Article pubs.acs.org/JAFC

Development of a Sandwich ELISA for Quantification of Gly m 4, a Soybean Allergen Tao Geng,* Kang Liu, Ronald Frazier, Lifang Shi, Erin Bell, Kevin Glenn, and Jason M. Ward Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, Missouri 63167, United States ABSTRACT: Gly m 4 is a key soybean allergen that causes allergic symptoms in the skin, gastrointestinal tract, or respiratory tract of sensitive individuals. To understand naturally variable levels of Gly m 4 among conventional soybean varieties, a sandwich ELISA was developed and validated using a mouse anti-Gly m 4 monoclonal antibody and a goat anti-Gly m 4 polyclonal antibody as capture and detection antibodies, respectively. The ELISA shows high specificity to Gly m 4 without any crossreactivity to other soybean proteins and has a quantification range of 7.8−250 ng/mL using an Escherichia coli-produced recombinant Gly m 4, with 2.1 ng/mL being the limit of detection. Within the quantification range, the coefficients of variation of the intra-assay and interassay precision are less than 5 and 12%, respectively. Moreover, extraction efficiency and dilutional parallelism experiments were completed to demonstrate the assay is accurate. The validated assay was used to quantify Gly m 4 levels in 128 soybean samples from 24 conventional soybean varieties grown at 8 distinct geographical locations. There was a 13fold difference between the least and greatest amounts of Gly m 4 concentrations among the samples, and the results demonstrate that the most significant sources of variability in Gly m 4 levels in the conventional varieties were related to location and variety. KEYWORDS: soybean, allergy, Gly m 4, ELISA, natural variability

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d 1),13 and cherry (54% with Pru av 1)14 pollen or food allergens. In addition to high amino acid sequence identity, Gly m 4 showed almost identical three-dimensional structure and cross-reaction to a major birch pollen allergen, Bet v 1,8,15−17 suggesting a possible causal association between exposure to birch pollen and soy food allergy. Although several studies have focused on Gly m 4 structure and its clinical implications, little is known about its expression levels in soybeans grown under different environmental conditions. Julka et al.18 developed and validated a twodimensional liquid chromatography approach with ultraviolet and mass spectrometry to quantify Gly m 4 from soybean seeds, but the method requires time-consuming processes and expensive equipment. Although Mittag et al.17 generated a rabbit anti-Gly m 4 antibody and performed a quantitative Western blot, the method is low throughput and hard to validate. The enzyme-linked immunosorbent assay (ELISA), the most common method used by food industries and food control agencies to identify and quantify allergens,2,19−21 could be the best approach to quantify a large amount of samples because of its sensitivity and high throughput. For these reasons ELISA methods for the detection of several soy allergens have been developed, including Gly m Bd 30K,22,23 Gly m Bd 28K,24,25 glycinin/Gly m 5,26 β-conglycinin,27,28 and Kunitz trypsin inhibitor.29 However, to date, there is no validated ELISA method to detect and quantify Gly m 4 levels from soybean seeds.

INTRODUCTION Soybean (Glycine max) seed has protein constituting 34.1− 56.8% of total content on a dry weight basis,1 which makes it a good source of nutritional protein for animal and human consumption. Soybean and its byproducts are widely used in the food, feed, and pharmaceutical industries because of their nutritional values and physicochemical properties.2 Because of the extensive use and need for protecting crop productivity, genetically modified (GM) soybeans have become the overwhelming majority of soybean varieties grown. In 2014, 82% (90.7 million of the 111 million hectares) of the soybeans planted globally were GM soybean. 3 To enable the commercialization of a GM soybean variety, an extensive safety assessment that includes an allergy safety assessment is completed.4 This assessment includes a comparison of endogenous allergen levels in the GM soybean and conventional control. To evaluate this comparison of endogenous allergen levels, an understanding of the variability in allergen levels would be useful for supporting eventual determinations of exposure levels. Therefore, quantification of endogenous allergens from variable non-GM varieties is needed. To date, eight soybean endogenous proteins have been shown to cause allergic symptoms in soybean-allergic individuals, including Gly m 3, Gly m 4, Gly m 5 (βconglycinin), Gly m 6 (glycinin), Gly m 8 (2S albumin), Gly m Bd 28K, Gly m Bd 30K, and Kunitz trypsin inhibitor.5 Gly m 4, also called SAM22, is a pathogenesis-related (PR) protein that is specifically expressed in response to plant pathogens or environmental factors that affect plant physiology.6 It belongs to the family of PR proteins that also includes several other pollen and food allergens7−10 and thus shows high amino acid sequence identity (>50%) to birch (53% with Bet v 1),11 hazelnut (58% with Cor a 1.0401),12 apple (53% with Mal © XXXX American Chemical Society

