Assessment of Natural Variability of Maize Lipid Transfer...

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Assessment of Natural Variability of Maize Lipid Transfer Protein Using a Validated Sandwich ELISA Xin Gu,* ,Thomas Lee, Tao Geng, Kang Liu, Richard Thoma, Kathleen Crowley, § Thomas Edrington, Jason M. Ward, # Yongcheng Wang, Sherry Flint-Garcia, Δ,Erin Bell, and Kevin C. Glenn Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, United States § Vasculox, 4320 Forest Park Avenue, Suite 304, St. Louis, Missouri 63108, United States # Royal Canin USA, 500 Fountain Lakes Boulevard, Suite 100, St. Charles, Missouri 63301, United States Δ Agricultural Research Service, U.S. Department of Agriculture, Columbia, Missouri 65211, United States Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, United States ABSTRACT: Lipid transfer protein (LTP) is the main causative agent for rare food allergic reactions to maize. This paper describes a new, validated ELISA that accurately measures maize LTP concentrations from 0.2 to 6.4 ng/mL. The levels of LTP ranged from 171 to 865 μg/g of grain, a 5.1-fold dierence, across a set of 49 samples of maize B73 hybrids derived from the Nested Association Mapping (NAM) founder lines and a diverse collection of landrace accessions from North and South America. A second set of 107 unique samples from 18 commercial hybrids grown over two years across 10 U.S. states showed a comparable range of LTP level (212751 μg/g of grain). Statistical analysis showed that genetic and environmental factors contributed 63 and 6%, respectively, to the variance in LTP levels. Therefore, the natural variation of maize LTP is up to 5-fold dierent across a diverse collection of varieties that have a history of safe cultivation and consumption. KEYWORDS: LTP, lipid transfer protein, maize, natural variation, ELISA INTRODUCTION Plant lipid transfer proteins (LTP) are a large family of small basic proteins. Over 100 protein family members from up to 50 dierent plant species have been identied, and their functions have been extensively studied. 13 The 9 kDa LTP1 protein contains eight conserved cysteine residues, which form four intramolecular disulde bonds, making LTP1 proteins heat stable and protease resistant. 2 Given the prevalence of LTP in a diverse array of safely consumed foods, consuming LTP for the majority of the population is safe. However, in some individuals, LTP have been reported to cause systemic food allergic reactions, especially for fruits and vegetables from the Rosaceae 46 and Vitaceae families. 7 With these food-allergic individuals, a high level of immune cross-reactivity has been noted for the LTP from dierent fruits and vegetables, even though limited amino acid sequence similarity exists between these LTP proteins. 8 Maize food allergy is very rarely reported. 9,10 It is for this reason that maize is not listed as an allergenic food in any national or international food allergy labeling regulations. 11,12 However, in the few reports of maize food allergy, it has been shown that some Rosaceae fruit LTP are immunologically cross-reactive with maize LTP. 13,14 One of the few publications on maize allergy noted that in their birth cohort study, only one child in a group of 969 food-allergic children showed a response when consuming maize. 15 It is well accepted that the few instances of maize allergic reactions are localized to a small population of patients, one in southern Europe (southern Italy) and a second in the United States. In both populations, the reaction is thought to be the result of prior consumption of LTP in Rosaceae fruits, such as plum, cherry, peach, apricot, or apple, which predisposed a very small subset of individuals to be sensitive to maize consumption. 1618 However, in the rare instances with maize food allergy, a 9 kDa lipid transfer protein, also known as Zea m 14, appears to be responsible for the allergic reaction, and observations of IgE reactivity to the other maize proteins were not considered to be clinically relevant. 19 Because maize LTP seems to be the only clinically relevant allergen in the few identied cases of maize allergy, there is interest in understanding the exposure level to LTP through consumption of maize grain. Two validated methods have been reported for maize grain LTP quantitation, one using liquid chromatographymass spectrometry (LC-UV/MS) 20 and one using tandem mass spectrometry. 21 The present study describes a new, high- throughput, validated sandwich ELISA for LTP quantitation in maize grain with high sensitivity, precision, and accuracy. The ELISA was used to measure LTP levels in two sets of grain samples. One set was from a genetically diverse collection of 49 hybrids of maize B73 crossed with varieties from the Nested Association Mapping (NAM) program 22 and a diverse collection of landrace accessions 23 from North and South America, all cultivated at a single location. The second set was from 18 commercially relevant maize hybrids, each cultivated in diverse environmental conditions (at least three locations Received: August 9, 2016 Revised: November 9, 2016 Accepted: February 5, 2017 Published: February 5, 2017 Article pubs.acs.org/JAFC © 2017 American Chemical Society 1740 DOI: 10.1021/acs.jafc.6b03583 J. Agric. Food Chem. 2017, 65, 17401749

Transcript of Assessment of Natural Variability of Maize Lipid Transfer...

Assessment of Natural Variability of Maize Lipid Transfer ProteinUsing a Validated Sandwich ELISAXin Gu,*,† Thomas Lee,† Tao Geng,† Kang Liu,† Richard Thoma,† Kathleen Crowley,§

Thomas Edrington,† Jason M. Ward,# Yongcheng Wang,† Sherry Flint-Garcia,Δ,⊥ Erin Bell,†

and Kevin C. Glenn†

†Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, United States§Vasculox, 4320 Forest Park Avenue, Suite 304, St. Louis, Missouri 63108, United States#Royal Canin USA, 500 Fountain Lakes Boulevard, Suite 100, St. Charles, Missouri 63301, United StatesΔAgricultural Research Service, U.S. Department of Agriculture, Columbia, Missouri 65211, United States⊥Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, United States

ABSTRACT: Lipid transfer protein (LTP) is the main causative agent for rare food allergic reactions to maize. This paperdescribes a new, validated ELISA that accurately measures maize LTP concentrations from 0.2 to 6.4 ng/mL. The levels of LTPranged from 171 to 865 μg/g of grain, a 5.1-fold difference, across a set of 49 samples of maize B73 hybrids derived from theNested Association Mapping (NAM) founder lines and a diverse collection of landrace accessions from North and SouthAmerica. A second set of 107 unique samples from 18 commercial hybrids grown over two years across 10 U.S. states showed acomparable range of LTP level (212−751 μg/g of grain). Statistical analysis showed that genetic and environmental factorscontributed 63 and 6%, respectively, to the variance in LTP levels. Therefore, the natural variation of maize LTP is up to 5-folddifferent across a diverse collection of varieties that have a history of safe cultivation and consumption.

