Chapter 6
Current Challenges and Advancements in Residue Analytical Methods Downloaded from pubs.acs.org by BETHEL UNIV on 04/06/19. For personal use only.
Endogenous Allergens from Genetically Modified Soybean: Background, Assessment, and Quantification Tao Geng,*,1 Yongcheng Wang,1 Lucy Liu,1 Bin Li,1 and Ryan C. Hill2 1Monsanto
Company, 700 Chesterfield Parkway West, Chesterfield, Missouri 63017, United States 2Corteva Agriscience, Agriculture Division of DowDupont, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States *E-mail:
[email protected].
Ensuring a safe food supply includes assessing the food allergenic potential of genetically modified (GM) plants. Approximately 4% of the U.S. population have food allergies. Soybeans are one of the eight foods that together elicit nearly 90% of food allergy cases. In Europe, where regulatory guidelines for GM crops include testing whether the transformation process has had unintended effects on the levels of endogenous allergens in crops such as soybeans, the average prevalence of soybean allergies based on food challenge has been reported to be 0.3%. To address requirements from the European Food Safety Authority (EFSA), endogenous allergen quantification is completed for GM soybean events and breeding stacks prior to commercialization. This chapter reviews the current methods available for identification and quantification of soybean allergens and discusses the advantages and disadvantages of each as they relate to the broader safety assessment of GM crops. These methods can accurately and reliably detect any differences in allergen expression levels between GM soybeans relative to the conventional control and commercially available conventional varieties. Importantly, the statistical differences between GM and non-GM soybeans should be assessed in the context of the range of natural variations for that parameter to determine its biological relevance.
© 2019 American Chemical Society
Food Allergy A food allergy is an adverse immunological response to a food or food component (1). Food allergic disease can induce gastrointestinal (vomiting, diarrhea, abdominal pain), skin related (urticaria, rashes, eczema), lower respiratory tract (wheezing, coughing), and upper respiratory tract (rhinitis, nasal congestion) symptoms. Although most reactions to foods are mild, in some cases, they can be life threatening (2). While these various types of reactions are often considered collectively as food allergies, true food allergies represent only a fraction of the individual adverse reactions to foods. Epidemiologic data suggest that almost 4% of the U.S. population have food allergy (3, 4). Once diagnosed, the current principle treatment for food allergic disease is total avoidance of the food responsible for the allergic reaction. Two types of allergic reactions occur as basic immunological mechanisms involved in food allergy. IgE-mediated reactions, also known as immediate hypersensitivity reactions, involve the formation of IgE antibodies that specifically recognize certain proteins in foods, and trigger an immediate response that includes the release of inflammatory agents such as histamines, cytokines, and proteases. Celiac disease is the predominant example of the second type of food allergic reaction that is a non-IgE, cell-mediated response. Gliadin and related protein fractions of wheat, barley, and rye are associated with initiation of celiac disease in susceptible individuals. Inflammatory damage to the absorptive intestinal epithelial cells involved in celiac disease is mediated by intestinal T lymphocytes (5). Plant Proteins and Allergens Although the diversity of the human diet is enormous, there are only eight commonly allergenic foods that are responsible for more than 90% of all food allergies: wheat, milk, soybeans, peanuts, tree nuts, shellfish, fish, and eggs (1, 6, 7). Peanuts, tree nuts, soybeans, and wheat are the only four plant-produced foods among the “big eight”. In Europe, the prevalence of soybean allergies based on food challenges has been reported to be 0.3% (8). Food allergens are almost always the result of proteins. Plant tissues that are consumed by humans contain thousands of different proteins. For example, the maximum number of different genes expressed at mid-endosperm development of wheat was estimated to be within the range of 4500 to 8000 (9). The most natural classification system might well be based on both structural and functional properties of proteins. Proteins are clustered together into families of related proteins. Proteins having low sequence identities but whose structures and functional features suggest a probable common evolutionary origin are placed together in superfamilies. In this context, it is worth noting that plant food allergens also fall into a small set of protein families (10). Many allergens belong to the cupin superfamily (7S and 11S seed storage proteins) or the prolamin superfamily (2S albumins, nonspecific lipid transfer proteins [nsLTPs], α-amylase/trypsin inhibitors, and prolamin storage proteins of cereals). The pathogenesis-related proteins (PRs) represent a heterogeneous collection of 74