Received: February 10, 2015 Revised: April 27, 2015 Accepted: May 6, 2015

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DOI: 10.1021/acs.jafc.5b00792 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Technology, Rochester, NY, USA). The final protein concentration was determined by total amino acid concentration on the average of five replicates. BSA standard (Sigma) with known concentration was used as control to evaluate the system suitability. The purified rGly m 4 protein was diluted with Laemmli buffer with β-mercaptoethanol and separated on a precast Tris−glycine 4−20% polyacrylamide gradient gel (Invitrogen). Gel was stained with colloidal Brilliant Blue G (Sigma). Gel image was captured and the protein purity analyzed using a GS-800 scanning densitometer equipped with Quantity One software (Bio-Rad, Hercules, CA, USA). N-terminal sequence analysis of rGly m 4 protein was performed for 15 cycles. The analysis was conducted using an Applied Biosystems 494 Procise Sequencing System (Applied Biosystems, Foster City, CA, USA). The purified rGly m 4 protein was also characterized by mass spectrometry. Briefly, the protein was run on a 4−20% SDS-PAGE Tris−glycine gel (Invitrogen), and the band corresponding to rGly m 4 at ∼17 kDa was excised. The protein was extracted, reduced, alkylated, and digested. The digest was applied to an Applied Biosystems Voyager DE-Pro Biospectrometry Workstation with the supplied Data Explorer software for the mass spectrometry (MS) analysis. Preparation of the Polyclonal Antibody. The production of goat polyclonal antibody (pAb) for Gly m 4 was conducted by Harlan Bioproducts for Science, Inc. (Madison, WI, USA). Briefly, two goats, G1195 and G1196, were immunized with the purified rGly m 4 by four subcutaneous injections. The first injection contained 1 mg of rGly m 4 emulsified in complete Freund’s adjuvant (FCA; Sigma). The three subsequent boosts contained 500 μg of rGly m 4 emulsified in incomplete Freund’s adjuvant (IFA; Sigma) and were given every 3 weeks. Blood was harvested from each goat 2 weeks after the final booster. The titers and the specificity of sera were determined by using the direct binding ELISA and Western blots as described below, respectively. The goat sera were then affinity purified by a 5 mL HiTrap protein G column (GE Healthcare, Piscataway, NJ, USA) with an AKTA FPLC (GE Healthcare) according to the manufacturer’s instruction. The purified total IgG was further purified by a rGly m 4 conjugated HiTrap NHS-Activated HP column (GE Healthcare) with an AKTA FPLC (GE Healthcare) according to the manufacturer’s instruction. Briefly, about 10 mg of rGly m 4 was conjugated by amide linkage to a 5 mL prepacked column of N-hydroxysuccinimide (NHS) cross-linked to HP Sepharose beads. The total IgG in PBS (pH 7.4) was flowed through the rGly m 4 column. Unbound IgG was washed out by using PBS, and Gly m 4 specific IgG was eluted out with 100 mM glycine (pH 2.5). To prevent antibody still binding to the column, 3.5 M MgCl2 in PBS was used to wash the column after elution. The specificity of the rGly m 4 column-purified IgG was assessed by Western blots by using soybean seed extract. The purified Gly m 4 specific IgG was dialyzed overnight by using 10K molecular weight cutoff (MWCO) dialysis cassettes (Thermo Scientific, Rockford, IL, USA) in the 4 L of PBS, with three changes of buffer, and then biotinylated by using an EZ-link Sulfo-NHS-LC-biotin kit (Thermo Scientific) according to the manufacturer’s instruction. The ratio of antibody and biotin was determined as 1:7 by using a Biotin Quantitation kit (Thermo Scientific). The biotin-conjugated pAb served as the detection antibody in the sandwich ELISA for Gly m 4. Preparation of the Monoclonal Antibody. The production of mouse monoclonal antibody (mAb) for Gly m 4 was conducted by Rockland Immunochemicals, Inc. (Gilbertsville, PA, USA). Prior to primary immunization, five BALB/c mice were fed a soybean-free diet for 2 weeks. The mice were then subcutaneously immunized with 50 μg of emulsified in CFA (Sigma). Two weeks later, the mice were reimmunized with 25 μg of rGly m 4 emulsified in IFA (Sigma). Three more boosts were given every 3 weeks with 25 μg of rGly m 4 emulsified in IFA. The titer of the test bleed of each boost was determined by using the direct coating ELISA, and the specificity of bleeds was assessed by using Western blots. The mouse with the highest antibody titer and specific to Gly m 4 was selected for cell fusion.