KEYWORDS: LTP, lipid transfer protein, maize, natural variation, ELISA

■ INTRODUCTION

Plant lipid transfer proteins (LTP) are a large family of smallbasic proteins. Over 100 protein family members from up to 50different plant species have been identified, and their functionshave been extensively studied.1−3 The 9 kDa LTP1 proteincontains eight conserved cysteine residues, which form fourintramolecular disulfide bonds, making LTP1 proteins heatstable and protease resistant.2 Given the prevalence of LTP in adiverse array of safely consumed foods, consuming LTP for themajority of the population is safe. However, in someindividuals, LTP have been reported to cause systemic foodallergic reactions, especially for fruits and vegetables from theRosaceae4−6 and Vitaceae families.7 With these food-allergicindividuals, a high level of immune cross-reactivity has beennoted for the LTP from different fruits and vegetables, eventhough limited amino acid sequence similarity exists betweenthese LTP proteins.8

Maize food allergy is very rarely reported.9,10 It is for thisreason that maize is not listed as an allergenic food in anynational or international food allergy labeling regulations.11,12

However, in the few reports of maize food allergy, it has beenshown that some Rosaceae fruit LTP are immunologicallycross-reactive with maize LTP.13,14 One of the few publicationson maize allergy noted that in their birth cohort study, only onechild in a group of 969 food-allergic children showed a responsewhen consuming maize.15 It is well accepted that the fewinstances of maize allergic reactions are localized to a smallpopulation of patients, one in southern Europe (southern Italy)and a second in the United States. In both populations, thereaction is thought to be the result of prior consumption of

LTP in Rosaceae fruits, such as plum, cherry, peach, apricot, orapple, which predisposed a very small subset of individuals tobe sensitive to maize consumption.16−18 However, in the rareinstances with maize food allergy, a 9 kDa lipid transfer protein,also known as Zea m 14, appears to be responsible for theallergic reaction, and observations of IgE reactivity to the othermaize proteins were not considered to be clinically relevant.19

Because maize LTP seems to be the only clinically relevantallergen in the few identified cases of maize allergy, there isinterest in understanding the exposure level to LTP throughconsumption of maize grain.Two validated methods have been reported for maize grain

LTP quantitation, one using liquid chromatography−massspectrometry (LC-UV/MS)20 and one using tandem massspectrometry.21 The present study describes a new, high-throughput, validated sandwich ELISA for LTP quantitation inmaize grain with high sensitivity, precision, and accuracy. TheELISA was used to measure LTP levels in two sets of grainsamples. One set was from a genetically diverse collection of 49hybrids of maize B73 crossed with varieties from the NestedAssociation Mapping (NAM) program22 and a diversecollection of landrace accessions23 from North and SouthAmerica, all cultivated at a single location. The second set wasfrom 18 commercially relevant maize hybrids, each cultivated indiverse environmental conditions (at least three locations

Received: August 9, 2016Revised: November 9, 2016Accepted: February 5, 2017Published: February 5, 2017

Article

pubs.acs.org/JAFC

© 2017 American Chemical Society 1740 DOI: 10.1021/acs.jafc.6b03583J. Agric. Food Chem. 2017, 65, 1740−1749

across 10 U.S. states), producing a total of 107 unique samplesthat can be used to address the contribution of genetic ×environment interaction (G × E).

■ MATERIALS AND METHODSReagents. All chemicals and reagents were purchased from Sigma-

Aldrich Chemical (St. Louis, MO,USA) unless specified otherwise.Maize Grain LTP Protein Standard Isolation. Conventional

maize grain of DKC55-11 (DEKALB, St. Louis, MO, USA) was usedfor the isolation of LTP. This purified lipid transfer protein served asan immunogen for both rabbit and goat antibody production, as wellas the protein standard for ELISA. LTP were isolated from maize grainusing a combination of anion exchange, cation exchange, and sizeexclusion chromatographies. Briefly, maize grain was ground to finepowder using a laboratory mill (Perten, Hag̈ersten, Sweden) in thepresence of dry ice and then extracted with 50 mM Bis-Tris-propanebuffer, pH 7.0. The clarified extract was loaded onto a Q-SepharoseFast Flow column (GE Healthcare, Marlborough, MA, USA) and theflow-through containing LTP was collected. After concentration, theresulting sample was loaded onto an S-Sepharose Fast Flow column(GE Healthcare) and eluted with a gradient (0−400 mM) of sodiumchloride. A single peak fraction containing the bulk of the LTP wascollected. The protein sample was concentrated and applied onto aSephacryl S-100 size exclusion column (GE Healthcare) equilibratedwith 50 mM phosphate buffer, pH 7.0, containing 150 mM sodiumchloride. The LTP peak, which eluted at approximately 9 kDa, wascollected. This preparation was characterized and used as the LTPprotein standard in all subsequent experiments. The characterizationsincluded protein purity assessment by SDS-PAGE, protein identity byMALDI-TOF mass spectrometry (Applied Biosystems, Foster City,CA, USA), and N-terminal amino acid sequence analysis by AppliedBiosystems 494 Procise Sequencing System (Applied Biosystems).Maize Grain for LTP Natural Variability Assessment.

Genetically Diverse Maize Grain Samples from B73 Hybrids withNAM and Landrace Lines. The experimental design for generating themaize grain samples analyzed in this study, including selection andidentity of the maize lines, production of hybrid seed, and field design,is detailed in a previous paper.24 Briefly, seed of 49 maize hybrids wasproduced by crossing B73 with a set of 24 diverse landrace inbredlines23 and the 25 NAM founder lines.22,25 The 49 maize hybrids weregrown in 2012 as a randomized complete block design in Aurora, NY,USA, with three replicates and the plants self-pollinated to control forpollen effects. A total of 133 independent samples were available andanalyzed in this investigation.Environmentally Diverse Maize Grain Samples from Conven-

tional Commercial Maize Hybrids. Conventional maize grain samplesfrom U.S. field trials conducted over two years (2013 and 2014) werechosen to evaluate the effect of different environmental conditions onLTP levels. A total of 18 conventional maize hybrids were involvedacross these various field trials. In total, 107 unique samples (unique inyear and/or location and/or trial) were drawn from 20 locations across10 U.S. states (one location each in Arkansas, Indiana, Kansas,Missouri, Ohio, Wisconsin, two locations in Pennsylvania, threelocations in Nebraska, four locations in Iowa, and five locations inIllinois) over two years. Grain samples from each maize hybrid werecollected from a minimum of three location/years and threerandomized replicate plots for each unique entry for a total of 321individual biological samples. These samples were randomized prior toanalysis.Anti-LTP Polyclonal Antibody Production. Polyclonal antibod-

ies from both goat and rabbit were separately produced for establishingthe sandwich ELISA method. The goat anti-LTP antibodies wereraised in goats by inoculation with the purified maize LTP using acommercial antibody production service (Thermo Fisher/OpenBiosystem Inc., Rockford, IL, USA). The IgG portion was isolatedfrom goat antisera using a Protein G affinity column (GE Healthcare)and served as the capture antibody.Rabbit anti-LTP antibodies were raised in four New Zealand White

rabbits by inoculation with the purified maize LTP using a commercial

antibody production service (Thermo Fisher/Open Biosystem Inc.,Rockford, IL, USA). The IgG portion was also isolated from rabbitantisera using a Protein G affinity column and served as the detectionantibody.

Both goat and rabbit antisera were screened by Western blot, andthey showed a high specificity to LTP, with lack of cross-reactivity toother maize grain proteins.

Maize Grain Sample Extraction for ELISA. Each maize grainsample was ground to a fine powder. For assay validation anddetection of LTP in the 49 diverse maize hybrids, approximately 100mg of maize powder was mixed at a 1:100 ratio (w/v) with PBScontaining 0.1% (w/v) bovine serum albumin (BSA). The sample washomogenized by using a Harbil mixer (Fluid Management Inc.,Wheeling, IL, USA) with eight chrome steel beads (0.25 in. indiameter) for two cycles of 3.5 min at 1500 rpm. Insoluble materialwas removed by centrifugation. The resulting supernatant wascollected and used for the ELISA.

