ACS Publications. Most Trusted. Most Cited. Most Read
My Activity
CONTENT TYPES

Figure 1Loading Img

Simplified Tracking of a Soy Allergen in Processed Food Using a Monoclonal Antibody-Based Sandwich ELISA Targeting the Soybean 2S Albumin Gly m 8

  • Elke Ueberham*
    Elke Ueberham
    Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
    *Phone: +49 341 355 36 1290. E-mail: [email protected]
  • Holger Spiegel
    Holger Spiegel
    Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
  • Heide Havenith
    Heide Havenith
    Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
  • Paul Rautenberger
    Paul Rautenberger
    Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
  • Norbert Lidzba
    Norbert Lidzba
    Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
  • Stefan Schillberg
    Stefan Schillberg
    Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
  • , and 
  • Jörg Lehmann
    Jörg Lehmann
    Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
Cite this: J. Agric. Food Chem. 2019, 67, 31, 8660–8667
Publication Date (Web):July 12, 2019
https://doi.org/10.1021/acs.jafc.9b02717

Copyright © 2019 American Chemical Society. This publication is licensed under CC-BY-NC-ND.

  • Open Access

Article Views

4135

Altmetric

-

Citations

LEARN ABOUT THESE METRICS
PDF (1 MB)
Supporting Info (4)»

Abstract

Soybean allergens in food samples are currently detected in most cases using enzyme-linked immunosorbent assays (ELISAs) based on antibodies raised against bulk soybean proteins or specifically targeting soybean trypsin inhibitor, conglycinin, or glycinin. The various commercial ELISAs lack standardized reference material, and the results are often inaccurate because the antibodies cross-react with proteins from other legumes. Furthermore, the isolation of allergenic proteins involves laborious denaturing extraction conditions. To tackle these challenges, we have developed a novel sandwich ELISA based on monoclonal antibodies raised against the soybean 2S albumin Gly m 8 and a recombinant Gly m 8 reference protein with native-analogous characteristics. The antibodies do not cross-react with other legume proteins, and the extraordinary stability and solubility of Gly m 8 allows it to be extracted even from complex matrices after processing. The Gly m 8 ELISA therefore achieves greater specificity and reproducibility than current ELISA tests.

Introduction

ARTICLE SECTIONS
Jump To

Soy allergy is among the eight most common forms of food allergy, and in severe cases it can trigger life-threatening anaphylaxis. The increasing use of soy flour and soy protein as food additives means that vigilance is increasingly necessary to exclude unintentional entering of soybean (Glycine max) allergens from the diet, and this requires accurate and sensitive test methods. Soy allergy can be divided in both mild forms related to food-pollen allergy syndrome mainly caused by the soy allergen Gly m 4 and substantial food allergy resulting from sensitization against the main storage proteins Gly m 5 and Gly m 6 and the minor storage protein Gly m 8. Recently, the estimation of sensitization level against a combination of Gly m 5 and Gly m 8 for diagnosing soy allergy in children was suggested, emphasizing the great significance of these two allergenic soy proteins. (1)
Gly m 4 is a PR10 protein, which represents a homologue of birch pollen allergen Bet-v1 and is a water-soluble molecule with weak resistance against heat, acids, and proteases. The physicochemical properties of Gly m 4 drive its loss during manufacturing processes of soy in processed food, for example, roasting, acid precipitation, or pasteurization. Therefore, processed food can scarcely be characterized by Gly m 4 content. Other allergenic proteins of soy include Gly m 1 and Gly m 2, which are hull proteins responsible for severe allergic Barcelona asthma outbreaks. (2) Peeling of soybeans, however, removes hull proteins in modern food processing. Therefore, they play a minor role in allergen detection of processed food. Other allergenic soy proteins are Gly m 7 (3) and Gly m Bd28K, (4) Gly m Bd30K, (5) and Gly m Bd39K. (6)
Currently, the detection of soy protein in food is realized in most cases using enzyme-linked immunosorbent assays (ELISAs) commonly based on polyclonal antibodies raised against whole soybean protein extracts or isolated components that are particularly stable. (7) Over decades, intensive effort has been made to detect allergenic soy proteins in food. An impressive number of ELISAs for these components have been published, (8−15) but all currently commercially available soy ELISA kits use antibodies that detect one of three soybean proteins: trypsin inhibitor (Gly m TI) or the abundant storage proteins conglycinin (Gly m 5) and glycinin (Gly m 6). (11) However, often the antibodies are not particularly specific, and false positive results might occur. For example, the Gly m TI antibody is unable to distinguish trypsin inhibitors from soybeans and other bean species, and the Gly m 6 antibody cross reacts with pea (Pisum sativum) storage proteins. (12) Furthermore, the extraction of these allergens requires harsh denaturing conditions, which can precipitate the proteins and prevent their detection, resulting in false negative results. Reliable extraction methods are very laborious. That applies to extraction procedures for detection of allergens by mass spectrometry too. Furthermore, mass spectrometry approaches are currently less established compared to allergen detection by ELISA (16) or polymerase chain reaction (PCR). (17) Since PCR targets DNA of the allergen source and not the allergenic protein by itself, PCR based methods are not relevant to assessing highly processed protein isolates and concentrates containing hardly detectable DNA.
Referring to a sophisticated extraction procedure prepared for food proteins, a more suitable target allergen is required for reliable detection by ELISA
Gly m 8 is a soybean 2S albumin. This protein has not been used before in the context of allergen detection, but it is the best known predictor of severe soy allergy in children. (18) The three-dimensional structure of 2S albumins is considered highly allergenic (19,20) together with the thermal stability of this protein family and its resistance to complete digestion. (21) Gly m 8 is also highly soluble in water and low-salt buffers, thus it is easier to extract even from complex food matrices and processed food samples compared to Gly m 5 and Gly m 6, making it particularly suitable for ELISA-based tests. For example, we recently established a sandwich ELISA based on antibodies against Gly m 5, (22) but the quantities of native and denatured Gly m 5 extracted from complex matrices and processed food using different extraction methods varied significantly, often underestimating the true Gly m 5 content.
Here, we set out to develop an ELISA based on antibodies specific for Gly m 8 and to confirm the ability of the assay to detect traces of soy proteins in food. We used recombinant Gly m 8 protein as a calibration standard and monoclonal antibodies to ensure the reproducibility of the assay. Detailed characterization of the activity and binding parameters of the antibodies using surface plasmon resonance (SPR) spectroscopy provided effective quality control of the test components. To the best of our knowledge, this is the first time that Gly m 8, which represents a soy storage molecule with a complex maturation cycle, (23) has been used as a target for the ELISA-based detection of soy protein in food products.

Materials and Methods

ARTICLE SECTIONS
Jump To

Recombinant Gly m 8 Protein

A synthetic gene coding for the Gly m 8 precursor (UniProt ID P19594) amino acids M1 to D158, including the signal peptide, the pro-peptide, and a 3′-terminal His6-tag, was codon optimized for Nicotiana benthamiana by Geneart (Invitrogen, Carlsbad, CA, USA). The synthetic gene was introduced into the binary plant expression vector pTRAkt-ER (24) at the NcoI/BamHI sites. The final construct pTRAkt_Gly m 8 was verified by sequencing. The pTRAkt_Gly m 8 vector was propagated in Escherichia coli DH5α cells (New England Biolabs, Frankfurt/Main, Germany). Plasmid DNA was purified and introduced into electrocompetent Agrobacterium tumefaciens cells for transient expression in N. benthamiana plants as previously described. (25)

Purification of Recombinant Gly m 8

The Gly m 8 protein was extracted from leaf tissue and isolated by immobilized metal ion affinity chromatography (IMAC) as previously described. (25) The Gly m 8 protein was purified by size exclusion chromatography (SEC) using a Superdex75 16/60 column (GE Healthcare, Freiburg, Germany). The integrity and purity of the recombinant Gly m 8 protein was verified by sodiumdodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) and liquid chromatography/mass spectrometry (LC/MS-MS).

Generation of Monoclonal Antibodies

Mouse anti-Gly m 8 monoclonal antibodies were generated by immunizing female BALB/c mice (Janvier Laboratories, Le Genest-Saint-Isl, France) with the recombinant Gly m 8 protein described above. The immunization experiments were approved by the State Animal Care and Use Committee (Landesdirektion Sachsen, Leipzig, Germany, V 07/14) and were carried out in accordance with the European Communities Council Directive (86/609/EEC) for the Care and Use of Laboratory Animals. Splenocytes were isolated from the mouse with the highest antibody titer specific for Gly m 8 and were fused to X63.Ag8.653 myeloma cells (ACC 43, DSMZ, Braunschweig, Germany). Hybridoma supernatants were screened by indirect ELISA on flat-bottom high protein-binding capacity 96-well ELISA plates (Nunc MaxiSorp, Thermo Fisher Scientific, Darmstadt, Germany) coated with either recombinant protein (2 μg/mL) or whole soy extract (10 μg/mL). Cross-reactivity to total protein extracts from other legumes, namely peas, lupin (Lupinus albus), peanuts (Arachis hypogaea), and different beans and nuts, as indicated in Figure 2B, was tested by indirect ELISA using 10 μg/mL seed protein extracts.

SPR Spectroscopy

Eleven IgG-positive clones were selected for SPR analysis on covalently coupled purified recombinant Gly m 8 protein using a Biacore T200 SPR biosensor instrument (GE Healthcare) as previously described, based on an Fc-specific antibody capture system. (26) The most promising Gly m 8-specific monoclonal antibodies anti-Gly m 8-3 (mAb3) and anti-Gly m 8-8 (mAb8) were used for calibration and further testing as described below.

Calibration-Free Concentration Analysis (CFCA)

CFCA (27) was used to determine the active concentration of recombinant Gly m 8 using a Biacore T200 instrument and a CM5-S-Series sensor chip with recombinant Protein A prepared as previously described. (28) The measurements were performed at 25 °C in HBS-EP running buffer (10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 150 mM NaCl, 3 mM EDTA, 0.005% (w/v) Tween 20). The surface was regenerated by pulsing with 30 mM HCl for 1 min.
To ensure a rapid initial binding rate, 2500 response units (RU) of mAb3 were captured in each assay step. Purified recombinant Gly m 8 was used at three different dilutions (1/3000, 1/4500, and 1/6000) to ensure an initial binding rate (IBR) between 0.5 and 5 RU/s at a flow rate of 5 μL/min. The IBR was measured at 5 and 100 μL/min using double referencing. The antigen-specific antibody concentration was determined using the CFCA module of the Biacore T200 Evaluation Software (GE Healthcare). The binding model was based on a molecular weight of 16 000 kDa and a diffusion coefficient of 9.16 × 10–11 m2/s.