In this study, a recombinant Gly m 4 (rGly m 4) protein was expressed, purified, and used as a reference standard protein for an ELISA. Mono- and polyclonal antibodies were generated using the rGly m 4 as an antigen and used to develop and validate a sandwich ELISA. The experiments completed during the assay validation demonstrated that the ELISA is sensitive, specific, accurate, and precise for Gly m 4 levels in soybean samples. Following the validation, the newly developed assay was used to quantify Gly m 4 levels from different soybean varieties grown under a variety of environmental conditions. The result showed that this key soybean food allergen has high variability across conventional soybean varieties grown in different locations.

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MATERIALS AND METHODS

Soybean Varieties. Conventional soybean varieties (24 varieties) were grown in a U.S. field trial during the 2010 growing season at eight geographically diversified sites (one site each in Arkansas, Illinois, Missouri, Indiana, and Pennsylvania and three sites in Iowa), with each site planted in a randomized complete block design with four replicated blocks. Normal agronomic practices were followed for each site. Seed was harvested at the R8 growth stage30 (95% of the pods had reached mature pod color) when the moisture content was approximately 12−15% and was stored at ambient temperature after harvest. Protein Extraction from Soybean Seeds. Soybean seed samples were ground using a mega grinder (Monsanto Co., St. Louis, MO, USA) for approximately 1 min. Protein was extracted from about 100 mg of each ground sample by using a Harbil Mixer (Fluid Management, Inc., Wheeling, IL, USA) with 10 mL (1:100 tissue to buffer ratio) of 0.01 M phosphate-buffered saline with 0.05% (v/v) Tween-20, pH 7.4 (PBST), and 8 chrome beads (0.25 in. in diameter) for 3.5 min at 1500 rpm. Insoluble material was removed from soybean seed extract using a 16 mm × 4 in. serum filter (catalog no. 02-681-51, Fisher Scientific, Pittsburgh, PA, USA). Expression, Purification, and Characterization of rGly m 4. The Gly m 4 cDNA sequence (GI 134194, NCBI) fused with a His tag at C-terminal was synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA) and then ligated into pET24 vector (Novagen, Madison, WI, USA) using NdeI/XhoI restriction sites. The Escherichia coli BL21 (DE3) strain harboring pET24 rGly m 4 was grown in 800 mL of Terrific Broth (Sigma, St. Louis, MO, USA) containing 50 μg/ mL kanamycin (Fisher Scientific). The cells were grown overnight at 37 °C with rotary shaking (250 rpm). The cultures were induced by the addition of IPTG (Invitrogen, Grand Island, NY, USA) to 200 μM and then grown for another 48 h at 15 °C. The cells were harvested by centrifugation at 20000g for 30 min. The cell paste was homogenized in 1 L of 50 mM Tris-HCl, 100 mM NaCl, pH 8, using a cell disruptor (Constant Systems Inc., Kennesaw, GA, USA) at 15000 psi. The cell lysate was centrifuged at 20000g for 30 min, and the supernatant was incubated with 25 mL of NiNTA resin (Qiagen, Valencia, CA, USA) on an orbital shaker for 1 h at 4 °C. The resin slurry was then poured into a column and washed with an additional 200 mL of lysis buffer. The resin was washed with 100 mL of lysis buffer containing 20 mM imidazole and then eluted with 75 mL of lysis buffer containing 200 mM imidazole. The eluted protein was dialyzed into 50 mM Tris-HCl, 100 mM NaCl, pH 8, and stored at −80 °C. The purified rGly m 4 protein concentration was determined by ACQUITY UPLC amino acid analysis using an AccQ-tag Ultra derivatization kit (Waters, Milford, MA, USA) and measured by a UPLC-UV detector (Waters). The protein was first acetone precipitated to get rid of the Tris-HCl buffer and then vapor phase acid hydrolyzed in 6 N HCl at 150 °C for 90 min. The hydrolyzed amino acids were precolumn derivatized with the AccQ-tag Ultra kit according to the manufacturer’s protocol and then injected into the ACQUITY UPLC. Individual amino acid concentration was determined by an amino acid standard H calibration curve (Life B