For assessing the LTP level in the 321 individual biological samplesfrom commercial hybrids, a 384-well format was used. The sampleamount was reduced to approximately 10 mg of each maize powder,and the extraction buffer volume was reduced to 1 mL. Thus, theextraction ratio (w/v) remained the same. The samples were extractedusing a Geno/Grinder with three to four chrome steel beads (0.125 in.in diameter) for one cycle of 4 min at 1750 rpm. The bridging testshowed that the extraction efficiencies of the two methods are thesame.

Sandwich ELISA for LTP Quantitation. For analyses using 96-well plates, all incubations were carried out for approximately 60 minat room temperature with a sample volume of 100 μL (unless specifiedotherwise). Between each incubation step, the plates were washedthree times with 200 μL of 1× PBST (PBS, pH 7.4, containing 0.05%Tween 20) per well using a plate washer. All samples were analyzed intriplicate, and the mean of the triplicates was reported. Goat anti-LTPantibody was used as the capture antibody and diluted into 1× PBS,pH 7.4, to the concentration of 2 μg/mL and then immobilized onto a96-well flat-bottom microtiter plate (Maxisorb, from NUNC,Wiesbaden, Germany) by incubation in a 4 °C refrigerator for ≥12h. Plates were blocked with 200 μL of 1× PBST containing 5% (w/v)nonfat dry milk (NFDM, Bio-Rad, Hercules, CA, USA) forapproximately 60 min at room temperature. Maize grain extractswere diluted 1:5000 (v/v) in 1× PBST containing 0.5% NFDM(referred to as the sample buffer) and loaded onto the plate along witheight LTP protein standards (0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, and 25.6 ng/mL of LTP) in the sample buffer, a 1.0 ng/mL LTP positive control,and a reagent blank as negative control. Rabbit anti-LTP antibody wasdiluted to 1 μg/mL in the sample buffer and loaded onto the plate.Anti-rabbit IgG−HRP conjugate (Vector Laboratories, Inc., Burlin-game, CA, USA) was diluted 1:4000 (v/v) in the sample buffer andused for detection. The HRP substrate, 3,3′,5,5′-tetramethylbenzidine(TMB; KPL, Gaithersburg, MD, USA), was added for colorimetricdetection, and the plates were developed for 8−9 min. The reactionwas stopped by the addition of 6 M phosphoric acid. Optical densities(OD) at 450 nm were measured with a microplate reader (MolecularDevices, Sunnyvale, CA, USA) equipped with SoftMax Pro softwarefrom Molecular Devices. An eight-point standard curve, ranging from0 to 25.6 ng/mL of LTP, was generated for each plate based on a four-parameter logistic curve fit of absorbance readings versus LTPstandard concentrations. The quantity of the LTP in sample extractswas calculated by interpolation from the protein standard curve, with aquantitative range of 0.2−6.4 ng/mL of LTP.

For analyses using 384-well plates, modifications to the 96-wellassay method were made as follows: Volumes used were reduced toone-fourth of the 96-well format (e.g., sample volumes were reducedfrom 100 to 25 μL, and wash volumes were reduced from 200 to 50μL). In addition, the number of plate washes between each incubationstep was increased from three to four times. All samples were run inquadruplicates (instead of triplicates) and the means of quadruplicatesreported. All other aspects of the 384-well format assay remained thesame as the 96-well assay format. A bridging assessment showed that

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both the assay format change and the changes made for sampleextraction did not affect the outcome of LTP quantitation.The unit of LTP level in the grain samples, in all cases, is expressed

as micrograms of LTP per gram of grain and abbreviated as μg/g.ELISA Validation. All assay validation runs were carried out with

96-well plates.Assay Precision. The degree of repeatability of the assay under

normal operating conditions was determined by repeated analysis ofthe LTP standard at the concentration of 1.0 ng/mL, along with eightconcentrations of the LTP standard. The assay precision wasdetermined from the results of four assays per analyst per day,performed by three independent analysts on three separate days, for atotal of 36 assays and 108 data points. Intra-assay and inter-assayprecisions were assessed. Precision was expressed as the percentagecoefficient of variation (%CV) of the reported LTP concentrationsunder the specified test conditions.Spike and Recovery. For determination of the accuracy of the

quantitation of LTP in maize sample matrix, maize grain powders(conventional maize hybrid MPA636B) were spiked with increasingamounts of the purified LTP standard in the extraction buffer beforeextraction. The optimal LTP concentrations were prequantified, andsuitable steps of dilution were chosen prior to spiking to fall within thequantitative range of the ELISA. Three concentrations of LTP werespiked at 20, 8, and 3.5 μg/mL (after 1:5000 sample dilution, theywere 4, 1.6, and 0.7 ng/mL) into the extraction buffer (PBS, pH 7.4,containing 0.1% (w/v) BSA) and then were added to maize powdersample pooled from five lots from the same maize line for extraction.Three sets of extraction were performed independently. Each setcontained the three spiked concentrations and a nonspiked sample asbaseline. The analysis was carried out by three analysts independently;each conducted analysis used all three sets of extracts. Becauseendogenous LTP was present in the maize grain prior to spiking, themean recovery of the spiked protein was calculated by subtracting theendogenous LTP baseline determined in the same assay run.

= − ×recovery %detected LTP level detected baseline LTP level

spiked LTP level100%

Dilutional Parallelism. When a test sample is diluted to result in aset of samples having protein concentrations that fall within thequantitative range of the assay, there should be no apparent trendtoward increasing or decreasing estimates of protein concentrations ofthe original sample over the range of dilutions. To assess whethersample dilution affects the quantitation of the target protein, five lotsfrom the same conventional maize hybrid (MPA636B) were extractedand used for the test at four different dilutions of 1:1000, 1:2000,1:5000, and 1:10,000 (v/v). Dilutional parallelism was determined bycomparing the observed LTP concentration for each individual maizeextract to the average concentrations for all those extracts.Statistical Analysis of the LTP Level in Environmentally

Diverse Maize Samples. Variance component analysis (VCA) wasconducted for LTP levels in the environmentally diverse maize hybridsto estimate the proportion of random effect contribution to the totalvariance, based upon the following analysis of variance (ANOVA)model by combining all locations:

= + + +Y U G E eijk i j ijk

Yijk is the unique individual observation, U is the overall mean, Gi is theith genetic effect, Ej is the jth environment effect, and eijk is the residualerror.In this application, all of the effects in the ANOVA model were set

as random. The SAS procedure PROC MIXED was employed toobtain covariance estimates for each effect in the model, which werethen divided by the total variance to obtain the proportion of varianceexplained by each effect. The same location from different years ortrials was treated as two locations and contributes as one of theenvironmental effects in the analysis. When setting all of the effects inthe ANOVA model as fixed, an overall F test was conducted to detectif there is a significant impact of each effect on the LTP levels.

