglandaceae family42−46 and (b) cashew and pistachio of the Anacardiaceae family.5,43,44,47−53 Cross-reactivity is likely due to the homologous linear amino acid sequence, three-dimensional structural similarity, or both, of different nut seed proteins. Several murine
sensitivity is comparable to the reported sensitivity by Romer (0.13 ppm) and Bio-Check (0.2 ppm) ELISA kits and the recently reported real-time PCR assay sensitivity (0.1 ppm).13 Compared to the Genon ELISA kit (10 ppm) and the PCR methods reported by Brežná et al.35 (4 ppm) and Barbieri and Frigeri36 (100 ppm), the MonoTrace pistachio ELISA kit assay exhibited better sensitivity. The LOQ1 (0.30 ppm) and LOQ2 (0.38 ppm) of the MonoTrace pistachio kit are comparable with the LOQ of a previously reported pistachio ELISA ( 0.99) is similar to the linear range (1−40 ppm) for the Romer kit. The concentration of pistachio soluble protein that registered 50% of the maximum signal is 152 ng/mL (7.9 ppm) (Figure 1), comparable to the value (∼137 ng/mL) reported by Lim.37 The sensitivity of an assay is considered sufficient when it can reliably detect the minimum dose of an allergen capable of eliciting a clinically measurable response. Many attempts have been made to determine the allergen threshold that causes an adverse reaction. The U.S. Food and Drug Administration established a Threshold Working Group in 2005 but failed to establish the threshold for the major food allergens due to insufficient data at the time.38 In 2010, the Australian Allergen Bureau first introduced defined allergen threshold levels for precautionary labeling. In their most recent update, the eliciting doses (EDp, dose of allergen at which p% of the allergic 9143
DOI: 10.1021/acs.jafc.5b03066 J. Agric. Food Chem. 2015, 63, 9139−9149
Article
Journal of Agricultural and Food Chemistry
also found to be cross-reactive with protein(s) from mango seeds48 and with proteins from seeds of taxonomically unrelated botanical families such as sunflower, peanut, and walnut.47 A previously observed cross-reactivity of pistachio ELISA (at 4 ppm level) using sheep antisera as the capture reagent and rabbit antisera as detection reagent with cashew nut seed proteins was therefore not unexpected.37 Recently, in a multiplex ELISA simultaneously detecting 14 different food allergens and gluten, cross-reactivity of pistachio detection assay with Brazil nut, cashew, and hazelnut seed proteins has also been reported.54 The MonoTrace pistachio ELISA kit, under the test conditions, is specific and did not display cross-reactivity (signal equivalent to 100 kDa.56 So far, four distinct pistachio 11S globulin protein sequences have been reported with predicted molecular masses ranging from 53.2 to 56.5 kDa.57−59 Among pistachio 11S acidic (35−40 kDa) and basic (22−27 kDa) subunits, the latter have been reported to exhibit a stronger reactivity, compared to the 11S acidic subunit and 2S albumin polypeptides, tested with pooled tree nut-allergic human sera.51 Therefore, pistachio 11S globulin basic subunit may serve as a good antigen for producing antibodies targeting human allergy relevant epitopes. Assay Reproducibility, Uncertainty, and Recovery. The intra- and inter-assay variability of the MonoTrace pistachio ELISA kit was
Pistachio (Pistacia vera L.) Detection and Quantification Using a Murine Monoclonal Antibody-Based Direct Sandwich Enzyme-Linked Immunosorbent Assay Changqi Liu, Guneet S. Chhabra, and Shridhar K. Sathe* Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, Florida 32306, United States ABSTRACT: A commercially available direct sandwich enzyme-linked immunosorbent assay (ELISA) (BioFront Technologies, Tallahassee, FL, USA) using murine anti-pistachio monoclonal antibodies (mAbs) as capture and detection antibodies was evaluated. The assay was sensitive (limit of detection = 0.09 ± 0.02 ppm full fat pistachio, linear