14 plant protein families involved in plant resistance to pathogens or adverse environmental conditions (11). Many plant food allergens are homologous to PRs (12, 13). Storage proteins are the cause of well-known allergic reactions to peanuts and cereals (14). PRs are responsible for pollen-fruit or latex-fruit cross-reactive syndromes. In addition, there are some unrelated families of structural and metabolic plant proteins that harbor allergenic proteins such as the profilins. Allergenicity Assessment of Foods Derived from Biotechnology Food crops that have been developed through agricultural biotechnology for commercial use must be thoroughly assessed for their safety. One aspect of this safety testing is a comprehensive examination of the allergenicity potential of the genetically modified (GM) crop. The allergenicity safety assessment used to assess GM crops has been developed and recommended by international organizations like Codex and FAO (15, 16). The allergy assessment of GM crops includes two elements: characterization of newly introduced protein(s) (which is discussed in Chapter 5) and comparison of the allergenic potential of GM food crops to their conventional counterpart (the focus of this chapter). For GM crops that are commonly allergenic, such as soybeans, assessing the effect of the transformation process on the abundance of endogenous allergens of the whole crop is recommended by Codex (2009). However, it has been found that the environment is the main factor in the variability of endogenous allergen levels in soybeans and the effects of transgenesis are negligible (17–19). The European Food Safety Authority (EFSA) requires quantification of endogenous soybean allergens in GM crops. This assessment addresses whether the transformation process potentially changes the endogenous allergen content in the GM crop in relation to a genetically matched non-GM control soybean line (Figure 1). Potential alterations in endogenous allergen levels could occur during the transformation process; however, to date, no biologically meaningful differences in endogenous allergen levels have been found between GM and non-GM varieties (20). This is expected and similar to other crop compositional components (21). To assess the overall allergenic potential of the GM food crop, the GM soybean variety is compared with conventional varieties to determine if the allergen expression levels are substantially similar (22). This comparison can be completed by using an IgE-binding assay, such as a validated, quantitative ELISA, to compare the IgE-binding capacity of proteins from GM and conventional soybean varieties (23). This type of assay requires a characterized serum from clinically diagnosed patients who are allergic to soybeans to ensure the quality of the results and reduce the possibility of false-positive or false-negative results. Recently, EFSA published a set of revised guidelines for the allergy assessment of GM crops (24, 25). As a replacement for measuring total endogenous allergen levels, EFSA has recommended that an assessment of individual allergens be performed by comparing IgE-binding from GM and conventional soybeans using an approach (such as two-dimensional gel electrophoresis [2-DE] IgE immunoblot [EFSA 2010]), or an absolute quantification method (such as IgG ELISAs or mass spectrometry [MS]) (24). 75
Figure 1. Flow chart of the endogenous allergenicity assessment of GM crops. The green color in the Allergen Potential bar represents foods that are either from non-allergenic crops, or have no changes to endogenous levels of allergens. The red color in the Allergen Potential bar represents a significant increase of endogenous allergen levels. Soybean Endogenous Allergens Soybean (Glycine max) seed is a good source of nutritional protein for animal and human consumption because protein constitutes 34.1% to 56.8% of its total content on a dry weight basis (26). As a major food ingredient, the soybean provides numerous health benefits to individuals with high plasma cholesterol, cancer, diabetes, and obesity (27–31). However, the soybean also ranks as the one of the “big 8” food allergens in the United States, and the “big 14” food allergens in Europe (6, 32). A review describing the identification and function of 15 potential soybean protein allergens has been published (22, 33). However, not all the sera used to identify these proteins were from individuals who have been clinically diagnosed with a soybean food allergy, and for some of the proteins, whole amino acid sequences are not known. Although these proteins are cross-reactive to human IgE antibodies in vitro, it is unclear if a subset of these proteins cause soybean allergic disease; therefore, additional reviews were conducted (34, 35). To date, ten soybean proteins have been confirmed as likely causes for allergic symptoms in soybean allergic individuals, including Gly m 1, Gly m 3, Gly m 4, Gly m 5 (β-conglycinin), Gly m 6 (glycinin), Gly m 7, Gly m 8 (2S albumin), Gly m Bd 28K, Gly m Bd 30K, and Kunitz trypsin inhibitor (KTI). Soybean allergic disease is typically manifested as a polyclonal IgE response to numerous soybean proteins unique to each sensitized individual (36, 37). The amount of soybean protein required to elicit symptoms in soybean allergic individuals has been reported, however a clear correlation between the severity of symptoms and the threshold level dose was not observed (36, 38–40). The amount of individual allergen needed to sensitize susceptible individuals or elicit an allergic reaction in sensitized individuals is unknown for most allergens and the exposure routes and timing varies from individual to individual (41). 76
Because the soybean is a commonly allergenic food, a comparison of the endogenous allergens in GM soybeans with their non-GM counterpart is currently required by the European Commission (EC) for the safety assessment of GM soybeans (EC Implementing Regulation No. 503/2013) (42). However, the food safety relevance of this requirement has been questioned since individuals with soybean allergies typically avoid eating foods containing soybean-derived ingredients (43–46). In addition, two soybean allergy studies showed a wider variation in IgE binding to proteins from different non-GM varieties than to proteins in GM soybeans (47, 48), suggesting that allergenicity of soybeans is more heavily affected by natural genetic variability than by the presence or absence of a GM trait. Further studies showed that environmental factors can affect the level of specific soybean allergens much more than the genetic differences among different conventionally-bred varieties (17, 20, 49–51).
