Pistachio (Pistacia vera L.) Detection and Quantification Using a

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
■
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.
■
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
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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 <0.09 ppm pistachio).
Inc., Waltham, MA, USA) for 10 min at room temperature. Aliquots of
the supernatant were analyzed (supernatants were stored at 4 °C prior to
analysis) within 48 h of preparation, and the remainder was stored in
plastic microcentrifuge tubes (1.5 mL capacity) at −20 °C until further
use.
To compare the antigen extraction efficiency of different buffers
under different conditions, full-fat pistachio flours were extracted (flourto-solvent ratio of 1:10 w/v) in BioFront extraction buffer (EXB;
containing 4.51 ± 0.37 mg protein/mL, pH 8.52 ± 0.01), borate saline
buffer (BSB; 0.1 M boric acid, 0.025 M sodium borate, 0.075 M sodium
chloride, pH 8.45), phosphate-buffered saline (PBS; 0.1 M sodium
phosphate, 0.9% w/v sodium chloride, pH 7.20), and sodium
bicarbonate buffer (SBC; 0.1 M sodium bicarbonate, 0.9% w/v sodium
chloride, pH 9.60) at two different conditions: (1) at room temperature
for 1 h with constant vortex mixing or (2) at 60 °C for 10 min with
vigorous manual vortex mixing every 2 min. The extracts were
centrifuged at 2000g for 10 min at room temperature, and the
supernatants were collected for soluble protein determination and
ELISA.
Osborne Fractionation. Pistachio protein fractions were prepared
according to the Osborne method24 as described in detail by Sze-Tao
and Sathe.25 Briefly, 5 g of defatted pistachio flour was extracted
sequentially with 100 mL of 1.0 M NaCl (albumin and globulin), 70%
aqueous ethanol (prolamin), and 0.1 M NaOH (alkaline glutelin) for 1 h
at room temperature with constant magnetic stirring. The slurry was
centrifuged at 12600g (J2-21 centrifuge, Beckman Coulter, Inc., Brea,
CA, USA) and 4 °C for 15 min following each extraction, and the
supernatant was vacuum filtered using Whatman no. 4 filter paper.
Residues from centrifugation and filtration were used for the next
extraction, whereas filtrates were dialyzed against distilled water for 24 h
with six water changes (4 L each). After dialysis, the albumin and
globulin fraction (1 M NaCl extract) was centrifuged (12600g, 4 °C, 15
min), and the precipitate (globulin) and supernatant (albumin) were
separately freeze-dried. Prolamin and glutelin fractions were lyophilized
directly after dialysis. All freeze-dried protein fractions were stored in
airtight plastic bottles at −20 °C until further analysis.
Protein Determination. The Bradford method26 using bovine serum
albumin (BSA) fraction V (Sigma Chemical Co., St. Louis, MO, USA) as
the standard protein (0−600 μg/mL) was used for soluble protein
determination.
ELISA Procedure. Ninety-six-well microtiter plates (coated with
murine antipistachio mAb and blocked), sample diluent, ready-to-use
pistachio standards (0, 0.5, 2, 6, 18, and 36 ppm pistachio), washing
buffer, murine anti-pistachio mAb conjugated with horseradish
peroxidase (HRP), 3,3′,5,5′-tetramethylbenzidine (TMB) substrate
solution, and HRP quench solution were provided in the ELISA kits.
Sample extracts were suitably diluted in sample diluent. Two hundred
microliters of diluted samples and ready-to-use standards were added to
each well and incubated at room temperature for 30 min. The plates
were washed three times with washing buffer and blot dried. After
washing, 100 μL of HRP-conjugated detection mAb was added to each
well, incubated in the dark at room temperature for 30 min, and again
washed three times with washing buffer. Subsequently, 100 μL of TMB
substrate solution was added and incubated in the dark at room
temperature for 10 min. The reaction was stopped by adding 100 μL of
quench solution to each well and mixed by gentle pipetting. The
absorbance was read at 450 nm by a BioTek PowerWave 200 microplate
scanning spectrophotometer (Winooski, VT, USA).