detection range = 0.5−36 ppm, 50% maximum signal concentration = 7.9 ± 0.7 ppm), reproducible (intra- and inter-assay variability < 24% CV), and rapid (post-extraction testing time ∼ 1.5 h). The target antigen was stable and detectable in whole pistachio seeds subjected to autoclaving (121 °C, 15 psi, 15, 30 min), blanching (100 °C, 5, 10 min), frying (191 °C, 1 min), microwaving (500, 1000 W, 3 min), and dry roasting (140 °C, 30 min; 168 °C, 12 min). No cross-reactivity was observed in 156 food matrices, each tested at 100,000 ppm, suggesting the ELISA to be pistachio specific. The pistachio recovery ranges for spiked (10 ppm) and incurred (10−50000 ppm) food matrices were 93.1−125.6% and 35.7−112.2%, respectively. The assay did not register any false-positive or -negative results among the tested commercial and laboratory prepared samples. KEYWORDS: pistachio, mAb, antibody, ELISA
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analysis of food allergen recalls for the fiscal years 2007−2012, representing the effective FALCPA years, indicates that bakery (31.5%), candy (10.0%), and snack (12.1%) foods, together, account for 53.6% of the total 732 recalls.12 Among the 933 recalls (counting each recall multiple times if the recall involved multiple allergens) covered in the analysis, milk (296), wheat (171), soy (153), tree nuts (119), and egg (108) were the top five allergens, representing 90.78% of the total recalls.12 Recent realtime PCR analysis of 229 commercial food products that did not declare pistachio or tree nut presence detected pistachio presence in 29 samples.13 With the likely increasing use of pistachio in food products, it is important to develop reliable pistachio detection assays. Several commercial polyclonal antibody (pAb)-based ELISA kits are available for pistachio detection including those from Astori Tecnica; Bio-Check (UK), Ltd.; Romer Laboratories, Inc.; Immunolab GmbH; and Genon Laboratories, Ltd. Typically, pAb-based assays exhibit measurable cross-reactivity.14−20 Antibody-based assays may encounter signal reduction/elimination for thermally processed foods/ingredients. This signal loss may result from epitope denaturation, destruction, or both. Such signal loss attributable to destruction of a clinically relevant and immunodominant conformational epitope, targeted by the assay detection antibody, is of interest. If, however, the tested food contains a heat stable human allergy relevant protein/epitope, targeting the heat stable protein/epitope is important for designing the detection method, regardless of the tested food/ ingredient/matrix and pAb/mAb use. Recently, a murine mAbbased direct sandwich ELISA kit for pistachio detection has been
INTRODUCTION Pistacia vera, a plant in the Anacardiaceae family, produces pistachio nut seeds with desirable sensory attributes and nutritional value1 that enjoy good consumer acceptability. Over the past three decades, pistachio production has increased >20 times. In 2013−2014, pistachio was the third largest produced tree nut in the United States with 235,000 metric tons (MT), following almond (1,732,800 MT) and walnut (482,000 MT).2 In 2013, the U.S. pistachio production (196,930 MT valued at U.S. $646,744,902) was second, behind that of Iran.3 Pistachio seeds are commonly consumed as a snack food or are used as an ingredient in ice cream and confections such as baklava, chocolate, halva, Turkish delight, and marzipan. Although safely enjoyed by most, upon exposure, sensitive individuals may experience adverse reactions to pistachios. The U.S. prevalence of tree nut allergy was estimated to be 0.6% of the population in 2008, of which 9% of the cases were attributed to pistachio sensitivity.4 The prevalence of pistachio allergy in Iran was reported to be 0.65% for people living in pistachio cultivation regions and 0.3% for those living outside those regions.5 