Kinetic Analysis

The kinetic properties of mAb3 and mAb8 were determined using the Biacore T200 instrument. We captured 500 RU of mAb8 on an a CM5 chip prepared with a mouse antibody capture kit (GE Healthcare), whereas mAb3 was captured on a Protein A surface prepared as described elsewhere. (28) To determine the kinetic binding constants, purified recombinant Gly m 8 was injected at a flow rate of 30 μL/min for 150 s (mAb8) or 180 s (mAb3), followed by dissociation for 900 s (mAb8) or 400 s (mAb3). Gly m 8 was used at CFCA-based concentrations of 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM. Between measurements, the surface was regenerated by pulsing for 1 min with 10 mM glycine/HCl. Buffer injections were used for double referencing. Binding curves were evaluated based on a 1:1 binding model using the Biacore T200 Evaluation Software.
To confirm the simultaneous binding of mAb3 and mAb8, mAb3 was captured on a Protein A-functionalized surface and saturated with recombinant Gly m 8. Then, mAb8 was injected to confirm the binding of mAb8 to Gly m 8 captured by mAb3.

ELISA

Gly m 8 was quantified by sandwich ELISA using mAb3 and mAb8. The capture antibody (mAb3) was immobilized onto 96-well plates (Nunc MaxiSorp) in 0.5 M carbonate buffer at 4 °C overnight. The plates were washed three times with phosphate-buffered saline (PBS) containing 154 mM NaCl and 0.05% Tween-20 (PBS-T) and blocked with Superblock blocking reagent (Thermo Fisher Scientific) for 1 h at room temperature. The plates were then sealed (Candor BioScience, Wangen, Germany), air-dried, shrink-wrapped, and stored at room temperature.
Both extracted samples and recombinant Gly m 8 standards were incubated for 10 min at room temperature in duplicate in PBS-T, Superblock mixture (Thermofisher, Massachusetts). After three washes in PBS-T, the horseradish peroxidase (HRP)-conjugated detection antibody (mAb8) was added, and the plates were incubated for 10 min at room temperature. HRP activity was determined after three further washes in PBS-T by incubating the plate with 3,3′,5,5′-tetramethylbenzidine (TMB-E) substrate (DUNN Labortechnik, Asbach, Germany). The yellow color generated by acidification with 0.5 M sulfuric acid represented the quantity of bound detection antibodies and was measured at 450 nm relative to a calibration curve consisting of eight known concentrations of pure Gly m 8. The ELISA was verified according to AOAC guidelines, Appendix M, (29) and DIN ISO11843-5.
The limit of detection (LOD) was determined by measuring eight different concentrations of purified recombinant Gly m 8 in extraction buffer. Recovery was calculated by spiking five different concentrations of recombinant Gly m 8 into three different matrices relevant for processed soy: almond-wheat muffin, rice cookie, and minced boiled sausage. The LOD and limit of quantification (LOQ) were calculated by checking sensitivity and specificity using the methodology for linear and nonlinear calibration (ISO 11843-5:2008). The ability of the anti-Gly m 8 sandwich ELISA to resist changes in results due to minor deviations in the experimental procedure was tested by deviations in time (two times ± 10% of recommended time of 10 min), volume (two volumes ±10% of set volume 100 μL), and temperature (ambient temperature, 20, 28, and 37 °C). Furthermore, two different individuals performed the test on three different days.
The specificity of the antibodies was tested by spiking the ELISA with recombinant Gly m 4, (30) Gly m TI (Fraunhofer IME), and native Gly m 6, Gly m 5, and Kunitz inhibitor (Kunitz, Sigma-Aldrich, Deisenhofen, Germany). The selectivity of the antibodies (the extent to which they can bind the antigen in complex mixtures without interference) was tested using the three different matrices described above. Samples (3.3 mg/mL, Supporting Information Table 1) were extracted by homogenizing and mixing for 30 min in 10 mM Tris at pH 9.0 and 0.5% sarcosyl at room temperature.

Purification of Native Gly m 8 Antigen by Immunoprecipitation

Extracts of hexane-defatted soy flakes (Fraunhofer IVV; prepared as previously described) (31) were preincubated with 0.5 mL of Protein G Sepharose 4 Fast Flow (90-μm particle size, GE Healthcare) for 1 h at room temperature. Afterward, the Protein G Sepharose was removed by filtration through a 30-μm polyethylene filter (Thermo Fisher Scientific). Preadsorbed protein extract was incubated with the capture antibody (mAb3) for 1 h at room temperature on a Stuart Tube Rotator SB3 (Cole-Parmer, Wertheim, Germany) before adding 1 mL of Protein G Sepharose 4 Fast Flow as above and mixing for another 1 h at room temperature. The mixture was filtered as above, forming a column matrix by gravity flow. This column was washed 10 times with 10 mL of PBS and eluted with 0.5 mL of 0.1 M glycine-HCl (pH 3.6). The eluate was neutralized with 50 μL of 1 M Tris (pH 9.0), and the proteins were separated by polyacrylamide gel electrophoresis using 16% (w/v) tricine gels. (32) The bands were analyzed by LC-MS/MS as previously described. (33)

Results

ARTICLE SECTIONS
Jump To

Plant Expression of Recombinant Protein

Recombinant Gly m 8 was produced in N. benthamiana by Agrobacterium-mediated transient expression. The native Gly m 8 sequence, including the N-terminal signal peptide, prepropeptide, and a C-terminal His6 tag, was codon-optimized for expression in N. benthamiana and transferred to an expression cassette in the binary plant expression vector pTRAkt-ER (Figure 1A). After proteolytic cleavage of the signal peptide and prepropeptide, the mature Gly m 8 protein consisted of two subunits joined by a disulfide bridge (Figure1B). Transient expression of Gly m 8 and subsequent purification by IMAC and SEC yielded a highly pure recombinant protein (Figure 1C). During expression in N. benthamiana, Gly m 8 underwent a complete maturation cycle as demonstrated by the presence of two bands representing the processed subunits on reducing gels and one band representing the 14 kDa complex of the two covalently linked subunits on nonreducing gels (Figure 1D). The molecular weight of the protein bands determined in the SDS-PAGE appears higher compared to the theoretical molecular weight. This is the case for both the recombinant protein (Figure 1) and the native protein represented by immunoprecipitation (Supporting Information Figure 1). Deviations between the calculated and the apparent molecular weight determined by SDS-PAGE is a common observation, since many different factors, besides incomplete processing or unexpected post-translational modifications, influence the running behavior of a protein in SDS-PAGE. The buffer system, the SDS concentration, and the pH in combination with the specific protein sequence may lead to such deviations. (34) Since the experimental data suggest successful processing of the pre-propeptide as well as the formation of disulfide bridges, the MW deviations are most likely such artifacts. Similar deviations have been described for various proteins, for example, for PyMsp1–19, a Plasmodium yoelii surface protein, which runs between 17 and 19 kDa in SDS-PAGE, while the calculated as well as MS-derived MW is around 12 kDa (35)

Figure 1

Figure 1. Plant expression construct and purity and integrity of recombinant Gly m 8. (A) Schematic presentation (not to scale) of the expression cassette Gly m 8. SAR, scaffold attachment region; CaMV 35S promoter and terminator, promoter with duplicated enhancer and terminator of the Cauliflower mosaic virus (CaMV) 35S gene; 5′ untranslated region, 5′-UTR of the chalcone synthase gene from Petroselinum crispum (CHS 5′ UTR); Gly m 8, coding sequence for Gly m 8, UniProt ID 19594; His6 tag, six histidine residues (affinity purification tag). (B) Schematic presentation (not to scale) of the Gly m 8 protein, including signal peptide (SP), propeptide (PP), and disulfide bond. (C) Analysis of purification of expressed recombinant Gly m 8 by SDS-PAGE under reducing conditions. Lane 1 = molecular weight marker. Crude filtered extracts of N. benthamiana leaves (lane 2) were loaded onto IMAC columns, and both the flow-through and wash-out samples were collected (lanes 3 and 4, respectively). In the eluate (lane 5), a protein band with the expected size of ∼12 kDa, respresenting the large subunit of Gly m 8 under reducing conditions, was detected. The small unit, with a molecular weight of ∼5 kDa, ran within the running front of the gel but was separately displayed in D. (D) SDS-PAGE analysis of SEC-polished Gly m 8 under nonreducing (lane 2) and reducing (lane 3) conditions. A 99% pure recombinant Gly m 8 protein was purified by SEC, which separates under reducing conditions into two subunits.

Monoclonal Antibodies

Immunization of mice with recombinant Gly m 8 protein led to the recovery of antibodies that bound with high affinity to both the native and recombinant protein. Indirect ELISA (Figure 2) captured the native protein from completely aqueous soy extracts. Clones with signal-to-noise ratios of OD 450 nm ≥ 10 corresponding to an OD of >0.1 were considered to be positive. Eleven antibodies showing no cross-reactivity against protein extracts isolated from legumes peas, peanuts, and lupins as well as various beans and nuts (Figure 2B) were preselected to develop a sandwich ELISA. Ranking the antibodies according to their binding affinity and stability on recombinant Gly m 8 covalently conjugated to the surface of Biacore CM 5 chips and proofing their compatibility with respect to pair production in a sandwich led to the selection of mAb3 and mAb8 (Figure 3). The kinetic analysis of these antibodies (Figure 4) revealed Kd values in the subnanomolar range: 3.92 × 10–10 for mAb3 and 1.57 × 10–10 for mAb8 (Figure 4 and Supporting Information Table 2). Furthermore, the captured mAb3 was characterized by the immunoprecipitation of native soy extract with Protein G Sepharose, because mAb3 does not bind to denatured protein under western-blot conditions. Analysis of the precipitate by polyacrylamide gel electrophoresis revealed two major protein species with molecular weights of 25 kDa and 15 kDa under nonreducing conditions (Supporting Information Figure 1). LC-MS/MS analysis confirmed the 25-kDa protein was the mouse kappa light chain from mAb3 (score 331, UniProt P01837), and the 15-kDa protein was Gly m 8 (score 896.03, UniProt ID P19594). Under reducing conditions, the 15-kDa band was converted into two bands of ∼11 kDa and 5 kDa (Supporting Information Figure 1) representing the linkage of the two subunits by disulfide bonds. The LC-MS/MS data confirmed the specific binding of mAb3 to Gly m 8. In addition, no cross-reactivity was observed for mAb3 and mAb8 either by indirect (screening) ELISA (Figure 2B) or by sandwich ELISA when tested against extracts derived from Triticum aestivum, Apium graveolens, Brassica nigra, Brassica juncea, Sinapis alba, Vigna angulariz, Vigna mungo, Phaseolus vulgaris, Phaseolus vulgaris Pinto Group, lupin beans, peanuts, peas, and field beans (Vicia faba).