■ RESULTSLTP ELISA Validation. Maize LTP Standard. LTP was

isolated from maize grain through extraction, anion exchange,cation exchange, and size exclusion chromatographies. Asshown in Figure 1, a single protein band migrating at

approximately 9 kDa was detected on a SDS-PAGE gel. Themolecular weight of this band corresponds to the major form ofmaize LTP.26 The purity of this preparation of maize LTPstandard was 99% as determined by densitometry of the SDS-PAGE gel. The identity of the isolated LTP used asimmunogen was confirmed as the major allergen identified byPastorello et al.,17 more specifically termed Zea m 14. MALDI-TOF mass spectrometry analysis of the intact protein showedthat the purified maize LTP contained two commonly observedmature maize LTP isoforms with [M + H]+ ions at m/z 9046and 9018 (Figure 2). On the basis of the published literature,both isoforms consist of 93 amino acids. One isoformcontaining an arginine at position 34, known as Zea m 14,has a calculated mass of [M + H]+ ion at m/z 9046Da26(GenBank accession no. J04176). The other isoformhaving a lysine at position 34, commonly referred to as LTPa,has a calculated mass of [M + H]+ ion at m/z 9019 Da27

(GenBank accession no. ACG24030). The experimentallyderived masses for the two isoforms were within 1 Da of thecalculated masses based on protein sequence and match themasses reported by Kuppanan and colleagues.20 Analysis of atryptic digest of the isolated LTP by MALDI-TOF massspectrometry showed that it contains fragments correspondingto both the arginine and lysine specific isoforms (data notshown). The identity of this isolated 9 kDa protein was alsoconfirmed by N-terminal amino acid sequence analysis through15 cycles (Figure 3). The N-terminal sequencing resultsdemonstrated that the first 15 amino acid residues matched thereported N-terminal sequence of the mature maize LTP.However, it should be noted that the two cysteine residuesexpected at positions 4 and 14 were not observed, as cysteineresidues are degraded during the Edmam degradation process.

Validation of an ELISA That Accurately Measures LTPLevels in Maize. To quantify LTP in maize with high precisionand accuracy, polyclonal antibodies were raised separately ingoat and rabbit using the purified maize LTP as immunogen,and a highly sensitive sandwich ELISA assay was developed andvalidated. The quantitative range of the ELISA covers LTP

Figure 1. SDS-PAGE gel analysis of purified LTP from maize grain.The purified proteins were separated on a 4−20% (w/v)polyacrylamide Tris−glycine gel and stained with colloidal CoomassieBlue G stain. Lanes: 1, molecular weight marker; 2, 1.95 μg of purifiedLTP; 3, 1.3 μg of purified LTP; 4, 0.65 μg of purified LTP.

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concentrations from 0.2 to 6.4 ng/mL. Data characterizing theprecision, accuracy, sensitivity, and specificity of this ELISAmethod for the detection of maize grain LTP validated theassay by conforming to all preset criteria.Assay Precision. The precision of the LTP ELISA was

evaluated using 1.0 ng/mL standard LTP protein. The detectedLTP concentration reported in Table 1 represented the averageof the assay triplicates interpolated from an eight-point LTPstandard curve from a total of 36 assays. The meanconcentration calculated from all of these assays was 1.021ng/mL. Inter- and intra-assay precision (%CV) values were alsocalculated and are presented in Table 1. The inter-assayprecision (%CV) was 15.8% and the intra-assay precision was4.0%, indicating that this ELISA can reproducibly and preciselymeasure the concentration of LTP in maize grain.Spike and Recovery. The high sensitivity of the LTP ELISA

requires considerable dilution of the grain extracts (dilution at

1:5000 v/v after 1:100 (w/v) extractions) prior to analysis,thereby significantly diluting all other components in thesample extract such that ELISA quantitation of LTP is unlikelyaffected by the matrix. Even so, the maize matrix effect on LTPquantitation was investigated. The samples were spiked withthree different concentrations of the LTP standard (20, 8, and3.5 μg/mL). The analytical recovery ranged from 99 to 112%(Table 2), demonstrating a high degree of LTP recovery andindicating that the maize matrix does not influence thequantitation of LTP by this ELISA method.

Dilutional Parallelism. To assess whether sample dilutionaffects the quantitation of LTP by the ELISA, four differentdilutions ranging from 1:1000 to 1:10,000 from the samesample extract were analyzed. The percentage of the observedLTP level for the maize samples versus the average LTP levelfor all those samples was calculated and is reported in Table 3.These observed percentages ranged from 88 to 116%, which

Figure 2. Intact mass spectrometry analysis of the purified maize LTP using MALDI-TOF mass spectrometry. Two main mass peaks of the [M +H]+ ion at m/z 9018 and 9046 Da were observed, representing the two common maize LTP isoforms, with one isoform having an arginine and theother isoform containing a lysine at position 34.

Figure 3. N-terminal amino acid sequence of the purified maize LTP. The purified LTP protein solution was transferred directly to a ProSorbmembrane cartridge prior to conducting 15 cycles of the Edman sequencing, and they were found to match the maize LTP sequence in GenBank(e.g., J04176, ACG24030). When Edman degradation is performed, the amino acids cysteine and tryptophan are degraded, so there is no signal inthe chromatography in that cycle. An “X” refers to undesignated calls, when a cysteine or tryptophan is expected in the amino acid sequence.

Table 1. Inter- and Intra-assay Precision Evaluation of the ELISA for Determination of LTP Level in Maize Graina

interpolated concn (ng/mL, mean of triplets)

analyst: A B C A B C A B C

day: 1 1 1 2 2 2 3 3 3

assay 1 0.911 1.113 1.017 0.932 1.031 0.961 1.163 1.111 0.952assay 2 1.001 1.087 1.027 0.893 1.128 0.986 0.994 1.069 1.007assay 3 0.938 1.162 1.074 0.902 1.063 0.954 1.059 1.087 0.955assay 4 0.968 1.166 1.059 0.829 1.116 0.991 1.003 1.086 0.959mean of 4 tests 0.954 1.132 1.044 0.889 1.084 0.973 1.054 1.088 0.968overall average 1.021 ng/mLinter-assay %CV 15.8%intra-assay %CV 4.0%

aIntra- and inter-assay precisions were determined by 36 independent assays performed by three analysts on three separate days with 1.0 ng/mLmaize LTP standard protein.

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demonstrated a very high level of dilutional parallelism for thisELISA method.

Assessment of Natural Variability for LTP. LTP Levels inGenetically Diverse Maize Grain Samples. To assess the effectof genetic diversity on the LTP level in maize grain, a total of133 grain samples from 49 diverse maize hybrids were tested.The samples were hybrids of B73 with 25 NAM founder linesand 24 landrace inbred lines. These 49 maize lines includedflint, tropical, temperate, and mixed maize varieties.The LTP level in each maize hybrid derived from the NAM

founder lines is presented in Table 4. The median LTP level ofall the samples from the NAM founder hybrids was 412 μg/g,and the LTP values from a single replicated field plot rangedfrom 278 to 759 μg/g (a 2.7-fold difference). Two hybrids, B73× P39 and B73 × IL14H, with flint ancestry (derived fromsweet corn inbred lines P39 and IL14H) showed higher thanaverage LTP levels of 570 and 531 μg/g, respectively. Theother hybrid from flint ancestry (derived from popcorn lineHP301), B73 × HP301, showed average LTP levels of 361μg/g, slightly below the median level from the NAM hybridpopulation.The levels of LTP in the landrace hybrids are presented in