ELISA Validation. 1. Limit of Detection (LOD). The LOD is defined
as the minimum concentration of analyte that can be reliably
distinguished from background. The LOD of the ELISA was calculated
using the following two commonly applied methods. LOD1 = 3σ/S,
where σ is the standard deviation of the blank (mean) and S is the slope
of the standard curve.27 LOD2 = M + 3σ, where M is the mean
absorbance of the blank and σ is the standard deviation of the blank.28
The resulting absorbance of LOD2 was converted to concentration using
the standard curve.
2. Limit of Quantification (LOQ). The LOQ is defined as the
minimum concentration of analyte that can be quantitatively
9141
DOI: 10.1021/acs.jafc.5b03066
J. Agric. Food Chem. 2015, 63, 9139−9149
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Journal of Agricultural and Food Chemistry
Pistachio-incurred corn flakes, sponge cakes, and sugar cookies were
prepared as described earlier. Pistachio full-fat flours were incurred at 10,
50, 100, 5000, 10000, 20000, and 50000 ppm levels prior to food
processing and were processed along with the other ingredients.
The spiked and incurred matrices were then subjected to protein
extraction as described under Protein Extract Preparation. The pistachio
content was measured and the percent recovery was determined using
the following formula:
determined with suitable precision and accuracy. The LOQ of the
ELISA was calculated using the following formulas. LOQ1 = 10σ/S,
where σ is the standard deviation of the blank (mean) and S is the slope
of the regression line for the linear range.27 LOQ2 = M + 10σ, where M is
the mean absorbance of the blank and σ is the standard deviation of the
blank.29 The resulting absorbance of LOQ2 was converted to
concentration using the standard curve.
3. Linear Detection Range. Two hundred microliters of pistachio
full-fat flour protein extract (8000 ng pistachio protein/mL diluent) was
added to the top row of the horizontally oriented (i.e., 8 rows ×12
columns/row) 96-well microtiter plate, and three times serially diluted
samples were processed down the plate. The ELISA was performed as
described earlier. A four-parameter curve was generated by KC4
software (version 2.0, Bio-Tek Instruments, Inc., Winooski, VT, USA)
to determine the linear detection range and the concentration for 50%
maximum signal in the assay.
4. Specificity and Cross-Reactivity. Assay specificity is defined as the
ability of an antibody to produce a measurable response only for the
analyte of interest (i.e., pistachio protein). Cross-reactivity is a
measurement of antibody response to substances other than the
analyte.30 A total of 156 commonly used foods and food ingredients at
100,000 ppm each were tested for cross-reactivity (Table 1). Samples
with signal equivalent to <0.09 ppm of pistachio were not considered
cross-reactive.
5. Reproducibility. Reproducibility, also called precision, describes
the ability of the assay to duplicate results in repeat determinations.
Reproducibility was determined by the percent coefficient of variation
(%CV) between replicates determined in the same assay (intra-assay
variability) and in different assays (inter-assay variability). Variation of
six pistachio standards was measured, each with 3 replicates within the
plate for intra-assay variability and 12 replicates in different plates for
inter-assay variability. The %CV was calculated by using the formula
recovery (%) = (measured pistachio concentration
/predicted pistachio concentration) × 100%
8. Assay Applicability and Robustness. Thermally processed
pistachio samples (described earlier), commercially sold, matched
samples (with and without pistachio), and laboratory-prepared samples
were analyzed to determine if the assay can detect processed pistachios
and if food matrices interfere with the assay. Each commercial food
analyzed was obtained in a single batch to ensure sample homogeneity.
Protein extracts of processed pistachios were normalized to 125 ng
pistachio protein/mL extraction buffer. The absorbance of the
processed samples was compared with the absorbance of the reference.
The relative immunoreactivity was calculated as follows:
relative immunoreactivity (%)
= [( ODsample − ODblank )/(ODreference − ODblank )] × 100%
where ODreference = OD generated by 25 ng of unprocessed pistachio
protein used as the reference and ODsample = OD generated by 25 ng of
protein from the sample assayed.
Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis
(SDS-PAGE). SDS-PAGE was performed as described by Fling and
Gregerson.33 BSB-solubilized tree nut and legume seed proteins as well
as pistachio protein fractions were heat denatured in SDS-PAGE sample
buffer (50 mM Tris-HCl, 1% w/v SDS, 0.01% w/v bromophenol blue,
and 30% v/v glycerol, pH 6.8) with and without 2% v/v βmercaptoethanol for 10 min by placing the microcentrifuge tubes
containing these samples in a boiling water bath (100 °C). A 4% stacking
gel with either a 12% separating gel (6 μg protein/lane) or an 8−25%
gradient gel (20 μg protein/lane) was used. Protein samples and
molecular mass markers were loaded on the stacking gel and
electrophoresed at a constant current of 8 mA/gel (12 h) followed by
30 mA/gel (4−5 h) until the tracking dye reached the separating gel
edge.
Western Blot. Following SDS-PAGE, the proteins were transferred
onto a 0.22 μm nitrocellulose membrane as described by Towbin et al.34
The transferred polypeptides were visualized by staining with (0.1% w/
v) Ponceau S solution prepared using distilled deionized water. The
unbound sites on the nitrocellulose membrane were blocked with 5%
(w/v) nonfat dry milk in Tris-buffered saline−Tween 20 (TBS-T, 10
mM Tris, 0.9% w/v sodium chloride, 0.05% v/v Tween 20, pH 7.6) at
room temperature for 1 h. The membranes were subsequently washed
with three changes of TBS-T, 5 min each. The membranes were then
incubated with the HRP-conjugated detection antibody provided in the
MonoTrace pistachio ELISA kit (1:10 or 1:50 v/v dilution in TBS-T)
overnight at 4 °C on a Lab-line thermal rocker (speed 2; Thermo Fisher
Scientific Inc., Waltham, MA, USA). After three washings with TBS-T
for 15 min each, the membranes were incubated with HRP-labeled goat
anti-mouse IgG polyclonal antibody (pAb, 1:10000 v/v dilution in TBST) for 1 h at room temperature on a rocker. The membranes were
washed again as described above and incubated with 1.3 mM luminol,
0.2 mM p-coumaric acid, and 0.01% (v/v) hydrogen peroxide in 0.1 M
Tris-HCl buffer (pH 8.5) for 5 min. The membranes were then exposed
(60 s) to an X-ray film for autoradiographic visualization. The molecular
mass of recognized polypeptide was determined by the ChemiDoc XRS
+ Image System (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Statistics. All ELISA experiments were performed at least in triplicate
on a single sample, and data are reported as the mean ± standard
deviation of the mean. One-way ANOVA was performed with SPSS
software (19.0, SPSS Inc., Chicago, IL, USA) to compare means for
CV (%) = (σ /X ) × 100%
where σ is the standard deviation and X is the mean of the replicate
determinations.20
6. Uncertainty Estimation. Measurement uncertainty of the
validated BioFront MonoTrace pistachio ELISA was estimated at each
concentration level of the pistachio standards. The bias uncertainty (uδ),
the combined uncertainty (uc) of bias and intralaboratory reproducibility, the expanded uncertainty (U), and the relative uncertainty (ur)
were calculated using the following formulas.31,32
SR 2 = S W 2 + SB 2
where SR2, SW2, and SB2 are the intralaboratory reproducibility variance,
intra-assay variance, and inter-assay variance, respectively.
p
SW 2 =
n
∑i = 1 ∑ j = 1 (xij − xi)2
p(n − 1)
n
with xi =
∑ j = 1 xij
n
p
SB 2 =
n
p
∑i = 1 ∑ j = 1 xij
∑i = 1 (xi − x ̅ )2
S 2
− W with x ̅ =
p−1
n
pn
where x is the analytical result (ppm pistachio), p is the number of kits
analyzed, and n is the number of replications within a kit.
uδ =
(
SR 2 1 − γ +
U = k × uc
γ
n
)
p
ur =
with γ =
SW 2
SR 2
uc =
SR 2 + uδ 2
U
× 100%
tested concentration
A coverage factor of k = 2 was used to define a 95% confidence
interval (assuming the values are normally distributed) where the
unknown true value is believed to lie.31
7. Recovery. Diluted pistachio soluble protein extracts were spiked to
finely ground cereal, corn flake, cookie, dark chocolate, milk chocolate,
white chocolate, ice cream, and sponge cake matrices to reach a spiking
level equivalent to 10 ppm of pistachio flour.