Figure 2

Figure 2. Screening of antibody-producing hybridoma clones by indirect ELISA using plates coated with soy extract (native) or recombinant Gly m 8 and extracts of legumes and nuts (A). Supernatants of hybridoma cultures were tested for the presence of Gly m 8-specific IgG antibodies which bound to both native soy extracts (filled circle) and recombinant Gly m 8 (triangle) using an indirect ELISA. Binding of antibodies to the Gly m 8 antigen resulted in a high OD450nm signal as shown in the scatter plot of 2000 hybridoma clones. Read-outs higher than 0.1 OD identified high-affinity anti-Gly m 8 antibodies. Clones producing high-affinity antibodies were cryopreserved, and antibody-containing supernatants were collected for further analysis. The arrows (solid line mAb3 and dotted line mAb8) are tag specific signals for the clones finally used in ELISA. (B) Supernatants of selected hybridoma cultures (mAb1 to mAb11) which were tested for the presence of Gly m 8-specific IgG antibodies which bound to both native soy extracts using an indirect ELISA (Figure 2) were rescreened on both native soy extract (filled circle) and legume and nut extracts as indicated. OD values above 0.1 were assessed as positive according to signal-to-noise ratios above 10 in the appropriate ELISA.

Figure 3

Figure 3. Ranking of anti-Gly m 8 antibodies. Binding and stability of selected anti-Gly m 8 antibodies (mAb1 to mAb11) tested using recombinant Gly m 8 conjugated onto the surface of a CM5 chip with the SPR biosensor instrument Biacore T200. Response units (RU) indicate specific binding of the antibody to the recombinant Gly m 8 covalently coupled to the chip at the late association phase (binding) and late dissociation phase (stability). The plot shows these response units from the late association phase (binding) and late dissociation phase (stability) of 11 selected antibodies on a Gly m 8 surface in order to choose appropriate capture antibodies. The binding and stability are related to both the association and dissociation rates of the interaction. The red encircled antibodies were used in the sandwich ELISA as the capture (mAb3) and detection (mAb8) antibodies.

Figure 4

Figure 4. Representative SPR sensorgrams for the kinetic analysis of the Gly m 8-specific mAb8 and simultaneous binding of mAb3 and mAb8 to recombinant Gly m 8. (A) The affinity of mAb8 for recombinant Gly m 8 was determined by SPR spectroscopy. For each cycle, purified mAb8 was captured onto a Protein G-coated surface (500 response units (RUs). Subsequently, recombinant Gly m 8 was injected at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, or 0.15625 nM for 150 s to determine the on-rate (ka)) and dissociation was observed for 900 s to determine the off-rate dissociation (kd). The kD values were estimated by fitting the data to interaction models using the Biacore T200 evaluation software, applying the 1:1 Langmuir fit model. (B) Because mAb8 is an IgG isotype IgG1, it binds only weakly to Protein A, whereas mAb3 (IgG2A) can be efficiently captured on a Protein A functionalized CM5 sensor surface. Therefore, it was possible to illustrate the compatibility of the two antibodies with a sandwich ELISA format in the context of an SPR experiment. The figure shows the subsequent injection of mAb3 (captured onto a Protein A surface), followed by recombinant Gly m 8 and finally mAb8. The comparable response unit (RU) levels for the two antibodies (1500–1700 RU) indicate that each molecule of recombinant Gly m 8 can be simultaneously recognized by both antibodies, confirming the suitability of the antibody combination for the development of a sandwich ELISA for the quantification of Gly m 8.

Sensitivity, Specificity, and Robustness of ELISA

The new Gly m 8 sandwich ELISA achieved a LOD > 10 pg/mL Gly m 8 (determined from the average of 10 matrix blanks plus three standard deviations) and a LOQ of 65 pg/mL Gly m 8 (determined as the lowest concentration of spiked Gly m 8 in three different matrices or buffer that is still reliably detectable). As described in the Materials and Methods section, a regression curve fitted by a four-parameter logistic model was used as the nonlinear equation for the estimation of the lower quantification limit. The LOQ represents the lowest Gly m 8 concentration in picograms per milliliter, which is measurable with a coefficient of variance below 20% (Figure 5B). The interassay variance (robustness) was determined by analyzing the same samples on three different days by two different operators (Figure 5C). The precision of the assay was confirmed by processing 10 technical replicates of three different samples (Figure 5B). When spiking recombinant Gly m 8 into three different matrices, we achieved recovery rates of 98–109% (Table 1). Gly m 8 was detected in all three of the food matrices we tested. In complex processed food matrices, Gly m 8 is easily detectable by the sandwich antibodies below 1 ppm in soy protein and soy milk and below 10 ppm in tofu and texturized vegetable protein, which is often a challenge to detect. In roasted soy material, it is more poorly detectable, but this is rather due to limitations of whole protein isolation than a specific matter of Gly m 8 isolation, e.g., the higher the roasting degree, the lower the protein content of the extract (Supporting Table 3).

Figure 5

Figure 5. Calibration curve, precision profile, and robustness testing of the Gly m 8 ELISA. (A) Representative calibration curves of the Gly m 8 sandwich ELISA are depicted in gray with the regression curve fitted by a four-parameter logistic model in red (A). LOD and LOQ as functions of the analytical specificity of Gly m 8 ELISA were determined by the linear and nonlinear calibration methods on the basis of calibration curve (ISO 11843–5:2008). The blue curves represent the reaction of antibodies with potential interfering proteins naturally present in whole soy extracts, namely, recombinant proteins produced in N. benthamiana Gly m 4 and Gly m TI and commercially purified native proteins Gly m 5, Gly m 6, and Kunitz (Sigma-Aldrich). Two different operators performed the ELISA on three different days. (B) Precision profile shows the repeatability calculated by coefficients of intra-assay variance (gray lines) and intermediate precision calculated by coefficients of inter-assay variance (red line). (C) Representative calibration curves of the sandwich ELISA obtained by measuring recombinant Gly m 8 at three different temperatures (20, 28, and 37 °C; gray curves, circles), using two different incubation volumes (±10%; gray curves, triangles), and using an incubation time variation (±10%, gray curves, rectangles). The right axis of the ordinate presents the corresponding absorbance values (OD450nm). The corresponding precision profiles are depicted in the same coordinate system related to the left axis of the ordinate.

Table 1. Recovery of Recombinant Gly m 8 at Five Different Concentration Levels in Three Different Matricesa
spiked Gly m 8 [pg/mL]recovery in almond muffin matrix in pg/mL and [%]recovery in minced boiled sausage matrix pg/mL and [%]recovery in rice cookie matrix pg/mL and [%]recovery in extraction buffer pg/mL and [%]
50005206 ± 228 [104 ± 4.5]5409 ± 230 [108 ± 4.6]5507 ± 612 [110 ± 12.2]5077 ± 296 [101 ± 5.9]
25002546 ± 115 [101 ± 4.6]2620 ± 167 [104 ± 6.7]2749 ± 298 [109 ± 11.9]2621 ± 236 [109 ± 15.0]
500497 ± 39 [99 ± 7.8]540 ± 30 [108 ± 5.9]534 ± 67 [106 ± 13.4]545 ± 75 [109 ± 15.1]
10095 ± 15 [95 ± 14.7]99 ± 9 [99 ± 9.0]98 ± 18 [98 ± 18.4]103 ± 16 [103 ± 16.1]
6563 ± 7 [97 ± 10.3]68 ± 7 [105 ± 10.9]64 ± 13 [99 ± 20.4]70 ± 16 [107 ± 23.2]
a

Matrices produced by extraction of indicated processed food were spiked with recombinant Gly m 8 protein, and the recovery rates (percent of the spiked amount) were measured by Gly m 8 sandwich ELISA.

Discussion

ARTICLE SECTIONS
Jump To

The detection of allergens in food products by ELISA depends on efficient protein isolation during the preparation of samples from complex food matrices. (36) The limited solubility of globulins in legume extracts, which is often a desired techno-functional characteristic of protein isolates, concentrates, and extruded material, (37−40) reduces the reliability of ELISA results, as previously shown for the two main soybean storage proteins Gly m 5 and Gly m 6. (14) Soluble proteins such as albumins are therefore preferable targets because they are protease resistant and thermostable, and they retain their native protein structure. In addition, there is less cross-reactivity among the albumins of different legume species, whereas the more strongly conserved globulins such as Gly m 6 lead to false positive results. (12) Phylogenetic analysis of the legume 2S albumins (21) suggests that only lupin δ-conglutin and peanut Ara h6 should cross-react with Gly m 8, but the monoclonal antibodies we selected (mAb3 and mAb8) showed no cross-reactivity to extracts of lupin and peanut. Compared to the storage protein glycinin (Gly m 6), the other main storage protein, the 7S globulin Gly m 5, does not share sufficient sequence identity to its equivalents in other legumes to cause cross-reaction, but the detection of Gly m 5 requires labor-intensive heat extraction, which makes the assay more cumbersome. (41,42)
A key advantage of the Gly m 8 sandwich ELISA presented herein is the high affinity of the antibodies mAb3 and mAb8, which therefore bind the Gly m 8 antigen at very low concentrations. Detailed characterization by SPR analysis allowed us to perform stringent quality control of both antibodies and the recombinant Gly m 8 reference protein. Further Gly m 8-specific antibodies are available that could be used together with mAb3 and mAb8 to detect other epitopes either individually or together, thus further increasing the sensitivity of the assay.
The utilization of Gly m 8 as an antigen for the detection and quantification of soy allergens in food has a second important advantage because this protein is currently the best predictor of severe allergic reactions in children. (18,1) However, the allergenicity of Gly m 8 has been assessed using different methods in different studies. (18,43,44) Ebisawa and colleagues tested native Gly m 8 coupled to an immunocap device, (18) whereas the other study used a recombinant Gly m 8 protein produced in E. coli (43) or microarrays with overlapping peptides representing solely linear epitopes, thus not reflecting the three-dimensional structure of the protein. (44) The Gly m 8 ELISA is also advantageous because it provides information about the allergen content of processed soy proteins. The commercial ELISA kits are reliable if the samples of processed food achieve recovery rates of 50–150%. (29) The new Gly m 8 ELISA would therefore be particularly suitable for the detection of soy ingredients in chocolate, which often contains texturized vegetable protein and native soy protein. Moreover, we were able to detect soy in highly processed food such as roasted soy beans and minced boiled sausages with the newly developed Gly m 8 ELISA, even though there are some limitations regarding the quantification. Minced boiled sausage material of proficiency testing 2017 (LVU, Germany) clearly tested positive, though quantification was not exactly possible because the value was below the LQL of the ELISA (Supporting Information Table 3). Furthermore, the available material was difficult to quantify, and only 9 out of 34 participants released a quantitative statement with very high deviations according to the evaluation report (LVU, Germany).
Every 1 g of total soy protein contains 300–600 mg of Gly m 5 and Gly m 6 (45,46,8) and 60 mg Gly m TI (8) but only 1.1 mg of Gly m 8. Nevertheless, the new Gly m 8 ELISA was able to detect minimal amounts of soy protein in both rice cookies and minced boiled sausage using extraction conditions avoiding heating and denaturation, which produced negative results using antibodies to the other allergens. This indicates that the new Gly m 8 ELISA has an unprecedented sensitivity.
In summary, the Gly m 8 ELISA combines the advantages of monoclonal antibodies (which can be produced in unlimited quantities) and a robust, highly purified recombinant protein standard that can be used as reference material to ensure uniform and stable quality. The simple sample preparation method that is the effortless extraction method will also allow the antibodies to be used as an on-site test system compatible with food swabs.