Types of Detection Methods for Allergenic Proteins The principle effective treatment currently available for individuals who suffer from food allergy is total avoidance of the offending food. However, total avoidance can be difficult as many allergenic foods are present infood products or might be present as contaminants from food storage and/or processing (6). Several countries have developed legislation to establish a list of common allergenic foods that are required to be labeled on an ingredient list if they are present in food products (52, 53). For this reason, measuring allergen levels is of great interest to the food industry. Methods to measure allergen levels in food products have been developed and several reviews assess the ability of each type of method to quantify allergen content in food products (54, 55). Although many methods could be used for both measuring allergens for food labeling purposes and as part of the safety assessment of a GM crop, each method may not be the best in all instances. Before discussing the types of methods that are currently available to measure endogenous allergen levels, it is important to first consider what aspects of an assay are critical for the safety assessment of a GM crop. Unlike assays required for food labeling purposes that must accurately quantify the amount of the allergenic food (e.g. total peanut or peanut protein) present in multiple foods, the goal of the safety assessment for a GM crop is to compare specific allergen protein levels between the GM variety and the near isogenic non-GM control with commercially available conventional varieties in grain or seed (6, 16). When measuring endogenous allergen levels as part of the safety assessment of a GM crop, it is critical to include several commercially available conventional varieties in the study design to establish the natural range of allergen levels that are safely consumed by non-allergic individuals (43). Alternatively, a database of allergen levels for previously assayed varieties can be used to assess the natural variation in the crop. Without this range of allergen levels, the measurement of allergen levels in GM crops provides limited safety information since the allergen levels required for sensitization (and the exposure routes and timing) and elicitation of an allergic response are not known for most allergens and vary from 77
individual to individual (41). For this reason, a survey of natural variability of allergen levels is needed to provide context as to the level of allergens known to be safely consumed in the non-modified crop by non-allergic individuals. Because most food allergens are proteins, the most appropriate methods for measuring allergen levels in the context of the safety assessment of GM crops are protein-based. There are polymerase chain reaction (PCR)-based methods that can detect changes in RNA transcript levels between GM and non-GM crops, however there is often very poor correlation between RNA and protein expression levels. For this reason, methods that directly measure or assess protein levels are typically used in the safety assessment of a GM crop. There are several types of protein-based methods including: serological (human IgE binding), 2-DE and 2-DE western blot, enzyme-linked immunosorbent assay (ELISA), and liquid chromatography with mass spectrometry (LC-MS). Human serum IgE binding was initially used to measure key allergens in a comparison with a GM, a conventional control, and a set of non-GM reference soybeans (47, 56). Since human sera are accessible only in very limited amounts and have variable IgE reactivity profiles (unique for each patient), modern analytic approaches (such as 2-DE and 2-DE western blot, ELISA, and LC-MS/MS) were developed to quantify key allergens from soybeans (17, 51). Both IgE binding and allergen quantitation methods showed that significant variation was observed at endogenous allergen levels among conventional varieties (17, 47, 51, 56). Thus, the biological significance of any observed differences between GM and non-GM soybeans should be assessed in the context of the range of natural variations for that parameter to determine its biological significance (16). Although the methods described in this chapter to measure endogenous allergens in soybeans have advantages and disadvantages, they are functionally similar tools for assessing endogenous allergen levels in GM crops. 2-DE Method Protein separation methods, such as 2-DE, have been instrumental in the identification of several allergens. 2-DE has also been used as a proteomic tool to semi-quantitatively measure the expression levels of many proteins and has recently been used to compare allergen levels in wild-type and GM plants (57–59). Using this method, GM and non-GM control soybean extracts are run on separate gels. These proteins are separated into two dimensions, first by isoelectric point and then by molecular weight. The gels are stained with Coomassie Blue or a fluorescent stain and protein spot intensities are compared between gels. Another protein separation technique, called difference gel electrophoresis (DIGE), allows comparison of two protein extracts on the same gel (60). Using this method, protein extracts are labeled with different fluorescent dyes, mixed, and then separated by 2-DE. Protein spot intensities are measured for each fluorescent dye and then comparisons are made between individual protein spots from each extract. For both 2-DE and DIGE, individual protein spots can be identified using MS. Taken together, these two protein separation techniques can provide comparative analysis between protein extracts (i.e. GM and non-GM conventional soybean extracts) and the allergen spots can be identified. As a result, these 78
techniques could be used for a comparative endogenous allergen assessment as part of the safety assessment of GM crops (16). Although 2-DE and DIGE could be used to measure allergen levels as part of the safety assessment, the reproducibility and quantitative ability of these methods have been questioned even when the most sophisticated gel separation, image capture, and image analysis technologies are used (61, 62). For this reason, it is critical that the number of replicated gels be sufficient to ensure that any spot differences observed are not simply due to technical or operational variability (63). Several studies have measured the variability associated with protein extraction, separation, staining method, and image analysis (59, 61, 62, 64–66). From this work, it is clear that 2-DE is a valid tool for measuring large differences (≥3-fold) in protein expression when the proper number of replicates are included in the experimental design and the results are confirmed by other techniques (61). Furthermore, for inclusion in the allergy safety assessment of a GM soybean variety, it is critical to include multiple commercially available conventional soybean varieties in the study design so that the measurement of allergens can be placed into a context of natural variability relative to food safety. Allergen-Specific Human IgE Antibody-Based Method Food allergy is typically mediated through allergen-specific IgE antibodies. These IgE antibodies can be used in several different types of assays, including ELISA and immunoblotting, to detect and quantify allergens. For instance, IgE ELISAs can be used as part of the safety assessment to compare the IgE-binding capacity of proteins from GM and non-GM soybean varieties (67). In this type of assay, total protein is extracted from the grain of GM, the conventional control, and the commercially available conventional varieties and bound to microplate wells. The IgE serum obtained from individuals clinically diagnosed with soybean food allergies binds the allergens present in each well, and then this IgE-allergen binding is detected with a secondary anti-IgE antibody. In this type of assay, total IgE reactivity (total allergen expression levels) is measured from each variety. Although this assay does not measure individual allergen expression levels, measuring total allergen levels is appropriate for the safety assessment of endogenous soybean allergenicity because soybean allergic disease is manifested by a polyclonal IgE response to numerous proteins (33, 36, 37). Although it is an appropriate assay, there are a number of criteria that should be considered when performing the IgE ELISA as part of the safety assessment of GM soybean varieties. These criteria include obtaining sera tested through a double-blind, placebo-controlled food challenge (DBPCFC) approach to avoid false-positive or false-negative results and using a fully validated method to ensure precise, robust, and reproducible measurement of allergen levels (67). Measurement of individual soybean allergens using serological methods by IgE immunoblotting has also been recommended for the safety assessment of GM soybean varieties (68). In this assay, proteins isolated from the grain of the GM variety and its non-GM near-isogenic conventional control are separated by 2-DE. The proteins are then transferred to a membrane and probed with serum from an individual who has been clinically diagnosed with a soybean food allergy (Figure 79
2). Qualitative comparisons can be made between the GM variety and its non-GM isogenic control. Because this method uses large amounts of human sera from soybean allergic individuals, which can be difficult to obtain and are limited in quantity, commercially available non-GM varieties are not typically included in the analysis. Furthermore, replicated immunoblots can also be difficult to include in the experimental design because of the limited supply of soybean allergic serum. For these reasons, if any minor differences in protein spot intensity are observed between the GM and conventional varieties, the experimental design does not distinguish between differences caused by technical variability or those potentially caused by the insertion of the transgene.