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difference. Post hoc analysis was performed using Fisher’s least
significant difference (LSD) at P ≤ 0.05.
■
RESULTS AND DISCUSSION
Assay Sensitivity. The MonoTrace pistachio ELISA kit
registered a low background absorbance (0.073 ± 0.009). The
LOD1 (0.09 ppm) and LOD2 (0.19 ppm) for soluble pistachio
proteins suggested the assay is sensitive (Table 2). The assay
Table 2. Summary of the MonoTrace Pistachio ELISA Kit for
50% Maximum Signal Concentration, Linear Detection
Range, Limit of Detection (LOD), and Limit of
Quantification (LOQ)a
ppm pistacho full fat
flour
50% max signal concentration
(n = 3)
linear detection range (n = 3)
LOD1b (n = 3)
LOD2c (n = 3)
LOQ1d (n = 3)
LOQ2e (n = 3)
ng pistachio
protein/mL
7.9 ± 0.7
151.9 ± 14.7
0.5−36 (R > 0.99)
0.09 ± 0.02
0.19 ± 0.07
0.30 ± 0.06
0.38 ± 0.13
9.5−684 (R > 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 (<1 ppm).37
The kit linear detection range of 0.5−36 ppm (linear regression R
> 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
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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
<0.09 ppm of pistachio) with 156 tested commonly used food
ingredients at 100,000 ppm including certain Anacardiaceae
seeds such as cashew (Anacardium occidentale), charoli
(Buchanania lanzan), and mango (Mangifera indica) peel, pulp,
and seed (Table 1). The specificity of the kit is likely due to the
use of mAbs.
To partially test the aforementioned assumption, the crossreactivity of the kit detection murine mAb with select tree nut
and legume seed proteins was assessed using Western blot. The
detection mAb (1:10 v/v dilution in TBS-T) was found to be
reactive with the 51 kDa polypeptide of cashew and the 55 kDa
polypeptide of charoli in the absence of β-mercaptoethanol
(Figure 2). In the presence of β-mercaptoethanol, the detection
mAb recognized the 19 and 21 kDa polypeptides in cashew and
the 23 kDa polypeptide in charoli. The discrepancy between the
ELISA kit results (no cross-reactivity with cashew and charoli)
Table 3. Antigen Extraction Efficiency of Select Buffers under
Different Extraction Conditionsa
condition
60 °C, 10 min
room temperautre, 1 h
LSD (P ≤ 0.05, n = 3)
extraction buffer
EXB (pH 8.52)b
BSB (pH 8.45)c
PBS (pH 7.20)d
SBC (pH 9.60)e
EXB (pH 8.52)
BSB (pH 8.45)
PBS (pH 7.20)
SBC (pH 9.60)
soluble
protein
(mg/100 mg
pistachio fullfat flour)
antigen
recovery (%)
19.0 ± 2.0
15.8 ± 2.1
15.4 ± 1.0
20.4 ± 1.1
19.2 ± 1.9
17.0 ± 1.2
14.3 ± 1.5
17.2 ± 0.5
2.61
101.3 ± 1.8
88.4 ± 2.5
84.1 ± 2.7
98.4 ± 2.1
104.5 ± 8.6
92.2 ± 2.6
83.0 ± 1.5
95.3 ± 4.0
6.70
a
Samples were extracted from three different batches of pistachios and
tested using a single kit. Data are expressed as mean ± SD (n = 3).
b
EXB, BioFront MonoTrace pistachio ELISA kit extraction buffer (pH
8.52 ± 0.01). cBSB, borate saline buffer (0.1 M boric acid, 0.025 M
sodium borate, 0.075 M NaCl, pH 8.45). dPBS, phosphate-buffered
saline (0.1 M sodium phosphate, 0.9% w/v NaCl, pH 7.20). eSBC,
sodium bicarbonate buffer (0.1 M sodium bicarbonate, 0.9% w/v
NaCl, pH 9.60).