Pistachio-induced anaphylaxis6 and fatality7 have been documented. Pistachio oral allergy syndrome (OAS) has also been reported.8 Although clinical trial efforts are continuing to show how to desensitize food allergy patients, currently avoidance of the offending allergens remains the best course of action for sensitive consumers.9 According to 2014 FDA data, food allergens have become the single largest cause of food recalls, representing ∼47% of the total recalls.10 Available 2015 data (April 15−June 10 recalls) indicate that of the listed 34 recalls, 11 (32.35%) were food allergen related.10 Although allergen declaration is required according to the FDA Food Allergen Labeling and Consumer Protection Act (FALCPA) of 2004,11 undeclared allergens may still be present in food products due to cross-contamination, mislabeling, or both. A comprehensive © 2015 American Chemical Society
Received: Revised: Accepted: Published: 9139
June 21, 2015 September 17, 2015 September 28, 2015 September 28, 2015 DOI: 10.1021/acs.jafc.5b03066 J. Agric. Food Chem. 2015, 63, 9139−9149
Article
Journal of Agricultural and Food Chemistry
For the pistachio dough, 50 g of pistachio full-fat flour, 60 g of Publix confectionery sugar, and 1 g of McCormick cardamom were ground in an Osterizer blender (speed setting “grind”) for 1 min followed by the addition of 0.5 g of McCormick green food coloring and 8 g of water. The mixture was manually kneaded into dough. To prepare the rolls, 10 g of cashew dough was rolled into a flat sheet (∼3 mm thick) and cut into a rectangular shape, whereas pistachio dough (0.1, 0.6, and 1.2 g) was formed into log shapes (2−10 mm diameter) and placed in the center of the cashew sheets. The cashew sheets were tightly rolled around the pistachio logs to form the kaju rolls. Pistachio contents in rolls prepared according to recipe 1 (Nestlé recipe) were 0, 0.6, 3.4, and 6.3% (w/w) and in rolls prepared according to recipe 2 (laboratory recipe) were 0, 0.4, 2.4, and 4.5% (w/w). Corn Flakes. Forty-five grams of Publix sweet corn kernels were ground in an Osterizer blender (speed setting “grind”) for 1 min with 50 mL of distilled water and baked at 120 °C in a KitchenAid Architect II oven (KitchenAid, Benton Harbor, MI, USA) until dry. The moisture content of the sweet corn was 78% as determined according to AOAC Official Method 925.40.23 The final yield of the corn flakes was approximately 10 g. Pistachio full-fat flour was incurred at 10, 50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to baking. Sponge Cakes. Seventeen grams of egg was beaten by a KitchenAid hand mixer (speed setting “1”) for 4 min. Thirty-four grams of Publix sugar was added, and the mixture was beaten for another 4−5 min until light and fluffy. Twenty-three grams of Publix all-purpose flour, 0.7 g of Argo baking powder, and 0.2 g of Publix salt were sifted into the mixture, and 0.4 g of McCormick vanilla extract was added. In a sauce pan, 20 g of Publix whole milk and 4.7 g of Publix butter were heated on low heat until the butter was melted. The milk and butter were combined with the batter and baked in greased cake pans at 163 °C in a KitchenAid Architect II oven for 30 min. Pistachio full-fat flour was incurred at 10, 50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to baking. Sugar Cookies. Twenty-three grams of Publix butter was creamed with 30 g of Publix sugar using a KitchenAid hand mixer (speed setting “1”) and were then mixed with 6 g of egg and 0.3 g of McCormick vanilla extract. Subsequently, 40 g of Publix all-purpose flour, 0.5 g of Arm & Hammer baking soda, and 0.2 g of Argo baking powder were sifted into the batter and blended to form the dough. The dough was rolled to 0.69 cm thickness and baked at 190 °C in a KitchenAid Architect II oven for 12 min. Pistachio full-fat flour was incurred at 10, 50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to