Supporting Information

ARTICLE SECTIONS
Jump To

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02717.

  • Supporting Figure 1: Analysis of native Gly m 8 isolated from soy extracts by immunoprecipitation with mAb3 followed by PAGE (PDF)

  • Supporting Table 1: Soy-containing foods and food ingredients (PDF)

  • Supporting Table 2: Kinetic parameters derived from SPR-based interaction analysis (PDF)

  • Supporting Table 3: Amount of Gly m 8 measured in processed food (PDF)

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

ARTICLE SECTIONS
Jump To

  • Corresponding Authors
    • Elke Ueberham - Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, GermanyOrcidhttp://orcid.org/0000-0002-2121-0627 Email: [email protected]
    • Holger Spiegel - Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
  • Authors
    • Heide Havenith - Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
    • Paul Rautenberger - Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
    • Norbert Lidzba - Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
    • Stefan Schillberg - Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstrasse 6, 52074 Aachen, Germany
    • Jörg Lehmann - Fraunhofer Institute for Cell Therapy and Immunology IZI, Perlickstraße 1, 04103 Leipzig, Germany
  • Funding

    This work was funded by the Fraunhofer Zukunftsstiftung as part of the joint research project FoodAllergen 883126.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

ARTICLE SECTIONS
Jump To

The authors would like to thank Mrs. Ulrike Scholz and Mr. Leander Zitzmann (Fraunhofer IZI, Leipzig, Germany) for their help with the production of monoclonal antibodies and the development of the sandwich ELISA. We are grateful to Pia Meinlschmidt and Isabel Murany (Fraunhofer IVV, Freising, Germany) for providing soy flakes, soy flour, and protein isolates and Martin Röder (Institut für Produktqualität, Berlin, Germany) for providing the model cake and chocolate. We thank Andreas Pich, MHH Institute of Toxicology, Core Unit Proteomics (Hannover, Germany) for the LC-MS/Ms analysis. We thank Richard M. Twyman for manuscript editing.

Abbreviations Used

ARTICLE SECTIONS
Jump To

ELISA

enzyme-linked immunosorbent assay

CFCA

calibration-free concentration analysis

IBR

initial binding rate

IMAC

immobilized metal ion affinity chromatography

LOD

limit of detection

LOQ

limit of quantification

mAb

monoclonal antibody

RU

response unit

SEC

size exclusion chromatography

SDS-PAGE

sodium dodecyl sulfate polyacrylamide electrophoresis

LC/MS-MS

liquid chromatography coupled with tandem mass spectrometry

PBS

phosphate-buffered saline

TMB-E

3,3′,5,5′-tetramethylbenzidine ELISA substrate

References

ARTICLE SECTIONS
Jump To

This article references 46 other publications.