DOI: 10.1021/acs.jafc.5b00792 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry One week following cell fusion, the supernatant culture fluid from all wells was tested by the direct coating ELISA and Western blots. Cell lines with high affinity and specificity for Gly m 4 were selected for subcloning. Two stable subclones were injected intraperitoneally into five mice for each clone to produce ascites. Finally, the monoclonal antibodies were purified from the ascites by a 5 mL HiTrap protein G column (GE Healthcare) with an AKTA FPLC (GE Healthcare). After screening by direct plate binding ELISAs, the mAb with the highest titer was selected and applied as the capture antibody in the sandwich ELISA for Gly m 4. Direct Binding ELISA To Measure Gly m 4. A direct binding ELISA was carried out to determine the titers of goat sera, mouse sera, and the hybridoma cell lines. rGly m 4 was used as the plate-coating antigen. A 96-well microtiter plate (Thermo Fisher Scientific, Rochester, NY, USA) was coated with a solution (100 μL/well) containing rGly m 4 (10 μg/mL) in 0.05 M carbonate/bicarbonate buffer with 0.15 M NaCl (pH 9.6) and incubated overnight at 4 °C. The plates were washed three times with 300 μL/well washing buffer (PBST, PBS containing 0.05% (v/v) Tween-20, pH 7.4) using a plate washer (ELC405, Biotek, Winooski, VT, USA) to remove unbound antigen. The plates were then blocked by 200 μL/well 1% (w/v) casein in PBS (Thermo Fisher Scientific) for 1 h at 37 °C. All plates were subsequently washed three times. Serially diluted goat sera, mouse sera, and the supernatants of hybridoma cell lines in PBST with 0.1% (w/v) BSA were added (100 μL/well) and incubated at 37 °C for 1 h. After three washings of the plate with PBST, rabbit anti-goat IgG-HRP (Thermo Fisher Scientific), or goat anti-mouse IgG-HRP (Cell Signaling) diluted to 1:2000 using PBST with 0.1% (w/v) BSA was added (100 μL/well) and incubated at 37 °C for 1 h, followed by three washings with PBST; peroxidase substrate TMB solution (KPL, Gaithersburg, MD, USA) was added (100 μL/well) and incubated at room temperature for 10 min. The reaction was terminated by 6 M H3PO4 solution (100 μL/well), and the optical density (OD) was measured at 450 nm with reference wavelength at 620 nm by a plate reader (SpectraMax 384 Plus, Molecular Devices, Sunnyvale, CA, USA) with a SoftMax software (version 5.4). SDS-PAGE and Western Blot. Western blot was performed to assess the specificity of the goat sera, mouse sera, the hybridoma cell lines, monoclonal antibody, and polyclonal antibody for Gly m 4. The rGly m 4 and the soybean seed extract were diluted initially in PBST and further diluted 1:2 (v/v) in Bio-Rad 2X Laemmli Buffer and then heated in a 95 °C metal block for approximately 5 min. The denatured proteins and prestained protein ladder (BenchMark, Invitrogen) or Bio-Rad broad range protein markers were subjected to SDS-PAGE using a midi-gel apparatus (Invitrogen) with precast Novex Tris− glycine 4−20% (w/v) polyacrylamide gels (Invitrogen). The separated proteins were eletrophoretically transferred from the gels to a 0.45 μm polyvinylidene fluoride (PVDF) microporous membrane (Invitrogen) in a vertical midi-format electrophoresis cell (Criterion, Bio-Rad). Blotting was performed at 100 V for 60 min. The membrane was blocked overnight at 4 °C in PBS containing 0.3% (v/v) Tween-20. The membrane was rinsed three times for 10 min each in PBST and probed for 1 h at room temperature with either antisera or purified IgG in PBST with 0.1% (w/v) BSA. After three washings with PBST for 10 min each, the membrane was probed with rabbit anti-goat IgGHRP (Thermo Fisher Scientific) or goat anti-mouse IgG-HRP (Cell Signaling) for 1 h at room temperature. After three rinses with PBST, SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) was added onto the membrane. The luminescence signals were captured by photographic films (Amersham Hyperfilm ECL, GE Healthcare, Buckinghamshire, UK) in a dark room using a Konica SRX-101A automated film processor (Tokyo, Japan). Different exposures were taken, and a single exposure was scanned using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 4.6.7). Indirect Sandwich ELISA. An indirect sandwich ELISA was developed and validated to detect Gly m 4 from soybean seed quantitatively. Ninety-six-well microtiter plates were coated with 100 μL/well of purified mAb (2 μg/mL) in 0.05 M carbonate/bicarbonate buffer with 0.15 M NaCl (pH 9.6) and incubated overnight at 4 °C.