baking. Flour Preparation. All high moisture content ingredients and foods were dried as described earlier.17,18 Specifically, heat-sensitive foods (e.g., ice cream, cheese) were freeze-dried (VirTis BenchTop K freezedryer, SP Scientific, Warminster, PA, USA), whereas fresh produce (e.g., fruits and vegetables) and high-sugar dried fruit (e.g., raisins, dates) were oven-dried for 24 h at 50−60 °C. All seeds, food ingredients, and processed foods, in their dried from, were ground in an Osterizer blender (speed setting “grind”; Galaxy model 869-18R, Jaden Consumer Solutions, Boca Raton, FL, USA) to obtain uniform flours. As needed, the flours were defatted for 8 h using a Soxhlet apparatus (Thermo Fisher Scientific Inc., Waltham, MA, USA) with petroleum ether (boiling point range of 38.2−54.3 °C) as the solvent (flour-to-solvent ratio of 1:10 w/v). After overnight drying in a fume hood at room temperature, the powders were passed through a 40 mesh sieve and then stored in screw-capped plastic bottles at −20 °C until further use. Protein Extract Preparation. Protein extracts were prepared as per the ELISA kit instructions. Briefly, sample flours (100 mg each for pistachio and other food ingredients, 1 g each for commercially prepared foods, pistachio-spiked samples, and samples incurred with ≥5000 ppm of pistachio) were extracted in extraction buffer (provided with the ELISA kit; flour-to-solvent ratio of 1:10 w/v) at 60 °C for 10 min with vigorous manual vortex mixing every 2 min. For corn flakes, sponge cakes, and sugar cookies incurred with ≤100 ppm of pistachio, the entire batch of each sample was blended in an Osterizer blender (speed setting “grind”) with borate saline buffer (BSB, 0.1 M boric acid, 0.025 M sodium borate, 0.075 M sodium chloride, pH 8.45; sample-to-solvent ratio of 1:10 w/v) at room temperature for 1 min and then magnetically stirred at room temperature for 1 h. The extracts were subsequently centrifuged at 2000g (Centrific 225 centrifuge, Thermo Fisher Scientific
introduced into the market by BioFront Technologies (Tallahassee, FL, USA). The kit claims include targeting a thermally stable antigen, assay specificity, 0.3 ppm sensitivity, and good recovery (>85%) from spiked samples. We were interested in assessing the kit specificity, sensitivity, and robustness. Included in these investigations were (a) laboratory-prepared ingredients and foods, (b) commercially processed and sold foods with declared and undeclared pistachio presence, (c) pistachio seed/flour and pistachio-containing foods/matrices exposed to select food-processing methods in the laboratory, and (d) commercial and laboratory-prepared foods spiked with known amounts of pistachio proteins.
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MATERIALS AND METHODS
Materials. Dehulled raw pistachio seeds, food ingredients, and commercially processed foods were purchased from local grocery stores and restaurants and were processed as needed as earlier described in detail by Tiwari et al.18 The MonoTrace pistachio ELISA kits were purchased from BioFront Technologies (Tallahassee, FL, USA). Sources of chemicals, reagents, supplies, and instruments have been reported earlier.20 Methods. Pistachio Seed Processing. Dehulled whole pistachio raw seeds were processed as follows.21 1. Pressure cooking in a SterileMax benchtop autoclave (Barnstead International, Inc., Dubuque, IA, USA) at 121 °C, 15 psi, for 15 and 30 min. Autoclaved samples were air-dried at room temperature (∼25 °C) in a fume hood until constant weight. 2. Blanching in boiling water (94 °C) for 5 and 10 min. The ratio of nut seeds to water was 1:10 w/v. Samples were patted dry on paper towels and further air-dried at room temperature in a fume hood until constant weight. 3. Frying in Crisco vegetable oil at 191 °C for 1 min. Excess oil was allowed to drain completely on paper towels prior to further handling. 