Table 5. The median LTP level of all the samples from thelandrace hybrids was 416 μg/g, and the LTP values from eachindividual field plot ranged from 171 to 865 μg/g (a >5-folddifference). The range of LTP values for the landrace sampleswas approximately twice the range that was observed for theNAM hybrids (Tables 4 and 5). In the hybrid of B73 withPalomero de Jalisco (a popcorn landrace), the average LTPlevel was 631 μg LTP/g, which is much higher than the medianof the population of landrace hybrids.LTP Levels in Environmentally Diverse Maize Grain

Samples. The assessment of the NAM and landrace hybridsprovided an evaluation of LTP levels across a broad diversity of

maize hybrids. To further investigate the impact of genetics aswell as the contribution of environmental factors (i.e., year and/or location effects) on LTP levels, grain samples were analyzedfrom a total of 18 maize hybrids that either are currently orwere historically commercially cultivated. The environmentalvariability of the samples came from field trials conducted in atotal of 20 locations across 10 U.S. states over two years. Theanalyzed maize grain samples from each hybrid were collectedfrom a minimum of three location/years, a total of 107 uniquesamples, and three randomized replicate plots from each ofthese unique samples, resulting in a total of 321 individualbiological samples (Figure 4). Figure 4A shows the LTP levelsof all 18 hybrids. The highest average LTP level was 594 μg/gfrom maize hybrid 11, whereas the lowest average LTP level of281 μg/g of maize grain was from maize hybrid 10 (a >2-folddifference). The LTP levels in these individual biologicalsamples range from 212 to 751 μg/g (a 3.5-fold difference).The overall average and median values of LTP were 382 and370 μg/g, respectively. Whereas it was apparent that thevariation in LTP levels was larger between the hybrids thanwithin the hybrids, to further illustrate the apparent minimalrole of environment on LTP levels in maize, the LTP levels forall samples from a single maize hybrid (hybrid 01, DEKALBDKC59-34), are shown in Figure 4B. With hybrid 01, LTPlevels varied 1.3-fold when compared across the sevenenvironmental conditions in which it was cultivated. Thehighest average LTP level was 432 μg/g for samples from the

Table 2. Spike and Recovery Assessment of LTP in MaizeExtracta

recovery (%)

spike LTP spiked (μg/mL) analyst A analyst B analyst C mean

1 20 103 108 102 1042 8 102 99 101 1013 3.5 110 112 103 108

aData show the average percentage recovery of LTP by each of threeanalysts on three extractions. Each extraction was conductedindependently using the same maize grain sample spiked with threelevels of LTP.

Table 3. Dilutional Parallelism Assessment of Maize LTPELISA (Percentage of Overall Average)a

percentage

dilution 1:1000 1:2000 1:5000 1:10000

sample lot 1 95 98 91 116sample lot 2 88 99 106 107sample lot 3 91 97 102 110sample lot 4 97 101 103 99sample lot 5 99 98 111 92mean 94 99 102 105

aData represent the percentage of the observed LTP level of the maizegrain extract samples relative to the mean LTP level for all thosesamples.

Table 4. LTP Level in NAM Founder Hybridsa

LTP level (μg/g of grain)

B73 × MAN group rep 1 rep 2 rep 3 mean SD

HP301 flint 290 391 403 361 62Il14H flint 536 505 552 531 24P39 flint 428 612 669 570 126Mo18W mixed 297 327 459 361 86M37W mixed na 459 399 429 42Tx303 mixed 561 501 581 548 42Ky21 temperate 320 359 370 350 26Oh43 temperate 406 na 404 405 1B97 temperate 336 505 378 406 88MS71 temperate 450 470 484 468 17M162W temperate 412 550 529 497 74Oh7B temperate 419 na 759 589 240CML277 tropical na 357 278 318 56Tzi8 tropical 331 372 332 345 23CML333 tropical 300 379 405 361 55CML322 tropical 353 414 398 388 32CML69 tropical 329 380 466 392 69CML228 tropical 320 369 537 409 114CML103 tropical 319 506 418 414 94NC358 tropical 315 438 498 417 93NC350 tropical 321 473 484 426 91Ki3 tropical 342 569 379 430 122Ki11 tropical 361 483 495 446 74CML247 tropical 406 498 496 467 53CML52 tropical 409 527 474 470 59

mean overall 432median 412

aThe LTP levels in 25 varieties of maize hybrids derived from crossesof maize line B73 with NAM founder lines and analyzed with thenewly established ELISA method. na, not available, sample lost in thefield.

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Nebraska location NEYO-1, and the lowest average LTP levelwas 349 μg/g from the Iowa location IAJE-1, both of whichwere produced in 2014. The rest of the five samples wereproduced in 2013. LTP values for samples from 2014 (IAJE-1and NEYO-1) bracket the average LTP levels of samplesproduced in 2013 (376 μg/g) and the overall average LTP levelof hybrid 01 (380 μg/g).A VCA was used to evaluate the influence of genetic and

environmental factors on LTP levels from these samples(Figure 5) and confirmed what seemed apparent. This analysisindicated that the genetic diversity of the various maize hybridsin this sample set had significantly more effect on LTP levels inmaize grain (63%, p value < 0.0001) than the environmentaldifferences across the different locations/years, which con-tributed only 6% to LTP variability, although the environmentaleffect is statistically significant (p value = 0.0002).

■ DISCUSSION

Developing a Validated LTP ELISA. To accurately andconsistently measure the quantity of a molecule, large or small,an analytical method needs to be optimized and validated. Keyelements of developing reliable assays include minimizing thepotential for assay-to-assay and analyst-to-analyst variation,ensuring that the assay is conducted under well-definedconditions, and ensuring that the values obtained from theassay are consistent and within a predetermined acceptancerange.28 The data presented herein establish the precision,recovery, dilutional parallelism, and sensitivity of an ELISAmethod developed for the quantitation of LTP level in maize

grain, thus validating that this ELISA can reliably measure LTPlevels.

High Levels of Variability of LTP Seen in GeneticallyDiverse Maize Hybrids. This newly established LTP ELISAwas used to measure the LTP levels in a diverse collection ofgrain samples from 49 maize hybrids including flint, tropical,temperate, and mixed maize varieties crossed with B73. Thewide range of maize germplasm used in this study wasproduced by crossing maize line B73 with a diverse set ofinbred maize varieties either from 24 landrace lines23 or from25 NAM founder lines.25 The LTP level from all individualreplicates of these 49 maize hybrids showed an approximately5-fold difference (171−865 μg/g). Looking across the averageLTP values for each maize hybrid in this study, the landracehybrids showed a wider range (244−700 μg/g) than the NAMfounder hybrids (318−589 μg/g). The wider range of LTP inlandraces is consistent with higher genome-wide diversity levelsin landraces as compared to inbred lines.29 Interestingly, theindividual replicate values for LTP from the tropical varieties ofthe landrace hybrids accounted for the full range of individualLTP levels within this entire population of maize hybrids, alsoconsistent with greater genetic diversity in tropical germplasmbecause the center of origin of maize is tropical. From a reviewof the individual replicate field plots within the NAM hybrid, itis observed that the temperate hybrid had the highest LTP leveland the tropical hybrid had the lowest level of LTP. The fourhighest average LTP values within the NAM founder linehybrid group were not tropical (two were flint hybrids and oneeach of temperate and mixed hybrid). Although very largedifferences were seen across individual maize hybrids, these two