mAbs raised against cashew Ana o 1 have been reported to
recognize the recombinant Pis v 3 in a dot blot assay, indicating
that the antibodies are likely targeting the conserved regions of
the Anacardiaceae seed proteins.52 Pistachio seed proteins are
Figure 2. SDS-PAGE pattern (Ponceau S staining) and Western blot of the soluble protein extracts (6 μg/lane) from select tree nut and legume seeds in
the absence (A) and presence (B) of 2% (v/v) β-mercaptoethanol. MonoTrace murine anti-pistachio mAb (1:10 v/v dilution in TBS-T) was used as the
detection antibody, and goat anti-mouse IgG pAb−HRP (1:10000 v/v dilution in TBS-T) was used as the secondary antibody in the Western blot.
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Figure 3. Pistachio protein fractionations (A) and their immunoreactivity assessed by MonoTrace pistachio ELISA kit (B) and Western blot (C).
Protein content in ELISA and Western blot was 30 ng/well and 20 μg/lane, respectively. MonoTrace murine anti-pistachio mAb (1:50 v/v dilution in
TBS-T) was used as the detection antibody, and goat anti-mouse IgG pAb-HRP (1:10000 v/v dilution in TBS-T) was used as the secondary antibody in
the Western blot. Pistachio protein fractions were prepared using three different batches of pistachios. Fractions from all three batches were tested by
ELISA, and one batch was tested by Western blot.
Table 4. Intra- and Inter-assay Variability and Uncertainty Estimation for the MonoTrace Pistachio ELISA Kit Using the 0.5−36
ppm Pistachio Standardsa
uncertaintyb
ppm
intra-assay %CV
inter-assay %CV
back-calculated concentration (ppm)
recovery (%)
uδ (ppm)
uc (ppm)
U (ppm)
ur (%)
0.5
2
6
18
36
1.3−3.7
1.4−8.4
1.2−3.9
1.2−3.5
0.5−1.7
24.0
15.9
19.5
14.9
12.3
0.4
2.1
6.6
18.7
34.8
79.9
102.6
110.4
103.9
96.7
0.09
0.13
0.20
0.57
0.18
0.22
0.32
0.48
1.35
0.50
0.43
0.64
0.95
2.70
1.00
87
32
16
15
3
a
Pistachio standards from four different kits were tested three times in each kit. buδ = bias uncertainty, uc = combined uncertainty, U = expanded
uncertainty, and ur = relative uncertainty.
and Western blot results was likely due to (1) the capture
antibody not being reactive to cashew and charoli and/or (2)
SDS-induced protein unfolding in the Western blot exposing
buried epitopes that were inaccessible to the ELISA detection
antibody in cashew and charoli native proteins.
Identification of the Target Antigen. As observed in
Western blot (Figure 2), the detection mAb (1:10 v/v dilution in
TBS-T) of the MonoTrace pistachio ELISA kit recognized three
pistachio polypeptides (50, 40, and 31 kDa) in the absence of βmercaptoethanol. Two polypeptides, molecular masses of 79 and
145 kDa, were weakly recognized. Under reducing condition, the
detection mAb exhibited a strong reactivity to a 22 kDa
polypeptide and a weaker reaction to a 20 kDa polypeptide.
At 1:50 v/v dilution, the detection mAb recognized the 50 and
31 kDa polypeptides under nonreducing conditions and the 22
kDa polypeptide under reducing conditions (Figure 3). The
globulin protein fraction had more intense bands than the total
protein extract, indicating the globulins were mainly responsible
for the reaction. The detection mAb recognized the albumin
protein fraction, suggesting the target antigen was partially
soluble in water. Shokraii and Esen have noted the presence of
common pistachio globulin polypeptide(s) in the albumin
fraction.55 No polypeptide was recognized by the detection
mAb in the prolamin and glutelin fractions under both reducing
and nonreducing conditions. The ELISA and Western blot
results were consistent. Compared to the total protein extract
immunoreactivity designated 100%, the albumins, globulins,
prolamins, and glutelins, respectively, registered 39.0, 132, 0, and
0% immunoreactivity.