4. Microwave heating in a Panasonic microwave oven (Sears, Roebuck and Co., Hoffman Estates, IL, USA) at 50% (500 W) and 100% power (1000 W) for 3 min. 5. Dry roasting at 140 °C for 30 min and at 168 °C for 12 min. Samples were placed in aluminum pans and subjected to roasting in a TempCon oven (American Scientific Products, McGaw Park, IL, USA) previously heated to the desired temperatures and monitored using a thermometer. Kaju Roll. Kaju roll is a sweet, popular in India, made by rolling a cashew dough sheet around a pistachio dough log. Cashew and pistachio doughs were each prepared separately using two different recipes: (1) Nestlé recipe22 and (2) laboratory recipe. Doughs were prepared as described below. 1. Nestle ́ Recipe. For the cashew dough, 60 g of full-fat cashew nut seed flour, 40 g of Nestlé sweetened condensed milk, 1 g of Ziyad ghee (Indian clarified butter), and 1 g of Dabur rose water were manually mixed in a pan with a spatula and heated at medium-low heat [stove temperature = 130 °C, dough temperature = 77 °C, monitored by a Traceable infrared thermometer (VWR International, LLC, Radnor, PA, USA)] for 10 min to form the dough. Subsequently, 3 g of Nestlé nonfat dry milk powder was added to the dough and the dough was allowed to cool to room temperature. For the pistachio dough, 60 g of full-fat pistachio seed flour, 40 g of Nestlé sweetened condensed milk, 1 g of Ziyad ghee, 1 g of McCormick cardamom, and 0.5 g of McCormick green food coloring (FD&C Yellow 5 and Blue 1) were manually mixed in a pan with a spatula and heated at medium-low heat for 10 min to form the dough. Subsequently, 3 g of Nestlé nonfat dry milk was added to the dough and the dough was allowed to cool to room temperature. 2. Laboratory Recipe. For the cashew dough, 50 g of cashew full-fat flour and 60 g of Publix confectionery sugar were ground in an Osterizer blender (speed setting “grind”; Galaxy model 869-18R, Jaden Consumer Solutions, Boca Raton, FL, USA) for 1 min followed by the addition of 1 g of Dabur rose water and 8 g of water. The mixture was manually kneaded into dough. 9140
DOI: 10.1021/acs.jafc.5b03066 J. Agric. Food Chem. 2015, 63, 9139−9149
Article
Journal of Agricultural and Food Chemistry
Table 1. Food Ingredients (100000 ppm) Tested for Assessing Cross-Reactivity of the MonoTrace Pistachio ELISA Kita
a Food matrix from a single batch was weighed in triplicate, and each sample was extracted using buffer provided in the kit. No cross-reactivity was detected (i.e., signal equivalent to 0.99) 1.73 ± 0.32 5.76 ± 1.07 3.64 ± 1.36 7.20 ± 2.40
a Data are expressed as mean ± SD. The 50% maximum signal concentration and linear detection range were determined using protein extracts from three different batches of pistachios and repeated in two different kits. The limits of detection and quantification were determined in three different kits using pistachio standards provided in those kits. bLimit of detection 1 (LOD1) was determined on the basis of the standard deviation of the blank (σ) and the slope of the standard curve (S) according to the formula LOD1 = 3σ/S. cLimit of detection 2 (LOD2) was determined on the basis of the mean absorbance (M) and standard deviation of the blank (σ) according to the formula LOD2 = M + 3σ. dLimit of quantification 1 (LOQ1) was determined on the basis of the standard deviation of the blank (σ) and the slope of the standard curve (S) according to the formula LOQ1 = 10σ/S. eLimit of quantification 2 (LOQ2) was determined based on the mean absorbance (M) and standard deviation of the blank (σ) according to the formula LOQ2 = M + 10σ.
Figure 1. Typical MonoTrace pistachio ELISA kit four-parameter curve (A) and linear standard curve using pistachio protein standards (0, 0.5, 2, 6, 18, and 36 ppm pistachio) provided within the kit (B). The standard curves were repeated three times within a kit.