Table 5. LTP Level in Landrace Hybridsa

LTP level (μg/g of grain)

B73 × Landrace group rep 1 rep 2 rep 3 mean SD

Santo Domingo flint 418 543 588 516 88Assiniboine flint 457 580 553 530 65Havasupai flint na 542 na 542Longfellow Flint flint 549 704 667 640 81Chapalote intermediate 328 579 na 454 177Palomero de Jalisco intermediate 506 716 672 631 111Hickory King temperate 287 426 399 371 74Shoe Peg temperate 327 393 517 412 96Araguito tropical 171 300 260 244 66Comiteco tropical na 284 263 274 15Crystalino Norteno tropical 235 317 358 303 63Canilla tropical na 335 na 335Reventador tropical 283 357 392 344 56Pepetilla tropical 291 359 414 355 62Pollo tropical 321 409 358 363 44Cuban Flint tropical na 392 350 371 30Poropo tropical 332 362 455 383 64Tuxpeno tropical 354 441 379 391 45Costeno tropical na 533 353 443 127Tabloncillo tropical 368 na 541 455 122Bolita tropical na 482 528 505 33Cateto tropical 444 575 631 550 96Cravo Riogranense tropical 455 479 775 570 178Zapalote Chico tropical 865 579 657 700 148

mean (overall) 448median 416

aThe LTP levels in 24 varieties of maize hybrids derived from crosses of maize line B73 with landrace lines and analyzed with the newly establishedELISA method. na, not available, sample lost in the field.

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maize hybrid populations showed vary similar medium LTPlevels (416 and 412 μg/g, respectively) in the aggregate.Flint Maize Hybrid Shows above Median LTP Levels.

Four subsets of these B73 maize hybrids are flint, tropical,temperate, and mixed maize varieties. One group, flint, wasfound to be different from the others. In the landrace hybridpopulation, the average LTP level of each of the four flinthybrids was above the median for that population. The averageLTP levels in these four landrace flint hybrids were B73 ×Santo Domingo (516 μg/g), B73 × Assiniboine (530 μg/g),B73 × Havasupai (542 μg/g), and B73 × Longfellow flint (640μg/g). In the NAM founder line hybrid population, the averageLTP levels from two of three flint hybrids were also above themedian for that population. They were B73 × P39 (570 μg/g)and B73 × Il14H (531 μg/g). Both of these NAM flint lines are

consumed by humans as sweet corn. Sweet corn lines havereduced starch content due to the sugary1 mutation and,therefore, higher levels of protein and oil24 and many othermetabolites.30 One NAM flint hybrid, B73 × HP301 (alsoconsumed by humans as popcorn), had an LTP level of 361μg/g, which was lower the median for that population. Bycomparison, the LTP level of another popcorn line (a landracevariety), a hybrid of Palomero de Jalisco, was 631 μg/g, wellabove the median LTP levels for the landrace hybridpopulation. From a food safety perspective, it is noteworthythat several maize lines that are known for human consumptionshowed LTP levels considerably higher than the median LTPlevel of both hybrid populations. These results support aconclusion that a wide range of maize LTP levels have beensafely consumed over the long history of maize cultivation.

Figure 4. Effect of genetics and environmental factors on LTP level in maize grain. For both panels, the box height is the 75% IQR (interquartilerange), the horizontal line inside each box is the median LTP value, and the circles are the LTP values of individual biological samples. For panel A,the error bar at the end of the line is the 90% IQR. Panel A shows LTP level versus the 18 maize hybrids (grown across 10 states in the United Statesand/or years of 2013 and 2014). Panel B shows the LTP level of maize hybrid 01, DEKALB DKC59-34, at seven specific cultivation locations and/oryear (all but two are 2013; IAJE-1 and NEUT-1 are 2014).

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Relative Contribution of Environment and Geneticsto LTP Level in Maize Grain. Having established that maizegrain from different maize germplasm can express up to a 5-folddifference in LTP levels, the LTP levels in a second set of maizegrain samples were measured with our validated ELISA tobetter understand the relative contribution of genetic andenvironmental factors on maize LTP variance. This second setof maize grain samples included 18 genetically different hybrids,representative of conventional commercial commodity maize.Environmental variability came from field trials conducted atdifferent locations and over two years. A total of 107 uniquesamples (321 biological replicates) were included in this sampleset. The results from the 321 individual biological samples ofdiverse cultivation conditions showed that the LTP levels inthese maize grain samples range from 212 to 751 μg/g, whichoverlap the range of LTP described above from the first set of49 genetically diverse maize hybrids. The landrace hybrids stillshowed the highest variation among all maize hybridpopulations tested in this study. The average LTP level ofthe 18 modern commercial maize hybrids ranged from 281 to594 μg/g, which is a 2.1-fold difference. The level of LTP foreach maize hybrid varied when that hybrid was grown underdifferent environmental conditions (location and year).However, variance component statistical analysis indicatedthat the genetic differences across this set of maize grainsamples was the major contributor (63%) to variance in LTPlevels, whereas the influence of environmental factors (multiplelocations and years) was comparatively smaller (6%). Thisanalysis supports a conclusion that maize genetics play themajor role in LTP levels in maize grain. It is noteworthy,however, that although the environmental factors have arelatively small effect on the LTP level in maize grain, theenvironmental factors also have a statistically significant impacton LTP levels (p value = 0.0002). In this study, the hybrid withthe greatest variability in LTP level (hybrid 14) showedvariation up to 1.6-fold when grown at several locations overthe two years.

The effect of environmental and genetic factors on allergenexpression levels have been reported for other crops, such assoybeans,31,32 and in soybean environmental factors have beenreported to have a large impact on the allergen expression level.

Comparison of ELISA to LC-UV/MS Measurements ofMaize LTP. The present study used a newly developed andvalidated ELISA to measure LTP level in maize grain. The LTPvalues measured by this ELISA from all of these maize grainsamples ranged from 171 to 865 μg/g, which are comparable toLTP levels reported by researchers using a validated LC-UV/MS method.20 The LC-UV/MS method measured LTP andtwo other variant forms of LTP and showed that the combinedLTP levels ranged from 341 to 781 μg/g across the 14 testedmaize lines.20 This comparison supports the conclusion that thepresent validated ELISA accurately quantifies LTP levels inmaize grain similar to the validated LC-UV/MS method. Inaddition to our validated LTP ELISA and the previouslydescribed validated LC-UV/MS assay, an immuno dot-blotmethod has been used to measure LTP, and a 15-fold variationin LTP level was observed and reported.33 The wider range ofLTP levels reported in the dot-blot study might be related tothe set of the maize hybrids tested or to the intrinsic variabilityof the method, compared to ELISA and LC-UV/MS methodsthat are more quantitative. Despite the differences, all threemethods (LC-UV/MS, ELISA, and dot-blot) support theconclusion that there is a wide range of LTP levels in maizegrain, including those which have been consumed as food andanimal feed.Recently, a method for the absolute quantification of multiple

maize LTP isoforms using tandem mass spectrometry wasreported.21 Their results confirm that the predominant formsdetected in maize grain were Zea m 14 and LTPa, which werethe forms used to immunize goats and rabbits for antibodyproduction in the current investigation. Their method alsoquantified two minor isoforms, termed LTPb and LTPc, whichwere expressed at approximately 10- and 100-fold lower levelsthan the predominant Zea m 14 and LTPa forms,respectively.21 LTPb (mature protein isoform) shares at least90% homology with the Zea m 14/LTPa mature proteinisoforms we purified. The LTPc isoform shows much lesssequence identity with the Zea m 14, LTPa, and LTPb, andtherefore it is unlikely to be accurately detected by our ELISAmethod. However, because its expression level is extremely low(∼100-fold less), the impact of this is very minimal in terms ofthe applicability of this ELISA method. The authors of thetandem mass method reported their LTP values in the extractsas μg LTP/mg protein, not as μg/g of grain sample. Thus, theLTP expression levels cannot be directly compared.In this investigation, a high-throughput validated sandwich