  1. 1
    Maruyama, N.; Sato, S.; Cabanos, C.; Tanaka, A.; Ito, K.; Ebisawa, M. Gly m 5/Gly m 8 fusion component as a potential novel candidate molecule for diagnosing soya bean allergy in Japanese children, Clinical and experimental allergy. Clin. Exp. Allergy 2018, 48, 17261734,  DOI: 10.1111/cea.13231
  2. 2
    Maggio, P.; Monso, E.; Baltasar, M.; Morera, J. Occupational asthma caused by soybean hull. A workplace equivalent to epidemic asthma. Allergy 2003, 58, 350351,  DOI: 10.1034/j.1398-9995.2003.00089.x
  3. 3
    Riascos, J. J.; Weissinger, S. M.; Weissinger, A. K.; Kulis, M.; Burks, A. W.; Pons, L. The Seed Biotinylated Protein of Soybean (Glycine max). A Boiling-Resistant New Allergen (Gly m 7) with the Capacity To Induce IgE-Mediated Allergic Responses. J. Agric. Food Chem. 2016, 64, 38903900,  DOI: 10.1021/acs.jafc.5b05873
  4. 4
    Tsuji, H.; Bando, N.; Hiemori, M.; Yamanishi, R.; Kimoto, M.; Nishikawa, K.; Ogawa, T. Purification of characterization of soybean allergen Gly m Bd 28K. Biosci., Biotechnol., Biochem. 1997, 61, 942947,  DOI: 10.1271/bbb.61.942
  5. 5
    Ogawa, T.; Tsuji, H.; Bando, N.; Kitamura, K.; Zhu, Y.-L.; Hirano, H.; Nishikawa, K. Identification of the Soybean Allergenic Protein, Gly m Bd 30K, with the Soybean Seed 34-kDa Oil-body-associated Protein. Biosci., Biotechnol., Biochem. 1993, 57, 10301033,  DOI: 10.1271/bbb.57.1030
  6. 6
    Xiang, P.; Baird, L. M.; Jung, R.; Zeece, M. G.; Markwell, J.; Sarath, G. P39, a novel soybean protein allergen, belongs to a plant-specific protein family and is present in protein storage vacuoles. J. Agric. Food Chem. 2008, 56, 22662272,  DOI: 10.1021/jf073292x
  7. 7
    Tukur, H. M.; Lallès, J.-P.; Plumb, G. W.; Mills, E. N. C.; Morgan, M. R. A.; Toullec, R. Investigation of the Relationship between in Vitro ELISA Measures of Immunoreactive Soy Globulins and in Vivo Effects of Soy Products. J. Agric. Food Chem. 1996, 44, 21552161,  DOI: 10.1021/jf950141y
  8. 8
    Brandon, D. L.; Friedman, M. Immunoassays of Soy Proteins. J. Agric. Food Chem. 2002, 50, 66356642,  DOI: 10.1021/jf020186g
  9. 9
    Geng, T.; Stojšin, D.; Liu, K.; Schaalje, B.; Postin, C.; Ward, J.; Wang, Y.; Liu, Z. L.; Li, B.; Glenn, K. Natural Variability of Allergen Levels in Conventional Soybeans. Assessing Variation across North and South America from Five Production Years. J. Agric. Food Chem. 2017, 65, 463472,  DOI: 10.1021/acs.jafc.6b04542
  10. 10
    Liu, B.; Teng, D.; Wang, X.; Wang, J. Detection of the soybean allergenic protein Gly m Bd 28K by an indirect enzyme-linked immunosorbent assay. J. Agric. Food Chem. 2013, 61, 822828,  DOI: 10.1021/jf303076w
  11. 11
    Pedersen, M. H.; Holzhauser, T.; Bisson, C.; Conti, A.; Jensen, L. B.; Skov, P. S.; Bindslev-Jensen, C.; Brinch, D. S.; Poulsen, L. K. Soybean allergen detection methods--a comparison study. Mol. Nutr. Food Res. 2008, 52, 14861496,  DOI: 10.1002/mnfr.200700394
  12. 12
    Lacorn, M.; Dubois, T.; Siebeneicher, S.; Weiss, T. Accurate and Sensitive Quantification of Soy Proteins in Raw and Processed Food by Sandwich ELISA. Food Sci. Technol. 2016, 4, 6977,  DOI: 10.13189/fst.2016.040404
  13. 13
    Morishita, N.; Kamiya, K.; Matsumoto, T.; Sakai, S.; Teshima, R.; Urisu, A.; Moriyama, T.; Ogawa, T.; Akiyama, H.; Morimatsu, F. Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods. J. Agric. Food Chem. 2008, 56, 68186824,  DOI: 10.1021/jf8007629
  14. 14
    Scharf, A.; Kasel, U.; Wichmann, G.; Besler, M. Performance of ELISA and PCR methods for the determination of allergens in food. An evaluation of six years of proficiency testing for soy (Glycine max L.) and wheat gluten (Triticum aestivum L.). J. Agric. Food Chem. 2013, 61, 1026110272,  DOI: 10.1021/jf402619d
  15. 15
    Holzhauser, T.; Franke, A.; Treudler, R.; Schmiedeknecht, A.; Randow, S.; Becker, W.-M.; Lidholm, J.; Vieths, S.; Simon, J.-C. The BASALIT multicenter trial. Gly m 4 quantification for consistency control of challenge meal batches and toward Gly m 4 threshold data. Mol. Nutr. Food Res. 2017, 61, 1600527,  DOI: 10.1002/mnfr.201600527
  16. 16
    Nitride, C.; Lee, V.; Baricevic-Jones, I.; Adel-Patient, K.; Baumgartner, S.; Mills, E. N. C. Integrating Allergen Analysis Within a Risk Assessment Framework. Approaches to Development of Targeted Mass Spectrometry Methods for Allergen Detection and Quantification in the iFAAM Project. J. AOAC Int. 2018, 101, 8390,  DOI: 10.5740/jaoacint.17-0393
  17. 17
    Cucu, T.; Jacxsens, L.; De Meulenaer, B. de Analysis to support allergen risk management. Which way to go?. J. Agric. Food Chem. 2013, 61, 56245633,  DOI: 10.1021/jf303337z
  18. 18
    Ebisawa, M.; Brostedt, P.; Sjölander, S.; Sato, S.; Borres, M. P.; Ito, K. Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children. J. Allergy Clin. Immunol. 2013, 132, 976978.e5,  DOI: 10.1016/j.jaci.2013.04.028
  19. 19
    Offermann, L.; Perdue, M.; He, J.; Hurlburt, B.; Maleki, S.; Chruszcz, M. Structural Biology of Peanut Allergens; JCI, 2015.
  20. 20
    Han, Y.; Lin, J.; Bardina, L.; Grishina, G. A.; Lee, C.; Seo, W. H.; Sampson, H. A. What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2. Molecules 2016, 21, E622,  DOI: 10.3390/molecules21050622
  21. 21
    Moreno, F. J.; Clemente, A. 2S Albumin Storage Proteins. What Makes them Food Allergens?. Open Biochem. J. 2008, 2, 1628,  DOI: 10.2174/1874091X00802010016
  22. 22
    Meinlschmidt, P.; Ueberham, E.; Lehmann, J.; Schweiggert-Weisz, U.; Eisner, P. Immunoreactivity, sensory and physicochemical properties of fermented soy protein isolate. Food Chem. 2016, 205, 229238,  DOI: 10.1016/j.foodchem.2016.03.016
  23. 23
    Lin, J.; Fido, R.; Shewry, P.; Archer, D. B.; Alcocer, M. J. C. The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris. Biochim. Biophys. Acta, Proteins Proteomics 2004, 1698, 203212,  DOI: 10.1016/j.bbapap.2003.12.001
  24. 24
    Sack, M.; Paetz, A.; Kunert, R.; Bomble, M.; Hesse, F.; Stiegler, G.; Fischer, R.; Katinger, H.; Stoeger, E.; Rademacher, T. Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures. FASEB J. 2007, 21, 16551664,  DOI: 10.1096/fj.06-5863com
  25. 25
    Feller, T.; Thom, P.; Koch, N.; Spiegel, H.; Addai-Mensah, O.; Fischer, R.; Reimann, A.; Pradel, G.; Fendel, R.; Schillberg, S.; Scheuermayer, M.; Schinkel, H. Plant-based production of recombinant Plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate. PLoS One 2013, 8, e79920  DOI: 10.1371/journal.pone.0079920
  26. 26
    Schräml, M.; Biehl, M. Kinetic screening in the antibody development process. Methods Mol. Biol. (N. Y., NY, U. S.) 2012, 901, 171181,  DOI: 10.1007/978-1-61779-931-0_11
  27. 27
    Pol, E.; Roos, H.; Markey, F.; Elwinger, F.; Shaw, A.; Karlsson, R. Evaluation of calibration-free concentration analysis provided by Biacore systems. Anal. Biochem. 2016, 510, 8897,  DOI: 10.1016/j.ab.2016.07.009
  28. 28
    Boes, A.; Spiegel, H.; Delbrück, H.; Fischer, R.; Schillberg, S.; Sack, M. Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco. Biotechnol Bioeng. 2011, 108 (12), 28042814,  DOI: 10.1002/bit.23262
  29. 29
    Abbott, M.; Hayward, S.; Ross, W.; Godefroy, S. B.; Ulberth, F.; van Hengel, A. J.; Roberts, J.; Akiyama, H.; Popping, B.; Yeung, J. M.; Wehling, P.; Taylor, S. L.; Poms, R. E.; Delahaut, P. Validation procedures for quantitative food allergen ELISA methods. Community guidance and best practices. J. AOAC Int. 2010, 93, 442450
  30. 30
    Havenith, H.; Kern, K.; Rautenberger, P.; Spiegel, H.; Szardenings, M.; Ueberham, E.; Lehmann, J.; Buntru, M.; Vogel, S.; Treudler, R.; Fischer, R.; Schillberg, S. Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants. Biotechnol. J. 2017, 12, 1600441,  DOI: 10.1002/biot.201600441
  31. 31
    Meinlschmidt, P.; Sussmann, D.; Schweiggert-Weisz, U.; Eisner, P. Enzymatic treatment of soy protein isolates. Effects on the potential allergenicity, technofunctionality, and sensory properties. Food Sci. Nutr. 2016, 4, 1123,  DOI: 10.1002/fsn3.253
  32. 32
    Schagger, H.; von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368379,  DOI: 10.1016/0003-2697(87)90587-2
  33. 33
    Meinlschmidt, P.; Brode, V.; Sevenich, R.; Ueberham, E.; Schweiggert-Weisz, U.; Lehmann, J.; Rauh, C.; Knorr, D.; Eisner, P. High pressure processing assisted enzymatic hydrolysis – An innovative approach for the reduction of soy immunoreactivity. Innovative Food Sci. Emerging Technol. 2017, 40, 5867,  DOI: 10.1016/j.ifset.2016.06.022
  34. 34
    Rabilloud, T. Variations on a theme. Changes to electrophoretic separations that can make a difference. J. Proteomics 2010, 73, 15621572,  DOI: 10.1016/j.jprot.2010.04.001
  35. 35
    Ma, C.; Wang, L.; Webster, D. E.; Campbell, A. E.; Coppel, R. L. Production, characterisation and immunogenicity of a plant-made Plasmodium antigen--the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1. Appl. Microbiol. Biotechnol. 2012, 94, 151161,  DOI: 10.1007/s00253-011-3772-7
  36. 36
    Amponsah, A.; Nayak, B. Evaluation of the efficiency of three extraction conditions for the immunochemical detection of allergenic soy proteins in different food matrices. J. Sci. Food Agric. 2018, 98, 2378,  DOI: 10.1002/jsfa.8729
  37. 37
    Hager, D. F. Effects of extrusion upon soy concentrate solubility. J. Agric. Food Chem. 1984, 32, 293296,  DOI: 10.1021/jf00122a029
  38. 38
    Liu, K.; Hsieh, F.-H. Protein-protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems. J. Agric. Food Chem. 2008, 56, 26812687,  DOI: 10.1021/jf073343q
  39. 39
    Lee, K. H.; Ryu, H. S.; Rhee, K. C. Protein solubility characteristics of commercial soy protein products. J. Am. Oil Chem. Soc. 2003, 80, 8590,  DOI: 10.1007/s11746-003-0656-6
  40. 40
    Jiang, J.; Xiong, Y. L.; Chen, J. pH Shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions. J. Agric. Food Chem. 2010, 58, 80358042,  DOI: 10.1021/jf101045b
  41. 41
    Pavlicevic, M.; Stanojevic, S.; Vucelic-Radovic, B. Influence of extraction method on protein profile of soybeans. Hem. Ind. 2013, 67, 687694,  DOI: 10.2298/HEMIND120919115P
  42. 42
    Lin, J.; Alcocer, M. J. C. Food Allergens. Methods and Protocols; Humana Press: New York, 2017.
  43. 43
    Lin, J.; Shewry, P. R.; Archer, D. B.; Beyer, K.; Niggemann, B.; Haas, H.; Wilson, P.; Alcocer, M. J. C. The potential allergenicity of two 2S albumins from soybean (Glycine max). A protein microarray approach. Int. Arch. Allergy Immunol. 2006, 141, 91102,  DOI: 10.1159/000094535
  44. 44
    Han, Y.; Lin, J.; Bardina, L.; Grishina, G. A.; Lee, C.; Seo, W. H.; Sampson, H. A. What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2. Molecules 2016, 21, E622,  DOI: 10.3390/molecules21050622
  45. 45
    Nielsen, N. C.; Dickinson, C. D.; Cho, T. J.; Thanh, V. H.; Scallon, B. J.; Fischer, R. L.; Sims, T. L.; Drews, G. N.; Goldberg, R. B. Characterization of the glycinin gene family in soybean. Plant Cell 1989, 1, 313328,  DOI: 10.1105/tpc.1.3.313
  46. 46
    Shuttuck-Eidens, D. M.; Beachy, R. N. Degradation of -Conglycinin in Early Stages of Soybean Embryogenesis. Plant Physiol. 1985, 78, 895898,  DOI: 10.1104/pp.78.4.895

Cited By

ARTICLE SECTIONS
Jump To

This article is cited by 25 publications.