On the basis of the solubility profile and Western blot, the
target antigen of the detection antibody is likely to be the small/
basic subunit of the pistachio 11S globulin. The electrophoretic
pattern of the recognized pistachio polypeptides under nonreducing conditions was similar to those of purified soybean 11S
glycinin, namely, two major bands at 57 and 28 kDa and two light
bands at 85 and >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 <24% CV, demonstrating that the assay is
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pistachio recovery from dark chocolate to 93% (from 82%).
NFDM proteins may possibly interact with the chocolate
polyphenols and acids, thereby improving target antigen
solubilization.
To demonstrate how food processing along with the food
matrix affects the allergen detection and quantification,
laboratory-prepared corn flakes, sponge cakes, and sugar cookies
were incurred with full-fat pistachio flour and subjected to
thermal processing. Samples were incurred with 10, 50, and 100
ppm of pistachio to mimic the case of inadvertent contamination
with pistachio during food processing and with 5000 ppm (0.5%
w/w), 10000 ppm (1% w/w), 20000 ppm (2% w/w), and 50000
ppm (5% w/w) pistachio to simulate the case of intentional use
of pistachio as an ingredient in food processing. The pistachio
recovery ranges for corn flakes, sponge cakes, and sugar cookies
were, respectively, 37.3−44.8, 50.3−67.1, and 68.9−82.6% at
10−100 ppm incurring level, and 35.7−49.1, 71.2−99.2, and
78.6−112.2% at 5000−50000 ppm incurring level (Table 6). The
reproducible (Table 4). Measurement uncertainties of the assay
were estimated using the pistachio standards (Table 4). Among
the uncertainties, uδ estimates the uncertainty of the bias (the
difference between the ELISA result and the true pistachio
concentration), uc combines the uncertainties of the bias and the
reproducibility, U defines an interval about the ELISA result
where the true value is confidently believed to lie, and ur describes
the expanded uncertainty in proportion to the corresponding
concentration. The relative uncertainty (ur) had a range of 3−
87%, which decreased with increasing concentration. For the 36
ppm of pistachio standard, the unknown true value is located
between 35 and 37 ppm with a confidence level of 95%.
Food matrices that may come in contact with pistachio during
food manufacturing and processing were tested for their possible
assay interference. Previous studies have indicated that certain
food matrices may result in over- or under-estimation of the
target antigen.17,18,20 In the current investigation, a spiking level
of 10 ppm of pistachio was used. The pistachio recovery range for
spiked cereal, corn flake, cookie, dark chocolate, milk chocolate,
white chocolate, ice cream, and sponge cake was 81−125%
(Table 5). The highest pistachio recovery was observed in spiked
Table 6. Pistachio Recovery from Foods Incurred with
Pistachio Full-Fat Flour As Determined by the MonoTrace
Pistachio ELISA Kit
Table 5. Pistachio Recovery from Foods Spiked with Pistachio
Soluble Protein Extract As Determined by the MonoTrace
Pistachio ELISA Kit
food
manufacturer
ice cream
Häagen-Dazs
dark chocolate
Hershey’s
a
dark chocolate with
5% NFDM
milk chocolate
white chocolate
cereal
Post
corn flakes
laboratory made
sponge cake
cookie
LSD (P ≤ 0.05,n = 3)
spike level
(ppm)
recoveryb
(%)
0
10
0
10
10
NDc
92.6 ± 2.3
ND
81.8 ± 1.8
93.1 ± 1.1
0
10
0
10
0
10
0
10
0
10
0
10
ND
112.8 ± 3.7
ND
110.6 ± 8.3
ND
117.4 ± 5.1
ND
125.6 ± 1.7
ND
113.8 ± 2.2
ND
122.5 ± 6.2
8.53
recoverya (%)
pistachio incurred level (ppm)
corn flake
sponge cake
sugar cookie
10
50
100
5000
10000
20000
50000
LSD (P ≤ 0.05, n = 3)
LSD (P ≤ 0.05, n = 3)
37.3 ± 3.9
45.8 ± 3.5
44.8 ± 6.2
43.6 ± 6.2
35.7 ± 4.4
48.3 ± 5.1
49.1 ± 2.0
8.20
50.3 ± 5.1
78.6 ± 3.6
67.1 ± 3.0
71.2 ± 6.1
77.9 ± 18.9
88.2 ± 19.3
99.2 ± 16.8
21.96
9.55
68.9 ± 15.5
89.2 ± 13.4
82.6 ± 13.0
91.4 ± 19.7
78.6 ± 4.9
108.2 ± 13.8
112.2 ± 10.4
23.88
a
Samples were prepared and analyzed in triplicate. Data are expressed
as mean ± SD.