Figure 2. Schematic of 2-DE IgE immunoblots for endogenous allergen comparisons of a GM soybean and its non-GM conventional control. Protein was extracted from the grain of a GM soybean and a non-GM control. Proteins from each of these extracts were separated using 2-DE. The gels were stained with Sypro Ruby fluorescence stain and imaged for spot intensity analysis. Then the proteins were blotted onto nitrocellulose membrane for immunoblotting. Prior to immunoblotting, the membranes were stained with Sypro Ruby fluorescence stain and imaged. The blots were destained and probed with human serum containing soybean-specific IgE followed by anti-human IgE secondary antibody. Signals from these immunoblots were detected using chemiluminescence and images were visualized by exposure to x-ray film. Although 2-DE can be used to measure protein expression, 2-DE coupled with immunoblotting is not typically used to measure differences in protein expression. This may be due to the large technical variation that is attributed to immunoblotting. For instance, significant changes in protein spot intensity are observed when gel replications are performed using protein extracts from the same conventional soybean variety (Figure 3). These differences demonstrate that 2-DE IgE immunoblots cannot be used to accurately measure soybean allergen levels with reproducible results; therefore this technique provides limited 80
quantitative information as part of the allergy safety assessment of a soybean variety.
Figure 3. Visual comparison of replicate IgE immunoblots shows differences in protein spot intensity attributed to technical variability. 2-DE was performed and gels were stained with SYPRO Ruby protein stain. Proteins were then transferred to a nitrocellulose membrane and stained with SYPRO Ruby protein stain. Membranes were imaged for spot intensity analysis. IgE immunoblots were performed using serum-containing soybean-specific IgE from one individual as the primary antibody. The secondary antibody used was a HRP-conjugated monoclonal antihuman IgE antibody. IgE-binding proteins were visualized using SuperSignal Substrate. Immunoblots were imaged for spot intensity analysis. ELISAs In addition to serological studies that take advantage of the specificity of IgE antibodies to allergens, protein expression analyses that use IgG antibodies from the serum of an immunized animal could be used in the safety assessment. ELISA is the most common method used by food industries and food control agencies to identify and quantify allergens due to its sensitivity and high throughput (33, 69–71). The ELISA methods for the quantification of soybean endogenous allergens were developed for soybean allergens such as Gly m 1, Gly m 3, Gly m 4, Gly m Bd 30K, Gly m Bd 28K, glycinin/Gly m 6, β-conglycinin/Gly m 5, and KTI (72–81). Reference protein standards and antibodies for each allergen need to be generated to develop a validated ELISA. Each allergen protein standard can be either expressed in microbes such as Escherichia coli or purified from soybean seed. Each protein needs to be characterized using techniques such as N-terminal sequence analysis and MS, and its concentration and purity need to be determined using techniques such as amino acid analysis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antibodies, either monoclonal or polyclonal, should have specificity to avoid cross-reactions with other soybean proteins. Because of this, the generation and purification of antibodies are critical. Using two antibodies in a sandwich ELISA format could 81
increase the specificity of the assay (Table 1) (17). In the study reported here, the protein standards for abundant soybean allergens (Gly m 5 and Gly m 6) were purified from soybean seeds, whereas the proteins were expressed in Esterichia coli for less abundant soybean allergens (Gly m 4, Gly m Bd 28k, and Gly m Bd 30k). Once each of these five soybean allergens were isolated, their purity levels were determined through the calculation of the allergen bands’ intensity over total protein intensity within the same lane on SDS-PAGE gels. All five allergen protein standards had greater than 70% purity. The purity corrected concentration of each protein standard was used to prepare ELISA standard curves. The identity of each allergen included N-terminal sequence analysis and MS analysis of trypsin-digested fragments. For all five proteins, the N-terminal amino acid sequence exactly matched the predicted amino acid sequence for the respective protein. Also, peptide MS fingerprint analysis showed that the unique peptides identified from trypsin digestion of each of the five allergens corresponded to the predicted masses for each fragment and that assembly of a peptide map of each protein confirmed the identity to the predicted result for that protein. The specificity of each allergen ELISA was assessed using other soybean allergens or proteins. Except for the targeted allergen to be measured, ELISA methods showed less specificity than the LOQ for all other soybean proteins (17). Taken together as a combination of the specificity of both antibodies and ELISA, each allergen ELISA method was verified for its ability to specifically measure the target allergen from the soybean seed. Finally, ELISAs were validated for their precision, accuracy, linearity, robustness, ruggedness, and stability (50). The use of IgG antibodies to develop and validate quantitative ELISAs that are sensitive with reproducible results is a viable approach for measuring individual allergen levels as part of the safety assessment of GM soybeans (35). These methods also have advantages such as ease of use, high-throughput, and low costs. However, one limitation is that significant effort is needed for the initial generation and characterization of each allergenic protein standard and their respective antibodies. Since soybean endogenous allergen levels can be measured by ELISAs, statistical analyses can be conducted according to the guidance documents from EFSA (25, 42). However, careful interpretation of the statistical difference in the context of biological relevance is needed since two soybean allergy studies showed a wider variation in IgE binding to different conventional soybean varieties than IgE binding in GM soybeans (47, 48). This suggests that individuals are exposed to naturally highly variable levels of soybean allergenic proteins.