  1. Michael Wiederstein, Sabine Baumgartner, Kathrin Lauter. Soybean (Glycine max) allergens─A Review on an Outstanding Plant Food with Allergenic Potential. ACS Food Science & Technology 2023, 3 (3) , 363-378. https://doi.org/10.1021/acsfoodscitech.2c00380
  2. Yaozhong Hu, Yi Wang, Jing Lin, Sihao Wu, Serge Muyldermans, Shuo Wang. Versatile Application of Nanobodies for Food Allergen Detection and Allergy Immunotherapy. Journal of Agricultural and Food Chemistry 2022, 70 (29) , 8901-8912. https://doi.org/10.1021/acs.jafc.2c03324
  3. Norbert Lidzba, Verónica García Arteaga, Andreas Schiermeyer, Heide Havenith, Isabel Muranyi, Stefan Schillberg, Jörg Lehmann, Elke Ueberham. Development of Monoclonal Antibodies against Pea Globulins for Multiplex Assays Targeting Legume Proteins. Journal of Agricultural and Food Chemistry 2021, 69 (9) , 2864-2874. https://doi.org/10.1021/acs.jafc.0c07177
  4. Anguo Liu, Luqing Yang, Yuanhe Yang, Siqi Lei, Zhen Li, Pingli He. Simultaneous detection of glycinin and β-conglycinin in processed soybean products by high-performance liquid chromatography-tandem mass spectrometry with stable isotope-labeled standard peptides. Food Research International 2023, 173 , 113387. https://doi.org/10.1016/j.foodres.2023.113387
  5. Cassie R. Bakshani, Fiona Cuskin, Neil J. Lant, Hamish C.L. Yau, William G.T. Willats, J. Grant Burgess. Analysis of glycans in a Burnt-on/Baked-on (BoBo) model food soil using Microarray Polymer Profiling (MAPP) and immunofluorescence microscopy. Food Chemistry 2023, 410 , 135379. https://doi.org/10.1016/j.foodchem.2022.135379
  6. Stephanie Dramburg, Christiane Hilger, Alexandra F. Santos, Leticia de las Vecillas, Rob C. Aalberse, Nathalie Acevedo, Lorenz Aglas, Friedrich Altmann, Karla L. Arruda, Riccardo Asero, Barbara Ballmer‐Weber, Domingo Barber, Kirsten Beyer, Tilo Biedermann, Maria Beatrice Bilo, Simon Blank, Philipp P. Bosshard, Heimo Breiteneder, Helen A. Brough, Merima Bublin, Dianne Campbell, Luis Caraballo, Jean Christoph Caubet, Giorgio Celi, Martin D. Chapman, Maksymilian Chruszcz, Adnan Custovic, Rebecca Czolk, Janet Davies, Nikolaos Douladiris, Bernadette Eberlein, Motohiro Ebisawa, Anna Ehlers, Philippe Eigenmann, Gabriele Gadermaier, Mattia Giovannini, Francisca Gomez, Rebecca Grohman, Carole Guillet, Christine Hafner, Robert G. Hamilton, Michael Hauser, Thomas Hawranek, Hans Jürgen Hoffmann, Thomas Holzhauser, Tomona Iizuka, Alain Jacquet, Thilo Jakob, Bente Janssen‐Weets, Uta Jappe, Marek Jutel, Tanja Kalic, Sandip Kamath, Sabine Kespohl, Jörg Kleine‐Tebbe, Edward Knol, André Knulst, Jon R. Konradsen, Peter Korošec, Annette Kuehn, Gideon Lack, Thuy‐My Le, Andreas Lopata, Olga Luengo, Mika Mäkelä, Alessandro Maria Marra, Clare Mills, Martine Morisset, Antonella Muraro, Anna Nowak‐Wegrzyn, Roni Nugraha, Markus Ollert, Kati Palosuo, Elide Anna Pastorello, Sarita Ulhas Patil, Thomas Platts‐Mills, Anna Pomés, Pascal Poncet, Ekaterina Potapova, Lars K. Poulsen, Christian Radauer, Suzana Radulovic, Monika Raulf, Pierre Rougé, Joaquin Sastre, Sakura Sato, Enrico Scala, Johannes M. Schmid, Peter Schmid‐Grendelmeier, Denise Schrama, Hélène Sénéchal, Claudia Traidl‐Hoffmann, Marcela Valverde‐Monge, Marianne van Hage, Ronald van Ree, Kitty Verhoeckx, Stefan Vieths, Magnus Wickman, Josefina Zakzuk, Paolo M. Matricardi, Karin Hoffmann‐Sommergruber. EAACI Molecular Allergology User's Guide 2.0. Pediatric Allergy and Immunology 2023, 34 (S28) https://doi.org/10.1111/pai.13854
  7. Andrea Roman-Mateo, Esther Rodriguez-de Haro, Jose M. Berral-Hens, Sonia Morales-Santana, Jose C. Jimenez-Lopez. Comparative Analysis of Molecular Allergy Features of Seed Proteins from Soybean (Glycine max) and Other Legumes Extensively Used for Food. 2022https://doi.org/10.5772/intechopen.106971
  8. Xiaowen Pi, Yuxue Sun, Xiaomin Deng, Dawei Xin, Jianjun Cheng, Mingruo Guo. Investigation of differences in allergenicity of protein from different soybean cultivars through LC/MS-MS. International Journal of Biological Macromolecules 2022, 220 , 1221-1230. https://doi.org/10.1016/j.ijbiomac.2022.08.154
  9. Neda Mollakhalili-Meybodi, Masoumeh Arab, Leila Zare. Harmful compounds of soy milk: characterization and reduction strategies. Journal of Food Science and Technology 2022, 59 (10) , 3723-3732. https://doi.org/10.1007/s13197-021-05249-4
  10. Stephanie C. Filep, Kristina Reid Black, Bryan R.E. Smith, Denise S. Block, Anna Kuklinska-Pijanka, Max Bermingham, Maria A. Oliver, Catherine M. Thorpe, Zachary P. Schuhmacher, Sayeh Agah, Sabina Wuenschmann, Martin D. Chapman. Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array. Food Chemistry 2022, 389 , 132986. https://doi.org/10.1016/j.foodchem.2022.132986
  11. Jiayuan Xu, Yongli Ye, Jian Ji, Jiadi Sun, Xiulan Sun. Advances on the rapid and multiplex detection methods of food allergens. Critical Reviews in Food Science and Nutrition 2022, 62 (25) , 6887-6907. https://doi.org/10.1080/10408398.2021.1907736
  12. Liyan Zhu, Siyue Li, Lirui Sun, Jinlong Zhao, Jianlian Huang, Yinmei Jiang, Shuo Wan, Tushar Ramesh Pavase, Zhenxing Li. Development and validation of a specific sandwich ELISA for determination of soybean allergens and its application in processed foods. Process Biochemistry 2022, 117 , 134-141. https://doi.org/10.1016/j.procbio.2022.03.022
  13. Jun Xi, Lili Yao, Yuhan Fan, Yichao Wang, Yang Fu, Yuying Duan. Establishment of DAS-ELISA for the detection of antigenic changes in glycinin after heat processing. International Journal of Biological Macromolecules 2022, 208 , 1090-1095. https://doi.org/10.1016/j.ijbiomac.2022.03.205
  14. Xiaowen Pi, Yuxue Sun, Guiming Fu, Zhihua Wu, Jianjun Cheng. Effect of processing on soybean allergens and their allergenicity. Trends in Food Science & Technology 2021, 118 , 316-327. https://doi.org/10.1016/j.tifs.2021.10.006
  15. Martin Röder, Claudia Wiacek, Frauke Lankamp, Jonathan Kreyer, Wolfgang Weber, Elke Ueberham. Improved Sensitivity of Allergen Detection by Immunoaffinity LC-MS/MS Using Ovalbumin as a Case Study. Foods 2021, 10 (12) , 2932. https://doi.org/10.3390/foods10122932
  16. L. I. Kovalev, M. A. Kovaleva, L. A. Novikova, I. M. Chernukha. Proteomic Identification of Proteins as Potential Biomarkers of Nonmeat Components in Meat Products. Applied Biochemistry and Microbiology 2021, 57 (6) , 786-792. https://doi.org/10.1134/S0003683821060077
  17. Liping Hong, Mingfei Pan, Xiaoqian Xie, Kaixin Liu, Jingying Yang, Shan Wang, Shuo Wang. Aptamer-Based Fluorescent Biosensor for the Rapid and Sensitive Detection of Allergens in Food Matrices. Foods 2021, 10 (11) , 2598. https://doi.org/10.3390/foods10112598
  18. Öykü Üzülmez, Tanja Kalic, Vanessa Mayr, Nina Lengger, Angelika Tscheppe, Christian Radauer, Christine Hafner, Wolfgang Hemmer, Heimo Breiteneder. The Major Peanut Allergen Ara h 2 Produced in Nicotiana benthamiana Contains Hydroxyprolines and Is a Viable Alternative to the E. Coli Product in Allergy Diagnosis. Frontiers in Plant Science 2021, 12 https://doi.org/10.3389/fpls.2021.723363
  19. Xiaoyue Xiao, Song Hu, Xiaocui Lai, Juan Peng, Weihua Lai. Developmental trend of immunoassays for monitoring hazards in food samples: A review. Trends in Food Science & Technology 2021, 111 , 68-88. https://doi.org/10.1016/j.tifs.2021.02.045
  20. Wen‐Che Tsai, Hsin‐Yi Yin, Ssu‐Ning Chen, Hung‐Chi Chang, Hsiao‐Wei Wen. Development of monoclonal antibody‐based sandwich ELISA for detecting major mango allergen Man i1 in processed foods. Journal of Food Safety 2021, 41 (2) https://doi.org/10.1111/jfs.12884
  21. Jun Xi, Qiurong Yu. The development of lateral flow immunoassay strip tests based on surface enhanced Raman spectroscopy coupled with gold nanoparticles for the rapid detection of soybean allergen β-conglycinin. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2020, 241 , 118640. https://doi.org/10.1016/j.saa.2020.118640
  22. Tomasz Tuzimski, Anna Petruczynik. Review of New Trends in the Analysis of Allergenic Residues in Foods and Cosmetic Products. Journal of AOAC INTERNATIONAL 2020, 103 (4) , 997-1028. https://doi.org/10.1093/jaoacint/qsaa015
  23. N. I. Smirnova, E. A. Zvereva, A. V. Zherdev, B. B. Dzantiev. Development of Immunoenzyme Assay for Detection of Soybean Raw Material in Food Products. Applied Biochemistry and Microbiology 2020, 56 (4) , 483-487. https://doi.org/10.1134/S0003683820040158
  24. Andreas Schiermeyer. Optimizing product quality in molecular farming. Current Opinion in Biotechnology 2020, 61 , 15-20. https://doi.org/10.1016/j.copbio.2019.08.012
  25. Yikun Huang, Haomin Liu, Wilfred Chen, Mu-Ping Nieh, Yu Lei. Genetically engineered bio-nanoparticles with co-expressed enzyme reporter and recognition element for IgG immunoassay. Sensors and Actuators Reports 2019, 1 , 100003. https://doi.org/10.1016/j.snr.2019.100003
  • Abstract

    Figure 1

    Figure 1. Plant expression construct and purity and integrity of recombinant Gly m 8. (A) Schematic presentation (not to scale) of the expression cassette Gly m 8. SAR, scaffold attachment region; CaMV 35S promoter and terminator, promoter with duplicated enhancer and terminator of the Cauliflower mosaic virus (CaMV) 35S gene; 5′ untranslated region, 5′-UTR of the chalcone synthase gene from Petroselinum crispum (CHS 5′ UTR); Gly m 8, coding sequence for Gly m 8, UniProt ID 19594; His6 tag, six histidine residues (affinity purification tag). (B) Schematic presentation (not to scale) of the Gly m 8 protein, including signal peptide (SP), propeptide (PP), and disulfide bond. (C) Analysis of purification of expressed recombinant Gly m 8 by SDS-PAGE under reducing conditions. Lane 1 = molecular weight marker. Crude filtered extracts of N. benthamiana leaves (lane 2) were loaded onto IMAC columns, and both the flow-through and wash-out samples were collected (lanes 3 and 4, respectively). In the eluate (lane 5), a protein band with the expected size of ∼12 kDa, respresenting the large subunit of Gly m 8 under reducing conditions, was detected. The small unit, with a molecular weight of ∼5 kDa, ran within the running front of the gel but was separately displayed in D. (D) SDS-PAGE analysis of SEC-polished Gly m 8 under nonreducing (lane 2) and reducing (lane 3) conditions. A 99% pure recombinant Gly m 8 protein was purified by SEC, which separates under reducing conditions into two subunits.