decreased pistachio recovery from certain incurred samples was
in agreement with decreased antigen recovery from glutenincurred corn bread and milk-incurred cookies and was likely due
to (1) interference from other food ingredients, (2) thermally
induced antigen denaturation, (3) chemical modification (e.g.,
Maillard reaction) of the antigen, and/or (4) hindered antigen
solubility/extractability.60,61
Assay Robustness and Applicability. The assay kit
detected pistachio traces when protein extracts prepared from
thermally processed pistachio seeds were tested (Table 7).
Relative immunoreactivity of the processed pistachio samples
ranged from 86% (dry roasting, 168 °C, 12 min) to 182%
(blanching, 94 °C, 10 min). The elevated immunoreactivity
under certain processing conditions may be due to improved
accessibility of a possibly buried epitope.15 The target antigen,
possibly pistachio 11S globulin, is thermally stable, a finding
consistent with our previous observations for tree nut 11S
globulins including almond amandin 21,62,63 and cashew
anacardein.15,21,64 To test whether the MonoTrace kit targeted
antigen is a reliable marker or not, selected 20 paired commercial
samples, with and without declared pistachio, were examined. No
false-positive or false-negative results were observed for the
tested samples except for kaju roll (Table 8). No pistachio was
detected in commercially sold kaju roll that was declared to
contain pistachio. Kaju roll is a dessert made by rolling
rectangular-shaped cashew dough around a log-shaped pistachio
a
Commercial sample brands were used for comparison, not for
endorsement. bLaboratory samples were prepared in triplicate.
Commercial foods were obtained in a single batch. All samples were
analyzed in triplicate using a kit. Data are expressed as mean ± SD.
c
ND, not detected (signal equivalent to <0.09 ppm pistachio).
corn flakes, whereas the lowest was in dark chocolate. ELISA
recovery of 183% for 11S globulin (amandin) for almond-spiked
corn flakes has also been reported.18 The reduced recovery in
dark chocolate was consistent with previous observations and
was likely induced by the formation of pistachio protein−
chocolate polyphenol insoluble complexes.17,18,20 For foods
containing dark chocolate, the addition of 5% w/v nonfat dry
milk (NFDM) to the extraction buffer was suggested in the latest
version of the kit user’s manual. To test this, we added 5%
NFDM to the extraction buffer and repeated the assay. This
modification in the extraction step significantly improved
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pistachio protein could be recovered using the kit. It was unclear
whether commercial kaju rolls contained pistachio or perhaps
just green food colorant. Therefore, we prepared kaju rolls with
different pistachio contents (0.6−6.3% w/w for recipe 1, 0.4−
4.5% w/w for recipe 2) in our laboratory using two separate
recipes. The MonoTrace pistachio kit was able to detect pistachio
in all laboratory-prepared kaju rolls. The pistachio recovery
ranged from 83 to 89% for recipe 1 kaju rolls and 93−127% for
recipe 2 kaju rolls (Table 9). These results suggest that the
Table 7. Effects of Thermal Processing on Immunoreactivity
of Whole Pistachio Seeds As Determined by the MonoTrace
ELISA kit
processing
unprocessed
(control)
autoclaving
blanching
frying
microwaving
roasting
condition
relative immunoreactivitya
(%)
NA
100.0 ± 1.5
121 °C, 15 psi, 15 min
121 °C, 15 psi, 30 min
94 °C, 5 min
94 °C, 10 min
191 °C, 1 min
500 W, 3 min
1000 W, 3 min
140 °C, 30 min
168 °C, 12 min
129.4 ± 38.3
134.4 ± 23.3
172.7 ± 25.0
182.5 ± 23.5
144.8 ± 29.6
161.4 ± 17.9
104.8 ± 30.5
95.2 ± 3.7
86.2 ± 1.2
40.75
LSD (P ≤ 0.05, n = 3)
Table 9. Pistachio Detection in Laboratory Prepared Kaju
Rolls Using the MonoTrace Pistachio ELISA Kit
recipe 1 (Nestlé)
a
Pistachios were processed in triplicate using the same batch of seeds
and analyzed using one kit. Data are expressed as mean ± SD.