The plates were washed three times with 300 μL/well PBST using a plate washer (ELC405, Biotek) to remove unbound mAb. Excess binding sites were blocked with 200 μL/well of Poly-HRP dilution buffer (Thermo Scientific) for 1 h at 37 °C. The plates were subsequently washed three times, and 100 μL of rGly m 4 standards and samples diluted in PBST with 0.1% (w/v) BSA at various concentrations was added into each well and incubated for 1 h at 37 °C. After three washings of the plate with PBST, biotinylated pAb with 1:4000 dilution in PBST with 0.1% (w/v) BSA was added (100 μL/ well) and incubated for 1 h at 37 °C. Following three washings of the plates, a Poly-HRP streptavidin conjugate (Thermo Scientific) with 1:10000 dilution in Poly-HRP dilution buffer (Thermo Scientific) was added to each well (100 μL/well) and incubated for 0.5 h at 37 °C. Following three washings with PBST, peroxidase substrate 3,3′,5,5′tetramethylbenzidine (TMB) solution (KPL, Gaithersburg, MD, USA) was added (100 μL/well) and incubated at room temperature for 10 min. The reaction was terminated by 6 M H3PO4 solution (100 μL/ well), and the optical density (OD) was measured at 450 nm with reference wavelength at 620 nm. Calibration curve was obtained by plotting the OD values against Gly m 4 concentrations. A buffer blank, a negative control (QC−), and a positive control (QC+, 60 ng of rGly m 4 in PBST with 0.1% (w/v) BSA) were also included on every ELISA plate. All samples were run in triplicate. The sandwich ELISA was subsequently validated for its precision, limits of detection and quantitation (LOD and LOQ), dilutional parallelism, and extraction efficiency.6,31 Statistical Analysis of Gly m 4 Levels from Conventional Soybean Varieties. The Gly m 4 level was calculated according to the following equation:

ppm = (ng/mL of extract) × (dilution factor) × (buffer to tissue ratio mL/g) ÷ 1000 ng/μg Variance components analysis (VCA) was conducted for Gly m 4 from 24 soybean varieties to estimate the proportion of random effects contributing to the total variance, on the basis of the following analysis of variance (ANOVA) model by combining all eight sites: Yijk = U + Ti + Lj + B(L)jk + LTij + eijk Yijk is the unique individual observation, U is the overall mean, Ti is the soybean variety effect, Lj is the location effect, B(L)jk is the block within location effect, LTij is the location by variety interaction effect, and eijk is the residual error. In this application, all of the effects in the ANOVA model were set as random effects. The SAS procedure PROC MIXED was employed to run the analysis. The output table of covariance parameter estimates from SAS PROC MIXED procedure gives estimates of the variance component parameters.32 The variance component parameters of each model component were divided by the total variance to get the variance proportions for each of the components.