    Figure 2

    Figure 2. Screening of antibody-producing hybridoma clones by indirect ELISA using plates coated with soy extract (native) or recombinant Gly m 8 and extracts of legumes and nuts (A). Supernatants of hybridoma cultures were tested for the presence of Gly m 8-specific IgG antibodies which bound to both native soy extracts (filled circle) and recombinant Gly m 8 (triangle) using an indirect ELISA. Binding of antibodies to the Gly m 8 antigen resulted in a high OD450nm signal as shown in the scatter plot of 2000 hybridoma clones. Read-outs higher than 0.1 OD identified high-affinity anti-Gly m 8 antibodies. Clones producing high-affinity antibodies were cryopreserved, and antibody-containing supernatants were collected for further analysis. The arrows (solid line mAb3 and dotted line mAb8) are tag specific signals for the clones finally used in ELISA. (B) Supernatants of selected hybridoma cultures (mAb1 to mAb11) which were tested for the presence of Gly m 8-specific IgG antibodies which bound to both native soy extracts using an indirect ELISA (Figure 2) were rescreened on both native soy extract (filled circle) and legume and nut extracts as indicated. OD values above 0.1 were assessed as positive according to signal-to-noise ratios above 10 in the appropriate ELISA.

    Figure 3

    Figure 3. Ranking of anti-Gly m 8 antibodies. Binding and stability of selected anti-Gly m 8 antibodies (mAb1 to mAb11) tested using recombinant Gly m 8 conjugated onto the surface of a CM5 chip with the SPR biosensor instrument Biacore T200. Response units (RU) indicate specific binding of the antibody to the recombinant Gly m 8 covalently coupled to the chip at the late association phase (binding) and late dissociation phase (stability). The plot shows these response units from the late association phase (binding) and late dissociation phase (stability) of 11 selected antibodies on a Gly m 8 surface in order to choose appropriate capture antibodies. The binding and stability are related to both the association and dissociation rates of the interaction. The red encircled antibodies were used in the sandwich ELISA as the capture (mAb3) and detection (mAb8) antibodies.

    Figure 4

    Figure 4. Representative SPR sensorgrams for the kinetic analysis of the Gly m 8-specific mAb8 and simultaneous binding of mAb3 and mAb8 to recombinant Gly m 8. (A) The affinity of mAb8 for recombinant Gly m 8 was determined by SPR spectroscopy. For each cycle, purified mAb8 was captured onto a Protein G-coated surface (500 response units (RUs). Subsequently, recombinant Gly m 8 was injected at concentrations of 5, 2.5, 1.25, 0.625, 0.3125, or 0.15625 nM for 150 s to determine the on-rate (ka)) and dissociation was observed for 900 s to determine the off-rate dissociation (kd). The kD values were estimated by fitting the data to interaction models using the Biacore T200 evaluation software, applying the 1:1 Langmuir fit model. (B) Because mAb8 is an IgG isotype IgG1, it binds only weakly to Protein A, whereas mAb3 (IgG2A) can be efficiently captured on a Protein A functionalized CM5 sensor surface. Therefore, it was possible to illustrate the compatibility of the two antibodies with a sandwich ELISA format in the context of an SPR experiment. The figure shows the subsequent injection of mAb3 (captured onto a Protein A surface), followed by recombinant Gly m 8 and finally mAb8. The comparable response unit (RU) levels for the two antibodies (1500–1700 RU) indicate that each molecule of recombinant Gly m 8 can be simultaneously recognized by both antibodies, confirming the suitability of the antibody combination for the development of a sandwich ELISA for the quantification of Gly m 8.

    Figure 5

    Figure 5. Calibration curve, precision profile, and robustness testing of the Gly m 8 ELISA. (A) Representative calibration curves of the Gly m 8 sandwich ELISA are depicted in gray with the regression curve fitted by a four-parameter logistic model in red (A). LOD and LOQ as functions of the analytical specificity of Gly m 8 ELISA were determined by the linear and nonlinear calibration methods on the basis of calibration curve (ISO 11843–5:2008). The blue curves represent the reaction of antibodies with potential interfering proteins naturally present in whole soy extracts, namely, recombinant proteins produced in N. benthamiana Gly m 4 and Gly m TI and commercially purified native proteins Gly m 5, Gly m 6, and Kunitz (Sigma-Aldrich). Two different operators performed the ELISA on three different days. (B) Precision profile shows the repeatability calculated by coefficients of intra-assay variance (gray lines) and intermediate precision calculated by coefficients of inter-assay variance (red line). (C) Representative calibration curves of the sandwich ELISA obtained by measuring recombinant Gly m 8 at three different temperatures (20, 28, and 37 °C; gray curves, circles), using two different incubation volumes (±10%; gray curves, triangles), and using an incubation time variation (±10%, gray curves, rectangles). The right axis of the ordinate presents the corresponding absorbance values (OD450nm). The corresponding precision profiles are depicted in the same coordinate system related to the left axis of the ordinate.

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 46 other publications.