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Table 1. Summary of ELISA Format, Protein Standard, and Antibodies for Five Soybean Allergen ELISA. Reprinted with the permission from reference (17). Copyright 2017 American Chemical Society. Allergen
a
Gly m 4
Gly m 5
Gly m 6
Gly m Bd 28k
Gly m Bd 30k
ELISA format
Indirect sandwich ELISA
Indirect sandwich ELISA
Indirect sandwich ELISA
Indirect sandwich ELISA
Indirect sandwich ELISA
Protein standard
rGly m 4a
Native Gly m 5b
Native Gly m 6b
rGly m Bd 28ka
rGly m Bd 30ka
Capture antibody
Mouse mAbc
Goat peptide pAbd
Goat peptide pAbd
Goat pAbe
Rabbit pAbe
Detection antibody
Goat pAbe
Mouse mAbc
Mouse mAbe
Goat pAbe
Goat pAbe
rGly: recombinant soybean allergens produced from E. coli. b Native: allergens were purified from soybean seed. c mAb: monoclonal antibody. Peptide pAb: common peptides from subunits of Gly m 5 or Gly m 6 were used to produce antibodies. e pAb: polyclonal antibody.
d
83
MS Methods Due to the increasing number of endogenous soybean allergens being requested for the GM crop safety assessment, multiplexing protein detection methods such as MS have been examined. The advantages of MS methods include short development times, high specificity and selectivity, and a large dynamic range suitable for multiplex detection of a range of allergen protein abundances. These methods can be used for qualitative or quantitative analysis and range from singleplex detection to highly multiplexing in a single analysis. The most common quantitative proteomic technique measures small peptides as surrogates for target proteins and is termed bottom up proteomics. In this application, use of a protease with known specificity and knowledge of the protein primary sequence allow for targeted generation of peptides small enough to be accurately measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (82). Trypsin is the enzyme most commonly used due to the high preference toward C-terminal lysine and arginine residues and the relative low cost with respect to most large-scale enzymatic analyses. Commercially produced heavy isotope peptides (most commonly L-lysine-13C615N2 or L-arginine-13C615N4), which are structurally identical to the natural abundance peptides produced from the tryptic digestion, are typically employed for quantitative analysis. The heavy isotope peptide can be added preor post-digestion depending on whether experimental processing or normalization of results is required through experimental design. For the latter, the heavy isotope peptide should be added as late in the procedure as possible to confine variability to the instrumental analysis. The heavy isotope peptides coelute chromatographically with the natural abundance peptides normalizing variability caused by common technical pitfalls, such as matrix effects or ion drift, and provide discrete identification for accurate quantitation by LC-MS/MS. Due to the reliance on enzymatic processing for quantitative detection, peptide selection is the most critical step in surrogate-peptide assay method development. This approach is relatively straightforward for GM proteins where the protein sequence is foreign to the crop of interest, purified recombinant proteins are available for in silico digestion, control tissue is available for investigation, and production of unique peptides can be confirmed using public databases such as UniProt and NCBI. With regard to endogenous proteins (such as soybean allergens), this concept is much more challenging due to the possibility of multiple isoforms for each protein with varying degrees of homology. MS analysis is ideal for endogenous protein assays due its high specificity; however, modifications to the peptide sequence that would alter the expected mass, which are monitored by LC-MS/MS, would not be detected (83). For example, if the monitored peptide sequence for a target protein differed by one amino acid residue in an isoform, the isoform would not be detected in the quantitative measurement. As a result, peptide selection of endogenous proteins requires an initial bioinformatic assessment to identify all possible isoforms as well as the degree of homology between potential isoforms. Conserved peptide sequences across all isoforms should be identified to guarantee that the total protein population is accounted for prior to further downstream peptide selection 84
criteria such as stability from an overnight digestion, sensitivity at detection, and selectivity against isobaric interferences. Additionally, peptide sequences containing modifiable amino acid residues, such as methionine or cysteine, are typically excluded from most surrogate peptide selections. Yet, it may be unavoidable to have these residues when there are a limited number of tryptic sequences available across all isoforms of interest (Figure 4). The enzymatic digestion protocol is also a very important consideration in surrogate peptide analysis, particularly for multiplex analysis for a range of protein chemistries. Once peptide selection has identified the possible target peptides, stability from an overnight digestion should be evaluated. This is commonly performed with a timecourse digestion where aliquots of trypsin digests are acid quenched at time points encompassing the digestion interval. With an assay targeting many proteins, method parameters such as trypsin enzyme concentration, incubation temperature, and reaction components and conditions (such as pH or solvent polarity) should be held constant when identifying peptides for many target proteins as a shift in one parameter may favor a different subset of resulting peptides (84). Typically, the digestion protocol for a quantitative multiplex analysis will favor the stability of the least stable peptide. For GM crops, purified recombinant protein spiked into a control matrix (non-GM crop) allows for the generation of the tryptic peptide stability profile. The high abundance of endogenous allergens allows for this assessment with digested soybean seed, without the need for purified protein standards, and can be evaluated before synthesizing surrogate peptide standards. To be able to measure relative differences between conventional and GM soybeans, only one peptide may be monitored per protein as all samples will be treated similarly within the analytical protocol. Where absolute protein abundances are required, confirmatory peptides and purified allergen protein standards may be necessary to validate interpolated results. Two multiplex LC-MS/MS protocols have been developed and validated to measure soybean allergens from soybean seed. In 2011, Houston et al reported a LC-MS/MS method employing absolute quantification (termed AQUA) that monitored five endogenous soybean allergens (Gly m 5, Gly m 6, two different trypsin inhibitors, and Gly m Bd 28K) (85). This methodology was later adapted and refined to measure ten confirmed and putative endogenous soybean allergens (Gly m 1, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28K, Gly m Bd 30K, two trypsin inhibitors, and Gly m 8); it was also validated to meet endogenous soybean regulatory requirements (20). A ring trial was performed for the later study between six laboratories to demonstrate the accuracy, precision, and robustness of the methodology. While there are differences in the specific methodology between the two protocols, data generated spanning multiple years are in general agreement with regard to the concentration ranges of endogenous allergen proteins found in soybeans. In addition, endogenous allergen levels detected by the described LC-MS/MS protocols are in the range of data from published ELISA methodologies (17). This agreement between methodologies demonstrates the suitability of both technologies for collecting data to support endogenous allergen quantification to support EFSA requirements. 85
Figure 4. Example peptide selection for Gly m 1 across identified isoforms. The nonsimilar, conservative, identical, and weakly similar residues are highlighted in white, blue, yellow, and green, respectively. The Gly m 1 peptide SYPSNATCPR (arrow) shows the greatest coverage across isoforms and is a candidate surrogate peptide for further method development.