ELISA method is described that is capable of accuratelyquantifying LTP levels in maize grain. This validated ELISAwas successfully applied to measure the LTP levels in grainsamples from a wide variety of maize hybrids. The LTP ELISAanalysis results showed that conventional maize hybrids have atleast a 5-fold range of LTP levels in grain. Further evaluation ofgenetic and environmental factors indicated that the LTP levelin maize grain was largely dependent on the geneticbackground of the maize hybrid. By comparison, the diversityof environmental conditions in which maize grain samples wereproduced from multiple locations and over two years had arelatively smaller, albeit statistically significant, contribution toLTP variability. The large natural variation of the LTP level inboth NAM founder hybrids and landrace maize hybrids, as well

Figure 5. Statistical variance components analysis of the factors thataffect the LTP levels in maize grain. The maize samples analyzed werefrom maize varieties cultivated among 20 locations in 10 U.S. statesover two years. Environmental factors are defined as the cultivationlocation and year, genetic factors are defined as the specific maizehybrid, and residual factors represent all other experimental factorsthat contributed to LTP variability. The statistical analysis showed thatthe genetics made the major contribution to the LTP level variation inmaize grain.

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as in modern commercial hybrids, indicates that a broad rangeof LTP levels in maize grain has been safely consumed byhumans over the long history of maize cultivation.

■ AUTHOR INFORMATIONCorresponding Author*(X.G.) Phone: (314) 694-7685. E-mail: [email protected] Gu: 0000-0001-8194-0309Tao Geng: 0000-0002-7367-4693Kevin C. Glenn: 0000-0002-7600-2550NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Shantanu Roychowdhury and Jing Berry forproviding N-terminal sequencing analyses for the purifiedmaize LTP. We also thank George Harrigan and T. V.Venkatesh for helping us to obtain the 49 different maizehybrid samples used for LTP level natural variation assessment.

■ REFERENCES(1) Salcedo, G.; Sanchez-Monge, R.; Barber, D.; Diaz-Perales, A.Plant non-specific lipid transfer proteins: an interface between plantdefence and human allergy. Biochim. Biophys. Acta, Mol. Cell Biol. Lipids2007, 1771, 781−791.(2) Kader, J. C. Lipid-Transfer Proteins in Plants. Annu. Rev. PlantPhysiol. Plant Mol. Biol. 1996, 47, 627−654.(3) Jose-Estanyol, M.; Gomis-Ruth, F. X.; Puigdomenech, P. Theeight-cysteine motif, a versatile structure in plant proteins. PlantPhysiol. Biochem. 2004, 42, 355−365.(4) Diaz-Perales, A.; Garcia-Casado, G.; Sanchez-Monge, R.; Garcia-Selles, F. J.; Barber, D.; Salcedo, G. cDNA cloning and heterologousexpression of the major allergens from peach and apple belonging tothe lipid-transfer protein family. Clin. Exp. Allergy 2002, 32, 87−92.(5) Pastorello, E. A.; Farioli, L.; Pravettoni, V.; Ortolani, C.; Ispano,M.; Monza, M.; Baroglio, C.; Scibola, E.; Ansaloni, R.; Incorvaia, C.;Conti, A. The major allergen of peach (Prunus persica) is a lipidtransfer protein. J. Allergy Clin. Immunol. 1999, 103, 520−526.(6) Pastorello, E. A.; Farioli, L.; Pravettoni, V.; Giuffrida, M. G.;Ortolani, C.; Fortunato, D.; Trambaioli, C.; Scibola, E.; Calamari, A.M.; Robino, A. M.; Conti, A. Characterization of the major allergen ofplum as a lipid transfer protein. J. Chromatogr., Biomed. Appl. 2001,756, 95−103.(7) Pastorello, E. A.; Farioli, L.; Pravettoni, V.; Ortolani, C.;Fortunato, D.; Giuffrida, M. G.; Perono Garoffo, L.; Calamari, A. M.;Brenna, O.; Conti, A. Identification of grape and wine allergens as anendochitinase 4, a lipid-transfer protein, and a thaumatin. J. AllergyClin. Immunol. 2003, 111, 350−359.(8) Morales, M.; Lopez-Matas, M. A.; Moya, R.; Carnes, J. Cross-reactivity among non-specific lipid-transfer proteins from food andpollen allergenic sources. Food Chem. 2014, 165, 397−402.(9) Moneret-Vautrin, D. A.; Kanny, G.; Beaudouin, E. Food allergy tocorn − does it exist? Allergy Immunol. 1998, 30, 230.(10) OECD. Consensus Document on Compositional Considerations forNew Varieties of Maize (Zea mays): Key Food and Feed Nutrients, Anti-nutrients and Secondary Plant Metabolites; Organisation of EconomicCo-Operation and Development: Paris, France, 2002.(11) Gendel, S. M. Comparison of international food allergenlabeling regulations. Regul. Toxicol. Pharmacol. 2012, 63, 279−285.(12) Allen, K. J.; Turner, P. J.; Pawankar, R.; Taylor, S.; Sicherer, S.;Lack, G.; Rosario, N.; Ebisawa, M.; Wong, G.; Mills, E. N.; Beyer, K.;Fiocchi, A.; Sampson, H. A. Precautionary labelling of foods forallergen content: are we ready for a global framework? World AllergyOrgan. J. 2014, 7, 10.