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RESULTS Expression, Purification, and Characterization of rGly m 4. The soybean protein was expressed in E. coli with a histidine tag to facilitate purification (Figure 1). The cell lysates of pET24 rGly m 4/BL21 (DE3) showed a His-tagged rGly m 4 at 17 kDa with other proteins from E. coli (lane 3, Figure 1). After elution with imidazole in a NiNTA resin column, a single rGly m 4 band at the expected molecular weight was observed by SDS-PAGE (lanes 5 and 6, Figure 1). The rGly m 4 was stored in the buffer with 50 mM Tris-HCl, 100 mM NaCl, pH 8. By calculating rGly m 4 protein band intensity over total intensity on lane 6 (Figure 1), its purity was determined as 97%. The purity-corrected concentration was used to prepare ELISA standard curves. The N-terminal sequence of rGly m 4 showed exact matches of 15 amino acids to the predicted amino acid sequence. MS

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DOI: 10.1021/acs.jafc.5b00792 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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The Western blot results show both antibodies react with Histagged rGly m 4 (approximate 17 kDa band in lanes 2−6) and Gly m 4 in soybean extract (approximate 16 kDa band in lanes 7−10). The monoclonal antibody showed more band intensity compared to the polyclonal antibody as expected on the basis of their respective titers. There were very faint cross-reaction bands (lanes 2−3) at approximately 49 kDa (Figure 2a) or at 60 and 26 kDa (Figure 2b) from rGly m 4, indicating both antibodies could have very slight immunoreactions with proteins from E. coli. This could be due to the small amount of impurities present in the rGly m 4 that was used as an immunogen for the antibody productions. Most importantly, both antibodies showed negligible cross-reaction with other soy proteins in the soybean seed extracts (Figure 2, lanes 7−10), indicating they were specific for Gly m 4 and could be used for the development of a Gly m 4 sandwich ELISA for soybean seed samples. From these experiments a monoclonal antibody and a polyclonal antibody were selected as capture and detection antibodies, respectively. ELISA Method Optimization and Validation. A sandwich ELISA was optimized to use 2 μg/mL of mAb to capture Gly m 4 and a 1:4000 dilution of the pAb to detect Gly m 4. Using these antibody amounts, the assay showed low background values and high sensitivity. The quantification range of the standard curve was established from 7.8 to 125 ng/ mL with anchor points of 0 and 500 ng/mL (Figure 3). A 60 ng/mL positive quality control (QC+) was used to evaluate the accuracy of the assay. The precision of the assay across the entire working range of rGly m 4 protein levels was determined both within a run (intra-assay precision of technical replicates) and across runs (interassay precision of assay plates over a period of time). To do this the seven-point standard curve of rGly m 4 (at concentrations shown in Table 1) and the QC+ sample were assayed in triplicate wells over 36 assay runs carried out on three different days by three different analysts. Assay precision was calculated as the percentage coefficient of variation (%CV) using the interpreted concentrations of the QC+ samples and the standards observed over all assay runs (Table 1). The %CV of intra-assay and interassay precisions of standards and QC+ were much less than 15%, the commonly accepted limit in industry bioanalytical method validation.33 To ensure accurate detection of Gly m 4 in the soybean seed, it was also important to determine the lowest concentration of

Figure 1. SDS-PAGE analysis of the expression and purification of rGly m 4 protein from E. coli. Lanes: 1, molecular weight markers; 2, blank; 3, lysates of pET24 rGly m 4/BL21 (DE3); 4, NiNTA flowthrough; 5, elution with 20 mM imidazole; 6, elution with 200 mM imidazole. The purity of rGly m 4 was determined as 97%.

result showed 41.8% sequence coverage. Overall, the identity of the rGly m 4 was confirmed by N-terminal sequence analysis and mass spectrometry. Characterization of Antibodies. Monoclonal antibodies were purified from ascites of clone 46F8.B4 that showed the highest titer of 1:102,400. Similarly, polyclonal antibodies, purified from sera of two goats, were screened by using direct plate binding ELISA. The goat with the highest titer (1:6400) was selected for further characterization. Western blots were used to determine the specificity of both antibodies to the rGly m 4 and Gly m 4 in soybean extract (Figure 2). For these Western blots the rGly m 4 protein and soybean seed extract were serially diluted and loaded onto SDS-PAGE gels. Following the transfer of separated proteins onto the membranes, the blots were probed by using either monoclonal antibody (Figure 2a) or goat polyclonal antibody (Figure 2b).