Summary GM crops have been in the food supply for more than twenty years and in 2016 were planted on 185.1 million hectares in 26 countries (86). One public health question with these crops is the potential increase in allergenicity as an unintended consequence of transformation, especially in already allergenic crops such as soybeans. Numerous studies detailing the identification of soybean allergens have been completed, and, to date, ten potentially clinically relevant food allergens have been identified. Currently, several analytic methods such as 2-DE gel and 2-DE western blot, human IgE binding, ELISA, and LC-MS can be used to compare individual allergen levels among GM, conventional, and commercially available conventional soybean varieties. Since the goal of the allergenicity assessment for a GM soybean is to compare allergen levels between the GM variety and the near isogenic control, the absolute measurement of allergen amounts present in the GM crop is of low importance for the safety assessment. Instead the ability to accurately and reliably detect any differences in allergen expression levels between the GM soybean relative to the conventional control and commercially available conventional varieties is critical for meaningful interpretation. In addition, the environmental factors associated with the various growing seasons (Table 2) were the major contributor to the natural variability of most soybean allergens. Most importantly, the statistical significance of any observed differences between GM and non-GM soybean should be assessed in the context of the range of natural variations for that parameter to determine its biological relevance (16). 86
Table 2. Mean Values of Five Endogenous Allergens (mg/g fwa) across Seasons Associated with Soybean Field Trials. Reprinted with the permission from reference (17). Copyright 2017 American Chemical Society. Growing Seasons
a
Gly m 4
Gly m 5
Gly m 6
Gly m Bd 28k
Gly m Bd 30k
2009
0.13 a
37.9 a
218 a
0.17 b
1.23 c
2010
0.15 a
38.5 a
212 a
0.18 b
1.38 b
2012
0.05 b
35.0 a
158 b
0.18 b
2.34 a
2013
0.13 a
27.0 b
141 b
0.17 b
0.99 de
2013/2014
0.11 a
38.3 ab
128 b
0.28 a
0.84 e
Differencesb
*
*
*
*
*
fw = fresh weight.
b
Statistical difference at 0.05 significance level. Different letters indicate statistical difference among values of the same column.
87
References 1. 2. 3. 4.
5. 6. 7.
8.
9. 10.
11.
12. 13. 14. 15.
16. 17.
Hefle, S. L.; Nordlee, J. A.; Taylor, S. L. Allergenic Foods. Crit. Rev. Food Sci. Nutr. 1996, 36, S69–S89. Sampson, H. A. 9. Food Allergy. J. Allergy Clin. Immunol. 2003, 111, S540–S547. Sicherer, S. H.; Sampson, H. A. Food Allergy. J. Allergy Clin. Immunol. 2010, 125, S116–S125. Venter, C.; Pereira, B.; Grundy, J.; Clayton, C. B.; Roberts, G.; Higgins, B.; Dean, T. Incidence of Parentally Reported and Clinically Diagnosed Food Hypersensitivity in the First Year of Life. J. Allergy Clin. Immunol. 2006, 117, 1118–1124. Valenta, R.; Hochwallner, H.; Linhart, B.; Pahr, S. Food Allergies: The Basics. Gastroenterology 2015, 148, 1120–1131.e4. Food Allergen Labeling and Consumer Protection Act of 2004; Food and Drug Administration: Washington, DC, 2004. Food Allergies. Report of the Technical Consultation of the Food and Agriculture Organization of the United Nations; Food and Agriculture Organization of the United Nations: Rome, 1995. Nwaru, B.; Hickstein, L.; Panesar, S.; Roberts, G.; Muraro, A.; Sheikh, A.; Allergy, E. F.; Group, A. G. Prevalence of Common Food Allergies in Europe: A Systematic Review and Meta‐Analysis. Allergy 2014, 69, 992–1007. Clarke, B. C.; Hobbs, M.; Skylas, D.; Appels, R. Genes Active in Developing Wheat Endosperm. Funct. Integr. Genomics 2000, 1, 44–55. Jenkins, J. A.; Griffiths-Jones, S.; Shewry, P. R.; Breiteneder, H.; Mills, E. N. Structural Relatedness of Plant Food Allergens with Specific Reference to Cross-Reactive Allergens: An in silico Analysis. J. Allergy Clin. Immunol. 2005, 115, 163–170. Van Loon, L. C.; Van Strien, E. A. The Families of Pathogenesis-Related Proteins, Their Activities, and Comparative Analysis of PR-1 Type Proteins. Physiol. Mol. Plant Pathol. 1999, 55, 85–97. Breiteneder, H.; Ebner, C. Molecular and Biochemical Classification of Plant-Derived Food Allergens. J. Allergy Clin. Immunol. 2000, 106, 27–36. Hoffmann-Sommergruber, K. Pathogenesis-Related (PR)-Proteins Identified as Allergens. Biochem. Soc. Trans. 2002, 30, 930–935. Breiteneder, H.; Ebner, C. Atopic Allergens of Plant Foods. Curr. Opin. Allergy Clin. Immunol. 2001, 1, 261–267. FAO. Evaluation of Allergenicity of Genetically Modified Foods; Report of a Joint FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology: Rome, Italy, January 2001; pp 22–25. Codex. Foods Derived from Modern Biotechnology; Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme: Rome, 2009. 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 88
18.
