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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 ΔAgricultural 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 difference, 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 (212−751 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 different 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 different plant species have been identified, and their functions have been extensively studied.1−3 The 9 kDa LTP1 protein contains eight conserved cysteine residues, which form four intramolecular disulfide 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 Rosaceae4−6 and Vitaceae families.7 With these food-allergic individuals, a high level of immune cross-reactivity has been noted for the LTP from different 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.16−18 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 identified 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 chromatography−mass 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) program22 and a diverse collection of landrace accessions23 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, 1740−1749

    pubs.acs.org/JAFC http://dx.doi.org/10.1021/acs.jafc.6b03583

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

    ■ MATERIALS AND METHODS Reagents. 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 used for the isolation of LTP. This purified lipid transfer protein served as an immunogen for both rabbit and goat antibody production, as well as the protein standard for ELISA. LTP were isolated from maize grain using a combination of anion exchange, cation exchange, and size exclusion chromatographies. Briefly, maize grain was ground to fine powder using a laboratory mill (Perten, Hag̈ersten, Sweden) in the presence of dry ice and then extracted with 50 mM Bis-Tris-propane buffer, pH 7.0. The clarified extract was loaded onto a Q-Sepharose Fast Flow column (GE Healthcare, Marlborough, MA, USA) and the flow-through containing LTP was collected. After concentration, the resulting sample was loaded onto an S-Sepharose Fast Flow column (GE Healthcare) and eluted with a gradient (0−400 mM) of sodium chloride. A single peak fraction containing the bulk of the LTP was collected. The protein sample was concentrated and applied onto a Sephacryl S-100 size exclusion column (GE Healthcare) equilibrated with 50 mM phosphate buffer, pH 7.0, containing 150 mM sodium chloride. The LTP peak, which eluted at approximately 9 kDa, was collected. This preparation was characterized and used as the LTP protein standard in all subsequent experiments. The characterizations included protein purity assessment by SDS-PAGE, protein identity by MALDI-TOF mass spectrometry (Applied Biosystems, Foster City, CA, USA), and N-terminal amino acid sequence analysis by Applied Biosystems 494 Procise Sequencing System (Applied Biosystems). Maize Grain for LTP Natural Variability Assessment.

    Genetically Diverse Maize Grain Samples from B73 Hybrids with NAM and Landrace Lines. The experimental design for generating the maize grain samples analyzed in this study, including selection and identity of the maize lines, production of hybrid seed, and field design, is detailed in a previous paper.24 Briefly, seed of 49 maize hybrids was produced by crossing B73 with a set of 24 diverse landrace inbred lines23 and the 25 NAM founder lines.22,25 The 49 maize hybrids were grown in 2012 as a randomized complete block design in Aurora, NY, USA, with three replicates and the plants self-pollinated to control for pollen effects. A total of 133 independent samples were available and analyzed in this investigation. Environmentally Diverse Maize Grain Samples from Conven-

    tional Commercial Maize Hybrids. Conventional maize grain samples from U.S. field trials conducted over two years (2013 and 2014) were chosen to evaluate the effect of different environmental conditions on LTP levels. A total of 18 conventional maize hybrids were involved across these various field trials. In total, 107 unique samples (unique in year and/or location and/or trial) were drawn from 20 locations across 10 U.S. states (one location each in Arkansas, Indiana, Kansas, Missouri, Ohio, Wisconsin, two locations in Pennsylvania, three locations in Nebraska, four locations in Iowa, and five locations in Illinois) over two years. Grain samples from each maize hybrid were collected from a minimum of three location/years and three randomized replicate plots for each unique entry for a total of 321 individual biological samples. These samples were randomized prior to analysis. Anti-LTP Polyclonal Antibody Production. Polyclonal antibod-

    ies from both goat and rabbit were separately produced for establishing the sandwich ELISA method. The goat anti-LTP antibodies were raised in goats 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 isolated from 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., Rockf