    1. 1
      Maruyama, N.; Sato, S.; Cabanos, C.; Tanaka, A.; Ito, K.; Ebisawa, M. Gly m 5/Gly m 8 fusion component as a potential novel candidate molecule for diagnosing soya bean allergy in Japanese children, Clinical and experimental allergy. Clin. Exp. Allergy 2018, 48, 17261734,  DOI: 10.1111/cea.13231
    2. 2
      Maggio, P.; Monso, E.; Baltasar, M.; Morera, J. Occupational asthma caused by soybean hull. A workplace equivalent to epidemic asthma. Allergy 2003, 58, 350351,  DOI: 10.1034/j.1398-9995.2003.00089.x
    3. 3
      Riascos, J. J.; Weissinger, S. M.; Weissinger, A. K.; Kulis, M.; Burks, A. W.; Pons, L. The Seed Biotinylated Protein of Soybean (Glycine max). A Boiling-Resistant New Allergen (Gly m 7) with the Capacity To Induce IgE-Mediated Allergic Responses. J. Agric. Food Chem. 2016, 64, 38903900,  DOI: 10.1021/acs.jafc.5b05873
    4. 4
      Tsuji, H.; Bando, N.; Hiemori, M.; Yamanishi, R.; Kimoto, M.; Nishikawa, K.; Ogawa, T. Purification of characterization of soybean allergen Gly m Bd 28K. Biosci., Biotechnol., Biochem. 1997, 61, 942947,  DOI: 10.1271/bbb.61.942
    5. 5
      Ogawa, T.; Tsuji, H.; Bando, N.; Kitamura, K.; Zhu, Y.-L.; Hirano, H.; Nishikawa, K. Identification of the Soybean Allergenic Protein, Gly m Bd 30K, with the Soybean Seed 34-kDa Oil-body-associated Protein. Biosci., Biotechnol., Biochem. 1993, 57, 10301033,  DOI: 10.1271/bbb.57.1030
    6. 6
      Xiang, P.; Baird, L. M.; Jung, R.; Zeece, M. G.; Markwell, J.; Sarath, G. P39, a novel soybean protein allergen, belongs to a plant-specific protein family and is present in protein storage vacuoles. J. Agric. Food Chem. 2008, 56, 22662272,  DOI: 10.1021/jf073292x
    7. 7
      Tukur, H. M.; Lallès, J.-P.; Plumb, G. W.; Mills, E. N. C.; Morgan, M. R. A.; Toullec, R. Investigation of the Relationship between in Vitro ELISA Measures of Immunoreactive Soy Globulins and in Vivo Effects of Soy Products. J. Agric. Food Chem. 1996, 44, 21552161,  DOI: 10.1021/jf950141y
    8. 8
      Brandon, D. L.; Friedman, M. Immunoassays of Soy Proteins. J. Agric. Food Chem. 2002, 50, 66356642,  DOI: 10.1021/jf020186g
    9. 9
      Geng, T.; Stojšin, D.; Liu, K.; Schaalje, B.; Postin, C.; Ward, J.; Wang, Y.; Liu, Z. L.; Li, B.; Glenn, K. Natural Variability of Allergen Levels in Conventional Soybeans. Assessing Variation across North and South America from Five Production Years. J. Agric. Food Chem. 2017, 65, 463472,  DOI: 10.1021/acs.jafc.6b04542
    10. 10
      Liu, B.; Teng, D.; Wang, X.; Wang, J. Detection of the soybean allergenic protein Gly m Bd 28K by an indirect enzyme-linked immunosorbent assay. J. Agric. Food Chem. 2013, 61, 822828,  DOI: 10.1021/jf303076w
    11. 11
      Pedersen, M. H.; Holzhauser, T.; Bisson, C.; Conti, A.; Jensen, L. B.; Skov, P. S.; Bindslev-Jensen, C.; Brinch, D. S.; Poulsen, L. K. Soybean allergen detection methods--a comparison study. Mol. Nutr. Food Res. 2008, 52, 14861496,  DOI: 10.1002/mnfr.200700394
    12. 12
      Lacorn, M.; Dubois, T.; Siebeneicher, S.; Weiss, T. Accurate and Sensitive Quantification of Soy Proteins in Raw and Processed Food by Sandwich ELISA. Food Sci. Technol. 2016, 4, 6977,  DOI: 10.13189/fst.2016.040404
    13. 13
      Morishita, N.; Kamiya, K.; Matsumoto, T.; Sakai, S.; Teshima, R.; Urisu, A.; Moriyama, T.; Ogawa, T.; Akiyama, H.; Morimatsu, F. Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods. J. Agric. Food Chem. 2008, 56, 68186824,  DOI: 10.1021/jf8007629
    14. 14
      Scharf, A.; Kasel, U.; Wichmann, G.; Besler, M. Performance of ELISA and PCR methods for the determination of allergens in food. An evaluation of six years of proficiency testing for soy (Glycine max L.) and wheat gluten (Triticum aestivum L.). J. Agric. Food Chem. 2013, 61, 1026110272,  DOI: 10.1021/jf402619d
    15. 15
      Holzhauser, T.; Franke, A.; Treudler, R.; Schmiedeknecht, A.; Randow, S.; Becker, W.-M.; Lidholm, J.; Vieths, S.; Simon, J.-C. The BASALIT multicenter trial. Gly m 4 quantification for consistency control of challenge meal batches and toward Gly m 4 threshold data. Mol. Nutr. Food Res. 2017, 61, 1600527,  DOI: 10.1002/mnfr.201600527
    16. 16
      Nitride, C.; Lee, V.; Baricevic-Jones, I.; Adel-Patient, K.; Baumgartner, S.; Mills, E. N. C. Integrating Allergen Analysis Within a Risk Assessment Framework. Approaches to Development of Targeted Mass Spectrometry Methods for Allergen Detection and Quantification in the iFAAM Project. J. AOAC Int. 2018, 101, 8390,  DOI: 10.5740/jaoacint.17-0393
    17. 17
      Cucu, T.; Jacxsens, L.; De Meulenaer, B. de Analysis to support allergen risk management. Which way to go?. J. Agric. Food Chem. 2013, 61, 56245633,  DOI: 10.1021/jf303337z
    18. 18
      Ebisawa, M.; Brostedt, P.; Sjölander, S.; Sato, S.; Borres, M. P.; Ito, K. Gly m 2S albumin is a major allergen with a high diagnostic value in soybean-allergic children. J. Allergy Clin. Immunol. 2013, 132, 976978.e5,  DOI: 10.1016/j.jaci.2013.04.028
    19. 19
      Offermann, L.; Perdue, M.; He, J.; Hurlburt, B.; Maleki, S.; Chruszcz, M. Structural Biology of Peanut Allergens; JCI, 2015.
    20. 20
      Han, Y.; Lin, J.; Bardina, L.; Grishina, G. A.; Lee, C.; Seo, W. H.; Sampson, H. A. What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2. Molecules 2016, 21, E622,  DOI: 10.3390/molecules21050622
    21. 21
      Moreno, F. J.; Clemente, A. 2S Albumin Storage Proteins. What Makes them Food Allergens?. Open Biochem. J. 2008, 2, 1628,  DOI: 10.2174/1874091X00802010016
    22. 22
      Meinlschmidt, P.; Ueberham, E.; Lehmann, J.; Schweiggert-Weisz, U.; Eisner, P. Immunoreactivity, sensory and physicochemical properties of fermented soy protein isolate. Food Chem. 2016, 205, 229238,  DOI: 10.1016/j.foodchem.2016.03.016
    23. 23
      Lin, J.; Fido, R.; Shewry, P.; Archer, D. B.; Alcocer, M. J. C. The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris. Biochim. Biophys. Acta, Proteins Proteomics 2004, 1698, 203212,  DOI: 10.1016/j.bbapap.2003.12.001
    24. 24
      Sack, M.; Paetz, A.; Kunert, R.; Bomble, M.; Hesse, F.; Stiegler, G.; Fischer, R.; Katinger, H.; Stoeger, E.; Rademacher, T. Functional analysis of the broadly neutralizing human anti-HIV-1 antibody 2F5 produced in transgenic BY-2 suspension cultures. FASEB J. 2007, 21, 16551664,  DOI: 10.1096/fj.06-5863com
    25. 25
      Feller, T.; Thom, P.; Koch, N.; Spiegel, H.; Addai-Mensah, O.; Fischer, R.; Reimann, A.; Pradel, G.; Fendel, R.; Schillberg, S.; Scheuermayer, M.; Schinkel, H. Plant-based production of recombinant Plasmodium surface protein pf38 and evaluation of its potential as a vaccine candidate. PLoS One 2013, 8, e79920  DOI: 10.1371/journal.pone.0079920
    26. 26
      Schräml, M.; Biehl, M. Kinetic screening in the antibody development process. Methods Mol. Biol. (N. Y., NY, U. S.) 2012, 901, 171181,  DOI: 10.1007/978-1-61779-931-0_11
    27. 27
      Pol, E.; Roos, H.; Markey, F.; Elwinger, F.; Shaw, A.; Karlsson, R. Evaluation of calibration-free concentration analysis provided by Biacore systems. Anal. Biochem. 2016, 510, 8897,  DOI: 10.1016/j.ab.2016.07.009
    28. 28
      Boes, A.; Spiegel, H.; Delbrück, H.; Fischer, R.; Schillberg, S.; Sack, M. Affinity purification of a framework 1 engineered mouse/human chimeric IgA2 antibody from tobacco. Biotechnol Bioeng. 2011, 108 (12), 28042814,  DOI: 10.1002/bit.23262
    29. 29
      Abbott, M.; Hayward, S.; Ross, W.; Godefroy, S. B.; Ulberth, F.; van Hengel, A. J.; Roberts, J.; Akiyama, H.; Popping, B.; Yeung, J. M.; Wehling, P.; Taylor, S. L.; Poms, R. E.; Delahaut, P. Validation procedures for quantitative food allergen ELISA methods. Community guidance and best practices. J. AOAC Int. 2010, 93, 442450
    30. 30
      Havenith, H.; Kern, K.; Rautenberger, P.; Spiegel, H.; Szardenings, M.; Ueberham, E.; Lehmann, J.; Buntru, M.; Vogel, S.; Treudler, R.; Fischer, R.; Schillberg, S. Combination of two epitope identification techniques enables the rational design of soy allergen Gly m 4 mutants. Biotechnol. J. 2017, 12, 1600441,  DOI: 10.1002/biot.201600441
    31. 31
      Meinlschmidt, P.; Sussmann, D.; Schweiggert-Weisz, U.; Eisner, P. Enzymatic treatment of soy protein isolates. Effects on the potential allergenicity, technofunctionality, and sensory properties. Food Sci. Nutr. 2016, 4, 1123,  DOI: 10.1002/fsn3.253
    32. 32
      Schagger, H.; von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 1987, 166, 368379,  DOI: 10.1016/0003-2697(87)90587-2
    33. 33
      Meinlschmidt, P.; Brode, V.; Sevenich, R.; Ueberham, E.; Schweiggert-Weisz, U.; Lehmann, J.; Rauh, C.; Knorr, D.; Eisner, P. High pressure processing assisted enzymatic hydrolysis – An innovative approach for the reduction of soy immunoreactivity. Innovative Food Sci. Emerging Technol. 2017, 40, 5867,  DOI: 10.1016/j.ifset.2016.06.022
    34. 34
      Rabilloud, T. Variations on a theme. Changes to electrophoretic separations that can make a difference. J. Proteomics 2010, 73, 15621572,  DOI: 10.1016/j.jprot.2010.04.001
    35. 35
      Ma, C.; Wang, L.; Webster, D. E.; Campbell, A. E.; Coppel, R. L. Production, characterisation and immunogenicity of a plant-made Plasmodium antigen--the 19 kDa C-terminal fragment of Plasmodium yoelii merozoite surface protein 1. Appl. Microbiol. Biotechnol. 2012, 94, 151161,  DOI: 10.1007/s00253-011-3772-7
    36. 36
      Amponsah, A.; Nayak, B. Evaluation of the efficiency of three extraction conditions for the immunochemical detection of allergenic soy proteins in different food matrices. J. Sci. Food Agric. 2018, 98, 2378,  DOI: 10.1002/jsfa.8729
    37. 37
      Hager, D. F. Effects of extrusion upon soy concentrate solubility. J. Agric. Food Chem. 1984, 32, 293296,  DOI: 10.1021/jf00122a029
    38. 38
      Liu, K.; Hsieh, F.-H. Protein-protein interactions during high-moisture extrusion for fibrous meat analogues and comparison of protein solubility methods using different solvent systems. J. Agric. Food Chem. 2008, 56, 26812687,  DOI: 10.1021/jf073343q
    39. 39
      Lee, K. H.; Ryu, H. S.; Rhee, K. C. Protein solubility characteristics of commercial soy protein products. J. Am. Oil Chem. Soc. 2003, 80, 8590,  DOI: 10.1007/s11746-003-0656-6
    40. 40
      Jiang, J.; Xiong, Y. L.; Chen, J. pH Shifting alters solubility characteristics and thermal stability of soy protein isolate and its globulin fractions in different pH, salt concentration, and temperature conditions. J. Agric. Food Chem. 2010, 58, 80358042,  DOI: 10.1021/jf101045b
    41. 41
      Pavlicevic, M.; Stanojevic, S.; Vucelic-Radovic, B. Influence of extraction method on protein profile of soybeans. Hem. Ind. 2013, 67, 687694,  DOI: 10.2298/HEMIND120919115P
    42. 42
      Lin, J.; Alcocer, M. J. C. Food Allergens. Methods and Protocols; Humana Press: New York, 2017.
    43. 43
      Lin, J.; Shewry, P. R.; Archer, D. B.; Beyer, K.; Niggemann, B.; Haas, H.; Wilson, P.; Alcocer, M. J. C. The potential allergenicity of two 2S albumins from soybean (Glycine max). A protein microarray approach. Int. Arch. Allergy Immunol. 2006, 141, 91102,  DOI: 10.1159/000094535
    44. 44
      Han, Y.; Lin, J.; Bardina, L.; Grishina, G. A.; Lee, C.; Seo, W. H.; Sampson, H. A. What Characteristics Confer Proteins the Ability to Induce Allergic Responses? IgE Epitope Mapping and Comparison of the Structure of Soybean 2S Albumins and Ara h 2. Molecules 2016, 21, E622,  DOI: 10.3390/molecules21050622
    45. 45
      Nielsen, N. C.; Dickinson, C. D.; Cho, T. J.; Thanh, V. H.; Scallon, B. J.; Fischer, R. L.; Sims, T. L.; Drews, G. N.; Goldberg, R. B. Characterization of the glycinin gene family in soybean. Plant Cell 1989, 1, 313328,  DOI: 10.1105/tpc.1.3.313
    46. 46
      Shuttuck-Eidens, D. M.; Beachy, R. N. Degradation of -Conglycinin in Early Stages of Soybean Embryogenesis. Plant Physiol. 1985, 78, 895898,  DOI: 10.1104/pp.78.4.895
  • Supporting Information

    Supporting Information

    ARTICLE SECTIONS
    Jump To

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02717.

    • Supporting Figure 1: Analysis of native Gly m 8 isolated from soy extracts by immunoprecipitation with mAb3 followed by PAGE (PDF)

    • Supporting Table 1: Soy-containing foods and food ingredients (PDF)

    • Supporting Table 2: Kinetic parameters derived from SPR-based interaction analysis (PDF)

    • Supporting Table 3: Amount of Gly m 8 measured in processed food (PDF)


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

Pair your accounts.

Export articles to Mendeley

Get article recommendations from ACS based on references in your Mendeley library.

You’ve supercharged your research process with ACS and Mendeley!

STEP 1:
Click to create an ACS ID

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

MENDELEY PAIRING EXPIRED
Your Mendeley pairing has expired. Please reconnect