Figure 2. Western blot analyses of monoclonal antibody (a) and polyclonal antibody (b) with rGly m 4 and soybean seed extract. Lanes: M, prestained protein ladder; 1, mouse IgG (a) or goat IgG (b) as positive controls; 2, antibodies reacted with 32 ng of rGly m 4; 3, 16 ng of rGly m 4; 4, 8 ng of rGly m 4; 5, 4 ng of rGly m 4; 6, 2 ng of rGly m 4; 7, soybean extract with 1:200 total dilution; 8, soybean extract with 1:400 total dilution; 9, soybean extract with 1:800 total dilution; 10, soybean extract with 1:1600 total dilution. These Western blots demonstrate that the monoclonal and polyclonal antibodies are specific for both rGly m 4 and Gly m 4 from soybean. D

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detect Gly m 4 from soybean seed samples across a range of working concentrations.

Figure 3. Standard curve of sandwich ELISA for Gly m 4. Each point represents the optical density (OD) of each Gly m 4 concentration with mean ± standard deviation from three determinations. The coefficient of determination (R2) is 1.00. The quantification range of the standard curve is 7.8−125 ng/mL. Figure 4. Dilutional parallelism of the sandwich ELISA for Gly m 4. The recovery of Gly m 4 across the multiple dilutions of soybean seed extracts ranged from 94 to 103%, indicating that this assay can accurately detect Gly m 4 from soybean seed samples across a range of working concentrations.

Table 1. Gly m 4 ELISA Dose-Dependent Intrarun and Inter-run Precisions of Seven Standard Points and the QC+ Sample concentration of standard (ng/mL)

%CV intrarun

%CV inter-run

250.0 125.0 62.5 31.3 15.6 7.8 QC+

3.9 1.1 1.0 1.3 1.7 4.0 4.3

3.9 2.4 1.8 1.9 2.8 5.0 11.5

Finally, an extraction efficiency experiment was also performed to estimate the efficiency of the procedure used to extract Gly m 4 from soybean seed. This experiment is completed by performing successive extractions on the same material and analyzing each extract to determine the amount of Gly m 4 protein that remains after the first extraction. The extraction efficiency is expressed as the percent of Gly m 4 protein recovered in the first extraction (supernatant, E1) compared to the total recovered from the consecutive reextractions of the same sample. For this evaluation, Gly m 4 from three soybean seed samples was extracted, and the average extraction efficiency was calculated to be 76%. Overall, the validation results demonstrate that this sandwich ELISA is precise and accurate and provides sensitive and reliable reproducible quantification of Gly m 4 levels in soybean seed samples. Gly m 4 Levels in Soybean Varieties Grown at Eight Locations. The ELISA assay was used to measure Gly m 4 levels in 24 different soybean varieties grown across eight different commercial soybean growing locations in the United States. The results showed that the range of Gly m 4 levels was about 13-fold difference (from 24 to 311 ppm; Figure 5a). Variance component analysis showed location and substance as well as residual (from uncontrolled systematic variability, including biological variability) are the three main contributions to the total variability of Gly m 4 expression in soybeans, indicating that both environmental and germplasm differences do affect endogenous allergen levels (Figure 5b).

Gly m 4 that can reliably be detected above the assay’s background level. Because no negative matrix could be used to determine this lowest concentration, 21 replicate buffer samples (seven in one assay buffer, total three lots of the assay buffer) were analyzed in triplicate wells on three ELISA runs by three different analysts. The limit of detection (LOD) was defined as the average concentration of Gly m 4 that could be calculated from the background OD observed in the assay buffers plus three standard deviations of the concentration. By these criteria, the LOD for Gly m 4 in the soybean seed samples was determined to be 2.1 ng/mL. The limit of quantification (LOQ) for Gly m 4 was defined as the lowest concentration on the standard curve with a %CV ≤ 15% and was determined to be 7.8 ng/mL. In addition to assay precision using a standard curve and QC +, it is important to confirm that the Gly m 4 ELISA is accurate across a range of expression levels in soybean seed samples. Dilutional parallelism is a method used to demonstrate the accuracy of an ELISA assay. Dilutional parallelism was demonstrated by testing multiple dilutions of a soybean protein extract to demonstrate that the ELISA method is accurate regardless of the sample dilution being tested. To test dilutional parallelism of Gly m 4, five independently extracted samples were analyzed at four dilution factors (20-, 30-, 40-, and 50fold) that were within the quantitative range of the standard curve. Each result was then corrected for the appropriate dilution factor and compared to the average concentration of all dilutions for that sample. The recovery of Gly m 4 across the multiple dilutions of five soybean seed samples ranged from 89 to 104% (Figure 4), indicating that this assay can accurately