Table 8. Pistachio Detection in Commercially Prepared
Foods Using the MonoTrace Pistachio ELISA Kit
food
vanilla ice cream (−)c
pistachio ice cream (+)
Tahitian vanilla bean gelato
(−)
Sicilian pistachio gelato (+)
malai kulfli (−)
pista kulfli (+)
kaju katli (−)
kaju roll (+)
plain halawa (−)
pistachio halawa (+)
rose Turkish delight (−)
pistachio Turkish delight
(+)
vanilla instant pudding and
pie filling (−)
pistachio instant pudding
and pie filling (+)
soledad almond nut blend
(−)
pomegranate pistachio
almond blend (+)
walnut baklava (−)
pistachio baklava (+)
milk chocolate (−)
milk chocolate pistachio (+)
LSD (P ≤ 0.05, n = 3)
manufacturera
Häagen-Dazs
Talenti
Reena’s
Rangoli
Mounir Bissat
Haci Bekir
Jell-o
mg full fat pistachio
detected/g sampleb
recovery (%)
pistachio % (w/w)
recovery (%)
NDa
97.7 ± 17.3
82.5 ± 2.0
87.1 ± 2.6
88.6 ± 0.9
16.54
0
0.001 (spiking)
0.4
2.4
4.5
LSD (P ≤ 0.05,
n = 3)
23.65
ND
109.4 ± 15.0
93.0 ± 27.0
98.3 ± 10.6
127.1 ± 10.9
32.40
negative results obtained on the tested commercial kaju rolls may
represent (a) the presence of pistachio below the detection limit
of the test kit, (b) the use of a pistachio variety in the tested kaju
roll not amenable for detection by the test kit, or (c) a possible
example of intentional misbranding.
In conclusion, the results of investigations in this paper
indicate that under the test conditions the BioFront MonoTrace
pistachio ELISA kit was sensitive, robust, and specific for
pistachio detection and quantification.
175.0 ± 9.6
ND
166.6 ± 1.5
ND
ND
ND
298.6 ± 5.0
ND
147.0 ± 3.5
■
ND
AUTHOR INFORMATION
Corresponding Author
*(S.K.S.) Phone: (850) 644-5837. Fax: (850) 645-5000. E-mail:
ssathe@fsu.edu.
Funding
ND
We gratefully acknowledge financial support from the Department of Nutrition, Food and Exercise Sciences, Florida State
University, and the USDA-NIFA 2009-65503-05797, 201167017-20079.
369.2 ± 9.1
Little Athens Gyro
Pitaria
Hershey’s
Flicks
pistachio % (w/w)
0
0.001 (spiking)
0.6
3.4
6.3
LSD (P ≤ 0.05,
n = 3)
LSD (P ≤ 0.05,
n = 3)
a
Kaju rolls were prepared using two different recipes, each in triplicate.
ND = not detected (signal equivalent to <0.09 ppm pistachio).
ND
76.2 ± 2.9
ND
0.1 ± 0.0
Sahale Snacks
recipe 2 (laboratory developed)
ND
138.0 ± 17.8
ND
685.3 ± 17.1
10.66
Notes
The authors declare no competing financial interest.
■
■
ACKNOWLEDGMENTS
We thank Prof. K. H. Roux for his help in designing certain
experiments.
a
Commercial sample brands were used for comparison, not for
endorsement. bCommercial samples from a single batch were weighed
into three subsamples, each extracted in extraction buffer and analyzed
in a kit. Data are expressed as mean ± SD. ND = not detected (signal
equivalent to <0.09 ppm pistachio). c(−) = without declared pistachio;
(+) = with declared pistachio.
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