population would react) for an allergic reaction in 1% of the population estimated for hazelnut and peanut were 0.2 and 0.1 mg of protein, respectively, and the reference dose for cashew (lower 95% confidence interval of ED05) was 2.0 mg of protein.39,40 The reference dose for most allergenic foods ranged from 0.03 mg of protein (ED01 for egg) to 10 mg of protein (lower 95% confidence interval of ED05 for shrimp). Currently, no threshold data are available for pistachio and other tree nuts, and thus the tested kit detection range 0.5−36 ppm must be interpreted with caution. Assay Efficiency. The MonoTrace pistachio ELISA was rapid with an assay time of approximately 1.5 h (extraction, 10 min; incubation with antigen, 30 min; incubation with detection antibody, 30 min; and color development, 10 min). The updated version of the MonoTrace Pistachio ELISA claims improvement in the assay efficiency and short incubations with antigen/food sample extract, detection antibody, and substrate (10 min each). To determine if the antigen extraction was sufficient, pistachio full-fat flours were extracted in four different buffers (EXB, BSB, PBS, and SBC) under two separate conditions (60 °C, 10 min and room temperature, 1 h). Under both conditions, the kitprovided buffer, EXB, yielded the highest soluble protein extractability and antigen recovery (Table 3). Protein extractions by EXB at 60 °C for 10 min and at room temperature for 1 h were equivalent (P > 0.05). Assay Specificity. Individuals allergic to one tree nut often exhibit IgE reactivity to other edible nut seed proteins.41 Crossreactive tree nuts are usually found in the same botanical family. Strong cross-reactivities have been documented in nut seed proteins of (a) pecan and walnut species of the Juglandaceae family42−46 and (b) cashew and pistachio of the Anacardiaceae family.5,43,44,47−53 Cross-reactivity is likely due to the homologous linear amino acid sequence, three-dimensional structural similarity, or both, of different nut seed proteins. Several murine
sensitivity is comparable to the reported sensitivity by Romer (0.13 ppm) and Bio-Check (0.2 ppm) ELISA kits and the recently reported real-time PCR assay sensitivity (0.1 ppm).13 Compared to the Genon ELISA kit (10 ppm) and the PCR methods reported by Brežná et al.35 (4 ppm) and Barbieri and Frigeri36 (100 ppm), the MonoTrace pistachio ELISA kit assay exhibited better sensitivity. The LOQ1 (0.30 ppm) and LOQ2 (0.38 ppm) of the MonoTrace pistachio kit are comparable with the LOQ of a previously reported pistachio ELISA ( 0.99) is similar to the linear range (1−40 ppm) for the Romer kit. The concentration of pistachio soluble protein that registered 50% of the maximum signal is 152 ng/mL (7.9 ppm) (Figure 1), comparable to the value (∼137 ng/mL) reported by Lim.37 The sensitivity of an assay is considered sufficient when it can reliably detect the minimum dose of an allergen capable of eliciting a clinically measurable response. Many attempts have been made to determine the allergen threshold that causes an adverse reaction. The U.S. Food and Drug Administration established a Threshold Working Group in 2005 but failed to establish the threshold for the major food allergens due to insufficient data at the time.38 In 2010, the Australian Allergen Bureau first introduced defined allergen threshold levels for precautionary labeling. In their most recent update, the eliciting doses (EDp, dose of allergen at which p% of the allergic 9143
DOI: 10.1021/acs.jafc.5b03066 J. Agric. Food Chem. 2015, 63, 9139−9149
Article
Journal of Agricultural and Food Chemistry
also found to be cross-reactive with protein(s) from mango seeds48 and with proteins from seeds of taxonomically unrelated botanical families such as sunflower, peanut, and walnut.47 A previously observed cross-reactivity of pistachio ELISA (at 4 ppm level) using sheep antisera as the capture reagent and rabbit antisera as detection reagent with cashew nut seed proteins was therefore not unexpected.37 Recently, in a multiplex ELISA simultaneously detecting 14 different food allergens and gluten, cross-reactivity of pistachio detection assay with Brazil nut, cashew, and hazelnut seed proteins has also been reported.54 The MonoTrace pistachio ELISA kit, under the test conditions, is specific and did not display cross-reactivity (signal equivalent to 100 kDa.56 So far, four distinct pistachio 11S globulin protein sequences have been reported with predicted molecular masses ranging from 53.2 to 56.5 kDa.57−59 Among pistachio 11S acidic (35−40 kDa) and basic (22−27 kDa) subunits, the latter have been reported to exhibit a stronger reactivity, compared to the 11S acidic subunit and 2S albumin polypeptides, tested with pooled tree nut-allergic human sera.51 Therefore, pistachio 11S globulin basic subunit may serve as a good antigen for producing antibodies targeting human allergy relevant epitopes. Assay Reproducibility, Uncertainty, and Recovery. The intra- and inter-assay variability of the MonoTrace pistachio ELISA kit was