19.
20.
21.
22.
23.
24. 25.
26.
27.
28. 29.
America from Five Production Years. J. Agric. Food Chem. 2017, 65, 463–472. Hill, R. C.; Fast, B. J.; Herman, R. A. Transgenesis Affects Endogenous Soybean Allergen Levels less than Traditional Breeding. Regul. Toxicol. Pharmacol. 2017, 89, 70–73. Hill, R. C.; Oman, T. J.; Wang, X.; Shan, G.; Schafer, B.; Herman, R. A.; Tobias, R.; Shippar, J.; Malayappan, B.; Sheng, L. Development, Validation, and Interlaboratory Evaluation of a Quantitative Multiplexing Method to Assess Levels of Ten Endogenous Allergens in Soybean Seed and Its Application to Field Trials Spanning Three Growing Seasons. J. Agric. Food Chem. 2017, 65, 5531–5544. McClain, S.; Stevenson, S. E.; Brownie, C.; Herouet-Guicheney, C.; Herman, R. A.; Ladics, G. S.; Privalle, L.; Ward, J. M.; Doerrer, N.; Thelen, J. J. Variation in Seed Allergen Content From Three Varieties of Soybean Cultivated in Nine Different Locations in Iowa, Illinois, and Indiana. Frontiers in plant science 2018, 9, 1025–1035. Herman, R. A.; Price, W. D. Unintended Compositional Changes in Genetically Modified (GM) Crops: 20 Years of Research. J. Agric. Food Chem. 2013, 61, 11695–11701. OECD. Revised Consensus Document on Compositional Considerations for New Variety of Soybean (ENV/JM/MONO(2012)24); Consensus Document for Organization of Economic Cooperation and Development: Paris, 2012. Holzhauser, T.; Wackermann, O.; Ballmer-Weber, B. K.; BindslevJensen, C.; Scibilia, J.; Perono-Garoffo, L.; Utsumi, S.; Poulsen, L. K.; Vieths, S. Soybean (Glycine Max) Allergy in Europe: Gly m 5 (β-conglycinin) and Gly m 6 (Glycinin) are Potential Diagnostic Markers for Severe Allergic Reactions to Soy. J. Allergy Clin. Immunol. 2009, 123, 452–458.e4. EFSA. Guidance on Allergenicity Assessment of Genetically Modified Plants. EFSA Journal 2017, 15, e04862. EFSA Panel on Genetically Modified Organisms (GMO). Guidance for Risk Assessment of Food and Feed from Genetically Modified Plants. EFSA Journal 2011, 9, 2150. Wilson, R. F. Seed Composition. In Soybeans: Improvement, Production and Uses, 3rd ed.; Boerma, H. R., Specht, J. E., Eds.; ASA, CSSA, SSA: Madison, WI, 2004. Kim, H. K.; Nelson-Dooley, C.; Della-Fera, M. A.; Yang, J. Y.; Zhang, W.; Duan, J. H.; Hartzell, D. L.; Hamrick, M. W.; Baile, C. A. Genistein Decreases Food Intake, Body Weight, and Fat Pad Weight and Causes Adipose Tissue Apoptosis in Ovariectomized Female Mice. J. Nutr. 2006, 136, 409–414. Omoni, A. O.; Aluko, R. E. Soybean Foods and Their Benefits: Potential Mechanisms of Action. Nutr. Rev. 2005, 63, 272–283. Ali, A. A.; Velasquez, M. T.; Hansen, C. T.; Mohamed, A. I.; Bhathena, S. J. Effects of Soybean Isoflavones, Probiotics, and Their Interactions on Lipid Metabolism and Endocrine System in an Animal Model of Obesity and Diabetes. J. Nutr. Biochem. 2004, 15, 583–590. 89
30. Friedman, M.; Brandon, D. L. Nutritional and Health Benefits of Soy Proteins. J. Agric. Food Chem. 2001, 49, 1069–1086. 31. Carroll, K. K.; Kurowska, E. M. Soy Consumption and Cholesterol Reduction - Review of Animal and Human Studies. J. Nutr. 1995, 125, S594–S597. 32. European Union. Commission Directive 2007/68/EC of 27 November 2007, Amending Annex IIIa to Directive 2000/13/EC of the European Parliament and of the Council as Regards Certain Food Ingredients; Commission Directive for the European Union: November 2007. 33. L’Hocine, L.; Boye, J. I. Allergenicity of Soybean: New Developments in Identification of Allergenic Proteins, Cross-Reactivities and Hypoallergenization Technologies. Crit. Rev. Food Sci. Nutr. 2007, 47, 127–143. 34. Ladics, G. S.; Budziszewski, G. J.; Herman, R. A.; Herouet-Guicheney, C.; Joshi, S.; Lipscomb, E. A.; McClain, S.; Ward, J. M. Measurement of Endogenous Allergens in Genetically Modified Soybeans - Short Communication. Regul. Toxicol. Pharmacol. 