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DISCUSSION Gly m 4 has been identified as a key allergen in soybean.34 Here a Gly m 4-specific sandwich ELISA is described, using antibodies generated by immunization of a histidine-tagged recombinant form of soy Gly m 4 (Figure 1). The ELISA shows high specificity and sensitivity to Gly m 4 in soybean seed extract, without any cross-reactivity to other soybean seed proteins (Figures 2 and 3). The intra- and interassay precisions of this Gly m 4 ELISA were well within the limit of variability accepted by industry bioanalytical method validation criteria E

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β-conglycinin ranged from 3.99 to 14.78% of total weight from 469 soybean seed samples,36 and Chen et al. found that glycinin ranged from 2.72 to 19.56% of total weight from the same set of soybean seed samples.37 For Gly m Bd 30k (P34) determined by ELISA in 138 soybean cultivars from a diverse germplasm collection from 10 different countries, the lowest and highest levels were 2.32 and 27.75 mg/g defatted flour, respectively.38 The effects of environment and genotype on Gly m 4 levels from conventional soybeans are also consistent with previous studies on compositional differences.39−43 Further investigation of environmental and genotypic effects on allergenicity is needed for other endogenous allergens. In conclusion, a robust sandwich ELISA for Gly m 4 has been developed and validated for soybean seed extracts. Use of this ELISA has shown that this common soybean food allergen has high variability across conventional soybean varieties grown in different locations.

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AUTHOR INFORMATION

Corresponding Author

*(T.G.) E-mail: [email protected]. Phone: (314) 6947695. Notes

The authors declare no competing financial interest. Figure 5. Gly m 4 levels in soybean seed samples: (a) Gly m 4 levels from 24 soybean varieties from 8 locations representing the major U.S. commercial growing areas; (b) sources of variation estimates from variance component analysis (VCA). Location is the environmental effect from eight growing locations; variety is the variety effect soybean from 24 soybean varieties; block is the block effect within each location; location × variety is the location by variety interaction effect, and the residual is the residual error from uncontrolled systematically variation.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Luis Burzio, George Harrigan, and John Vicini for excellent technical review. We also thank Emily Barr, Holly Chapman, and Cunxi Wang for technical assistance.

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(Table 1).33 Additionally, extraction efficiency and dilutional parallelism experiments demonstrated the high level of accuracy that the ELISA method has in quantifying Gly m 4 (Figure 4) in soybean seed extracts. Taken together, the Gly m 4 ELISA described is shown to have the specificity, sensitivity, accuracy, and precision required for use in the assessment of this soybean allergen in samples from both GM and conventional soybean samples. The purpose of the endogenous allergen assessment for GM soybean varieties is to compare the endogenous allergen levels in the GM variety to those in non-GM varieties. To evaluate this comparison of endogenous allergen levels, an understanding of the variability in allergen levels safely consumed by nonallergic individuals must be established. For this reason, the developed ELISA method was used to measure Gly m 4 in 24 conventional soybean varieties planted in different locations. The result showed that Gly m 4 levels could vary up to 13-fold (from 24 to 311 ppm) for soybean seed samples from different varieties and grown in different locations. This high level of natural variability of Gly m 4 is similar to those previously reported by Mittag et al. using a quantitative Western blot method, which showed Gly m 4 level ranged from 8 to 224 ppm.17 Similar observations were obtained for other soybean allergens. By using SDS-PAGE, Martinez-Villaluenga et al. found β-conglycinin and glycinin of 15 soybean genotypes ranged from 0.0 to 35.7% and from 23.1 to 50.8% of total protein, respectively.35 By using ELISA, Hei et al. observed that F

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