(13) Asero, R.; Mistrello, G.; Roncarolo, D.; Amato, S.; Caldironi, G.;Barocci, F.; van Ree, R. Immunological cross-reactivity between lipidtransfer proteins from botanically unrelated plant-derived foods: aclinical study. Allergy 2002, 57, 900−906.(14) Guillen, D.; Barranco, P.; Palacin, A.; Quirce, S. Occupationalrhinoconjunctivitis due to maize in a snack processor: a cross-reactivitystudy between lipid transfer proteins from different cereals and peach.Allergy, Asthma Immunol. Res. 2014, 6, 470−473.(15) Venter, C.; Pereira, B.; Voigt, K.; Grundy, J.; Clayton, C. B.;Higgins, B.; Arshad, S. H.; Dean, T. Original article: Prevalence andcumulative incidence of food hypersensitivity in the first 3 years of life.Allergy 2008, 63, 354−359.(16) Scibilia, J.; Pastorello, E. A.; Zisa, G.; Ottolenghi, A.; Ballmer-Weber, B.; Pravettoni, V.; Scovena, E.; Robino, A.; Ortolani, C. Maizefood allergy: a double-blind placebo-controlled study. Clin. Exp. Allergy2008, 38, 1943−1949.(17) Pastorello, E. A.; Farioli, L.; Pravettoni, V.; Ispano, M.; Scibola,E.; Trambaioli, C.; Giuffrida, M. G.; Ansaloni, R.; Godovac-Zimmermann, J.; Conti, A.; Fortunato, D.; Ortolani, C. The maizemajor allergen, which is responsible for food-induced allergic reactions,is a lipid transfer protein. J. Allergy Clin. Immunol. 2000, 106, 744−751.(18) Pastorello, E. A.; Pompei, C.; Pravettoni, V.; Brenna, O.; Farioli,L.; Trambaioli, C.; Conti, A. Lipid transfer proteins and 2S albumins asallergens. Allergy 2001, 56 (Suppl. 67), 45−47.(19) Fasoli, E.; Pastorello, E. A.; Farioli, L.; Scibilia, J.; Aldini, G.;Carini, M.; Marocco, A.; Boschetti, E.; Righetti, P. G. Searching forallergens in maize kernels via proteomic tools. J. Proteomics 2009, 72,501−510.(20) Kuppannan, K.; Albers, D. R.; Schafer, B. W.; Dielman, D.;Young, S. A. Quantification and characterization of maize lipid transferprotein, a food allergen, by liquid chromatography with ultraviolet andmass spectrometric detection. Anal. Chem. 2011, 83, 516−524.(21) Stevenson, S. E.; McClain, S.; Thelen, J. J. Development of anisoform-specific tandem mass spectrometry assay for absolutequantitation of maize lipid transfer proteins. J. Agric. Food Chem.2015, 63, 821−828.(22) McMullen, M. D.; Kresovich, S.; Villeda, H. S.; Bradbury, P.; Li,H.; Sun, Q.; Flint-Garcia, S.; Thornsberry, J.; Acharya, C.; Bottoms, C.;Brown, P.; Browne, C.; Eller, M.; Guill, K.; Harjes, C.; Kroon, D.;Lepak, N.; Mitchell, S. E.; Peterson, B.; Pressoir, G.; Romero, S.;Oropeza Rosas, M.; Salvo, S.; Yates, H.; Hanson, M.; Jones, E.; Smith,S.; Glaubitz, J. C.; Goodman, M.; Ware, D.; Holland, J. B.; Buckler, E.S. Genetic properties of the maize nested association mappingpopulation. Science 2009, 325, 737−740.(23) Chia, J. M.; Song, C.; Bradbury, P. J.; Costich, D.; de Leon, N.;Doebley, J.; Elshire, R. J.; Gaut, B.; Geller, L.; Glaubitz, J. C.; Gore, M.;Guill, K. E.; Holland, J.; Hufford, M. B.; Lai, J.; Li, M.; Liu, X.; Lu, Y.;McCombie, R.; Nelson, R.; Poland, J.; Prasanna, B. M.; Pyhajarvi, T.;Rong, T.; Sekhon, R. S.; Sun, Q.; Tenaillon, M. I.; Tian, F.; Wang, J.;Xu, X.; Zhang, Z.; Kaeppler, S. M.; Ross-Ibarra, J.; McMullen, M. D.;Buckler, E. S.; Zhang, G.; Xu, Y.; Ware, D. Maize HapMap2 identifiesextant variation from a genome in flux. Nat. Genet. 2012, 44, 803−807.(24) Venkatesh, T. V.; Harrigan, G. G.; Perez, T.; Flint-Garcia, S.Compositional assessments of key maize populations: B73 hybrids ofthe Nested Association Mapping founder lines and diverse landraceinbred lines. J. Agric. Food Chem. 2015, 63, 5282−5295.(25) Yu, J.; Holland, J. B.; McMullen, M. D.; Buckler, E. S. Geneticdesign and statistical power of nested association mapping in maize.Genetics 2008, 178, 539−551.(26) Tchang, F.; This, P.; Stiefel, V.; Arondel, V.; Morch, M. D.;Pages, M.; Puigdomenech, P.; Grellet, F.; Delseny, M.; Bouillon, P.;et al. Phospholipid transfer protein: full-length cDNA and amino acidsequence in maize. Amino acid sequence homologies between plantphospholipid transfer proteins. J. Biol. Chem. 1988, 263, 16849−16855.(27) Alexandrov, N. N.; Brover, V. V.; Freidin, S.; Troukhan, M. E.;Tatarinova, T. V.; Zhang, H.; Swaller, T. J.; Lu, Y. P.; Bouck, J.; Flavell,R. B.; Feldmann, K. A. Insights into corn genes derived from large-scale cDNA sequencing. Plant Mol. Biol. 2009, 69, 179−194.

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03583J. Agric. Food Chem. 2017, 65, 1740−1749

1748

(28) Findlay, J. W.; Smith, W. C.; Lee, J. W.; Nordblom, G. D.; Das,I.; DeSilva, B. S.; Khan, M. N.; Bowsher, R. R. Validation ofimmunoassays for bioanalysis: a pharmaceutical industry perspective. J.Pharm. Biomed. Anal. 2000, 21, 1249−1273.(29) Hufford, M. B.; Xu, X.; van Heerwaarden, J.; Pyhajarvi, T.; Chia,J. M.; Cartwright, R. A.; Elshire, R. J.; Glaubitz, J. C.; Guill, K. E.;Kaeppler, S. M.; Lai, J.; Morrell, P. L.; Shannon, L. M.; Song, C.;Springer, N. M.; Swanson-Wagner, R. A.; Tiffin, P.; Wang, J.; Zhang,G.; Doebley, J.; McMullen, M. D.; Ware, D.; Buckler, E. S.; Yang, S.;Ross-Ibarra, J. Comparative population genomics of maize domes-tication and improvement. Nat. Genet. 2012, 44, 808−811.(30) Venkatesh, T. V.; Chassy, A. W.; Fiehn, O.; Flint-Garcia, S.;Zeng, Q.; Skogerson, K.; Harrigan, G. G. Metabolomic assessment ofkey maize resources: GC-MS and NMR profiling of grain from B73hybrids of the Nested Association Mapping (NAM) founders and ofgeographically diverse landraces. J. Agric. Food Chem. 2016, 64, 2162−2172.(31) Geng, T.; Liu, K.; Frazier, R.; Shi, L.; Bell, E.; Glenn, K.; Ward,J. M. Development of a sandwich ELISA for quantification of Gly m 4,a soybean allergen. J. Agric. Food Chem. 2015, 63, 4947−4953.(32) Stevenson, S. E.; Woods, C. A.; Hong, B.; Kong, X.; Thelen, J. J.;Ladics, G. S. Environmental effects on allergen levels in commerciallygrown non-genetically modified soybeans: assessing variation acrossnorth america. Front. Plant Sci. 2012, 3, 196.(33) Panda, R.; Ariyarathna, H.; Amnuaycheewa, P.; Tetteh, A.;Pramod, S. N.; Taylor, S. L.; Ballmer-Weber, B. K.; Goodman, R. E.Challenges in testing genetically modified crops for potential increasesin endogenous allergen expression for safety. Allergy 2013, 68, 142−151.

Journal of Agricultural and Food Chemistry Article

DOI: 10.1021/acs.jafc.6b03583J. Agric. Food Chem. 2017, 65, 1740−1749

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