2014, 70, 75–79. 35. Selb, R.; Wal, J. M.; Moreno, F. J.; Lovik, M.; Mills, C.; HoffmannSommergruber, K.; Fernandez, A. Assessment of Endogenous Allergenicity of Genetically Modified Plants Exemplified by Soybean - Where Do We Stand? Food Chem. Toxicol. 2017, 101, 139–148. 36. Ballmer-Weber, B. K.; Holzhauser, T.; Scibilia, J.; Mittag, D.; Zisa, G.; Ortolani, C.; Oesterballe, M.; Poulsen, L. K.; Vieths, S.; Bindslev-Jensen, C. Clinical Characteristics of Soybean Allergy in Europe: A Double-Blind, Placebo-Controlled Food Challenge Study. J. Allergy Clin. Immunol. 2007, 119, 1489–1496. 37. Batista, R.; Martins, I.; Jeno, P.; Ricardo, C. P.; Oliveira, M. M. A Proteomic Study to Identify Soya Allergens--The Human Response to Transgenic Versus Non-Transgenic Soya Samples. Int. Arch. Allergy Immunol. 2007, 144, 29–38. 38. Magnolfi, C. F.; Zani, G.; Lacava, L.; Patria, M. F.; Bardare, M. Soy Allergy in Atopic Children. Ann. Allergy Asthma Immunol. 1996, 77, 197–201. 39. Wensing, M.; Penninks, A. H.; Hefle, S. L.; Koppelman, S. J.; BruijnzeelKoomen, C. A.; Knulst, A. C. The Distribution of Individual Threshold Doses Eliciting Allergic Reactions in a Population with Peanut Allergy. J. Allergy Clin. Immunol. 2002, 110, 915–920. 40. Zeiger, R. S.; Sampson, H. A.; Bock, S. A.; Burks, A. W., Jr.; Harden, K.; Noone, S.; Martin, D.; Leung, S.; Wilson, G. Soy Allergy in Infants and Children with IgE-Associated Cow’s Milk Allergy. J. Pediatr. 1999, 134, 614–622. 41. Taylor, S. L.; Hefle, S. L.; Bindslev-Jensen, C.; Bock, S. A.; Burks, A. W.; Christie, L.; Hill, D. J.; Host, A.; Hourihane, J. O.; Lack, G.; Metcalfe, D. D.; Moneret-Vautrin, D. A.; Vadas, P. A.; Rance, F.; Skrypec, D. J.; Trautman, T. A.; Yman, I. M.; Zeiger, R. S. Factors Affecting the Determination of Threshold Doses for Allergenic Foods: How Much is Too Much? J. Allergy Clin. Immunol. 2002, 109, 24–30. 90
42. Commission Implementing Regulation (EU); No 503/2013 on Applications for Authorisation of Genetically Modified Food and Feed in Accordance with Regulation (EC) No 1829/2003 of the European Parliament and of the Council and amending Commission Regulations (EC) No 641/2004 and (EC) No 1981/2006; Report for Official Journal of the European Union: 2013. 43. Goodman, R. E.; Vieths, S.; Sampson, H. A.; Hill, D.; Ebisawa, M.; Taylor, S. L.; van Ree, R. Allergenicity Assessment of Genetically Modified Crops-What Makes Sense? Nat. Biotechnol. 2008, 26, 73–81. 44. Goodman, R. E.; Panda, R.; Ariyarathna, H. Evaluation of Endogenous Allergens for the Safety Evaluation of Genetically Engineered Food Crops: Review of Potential Risks, Test Methods, Examples and Relevance. J. Agric. Food Chem. 2013, 61, 8317–8332. 45. Herman, R. A.; Ladics, G. S. Endogenous Allergen Upregulation: Transgenic vs. Traditionally Bred Crops. Food Chem. Toxicol. 2011, 49, 2667–2669. 46. Dunn, S. E.; Vicini, J. L.; Glenn, K. C.; Fleischer, D. M.; Greenhawt, M. J. The Allergenicity of Genetically Modified Foods from Genetically Engineered Crops. Ann. Allergy Asthma Immunol. 2017, 119, 214–222.e3. 47. Sten, E.; Skov, P. S.; Andersen, S. B.; Torp, A. M.; Olesen, A.; BindslevJensen, U.; Poulsen, L. K.; Bindslev-Jensen, C. A Comparative Study of the Allergenic Potency of Wild-Type and Glyphosate-Tolerant Gene-Modified Soybean Cultivars. APMIS : Acta Pathologica, Microbiologica, et Immunologica Scandinavica 2004, 112, 21–28. 48. Codina, R.; Ardusso, L.; Lockey, R. F.; Crisci, C.; Medina, I. Allergenicity of Varieties of Soybean. Allergy 2003, 58, 1293–1298. 49. Stevenson, S. E.; Woods, C. A.; Hong, B.; Kong, X.; Thelen, J. J.; Ladics, G. S. Environmental Effects on Allergen Levels in Commercially Grown NonGenetically Modified Soybeans: Assessing Variation Across North America. Frontiers in Plant Science 2012, 3, 196. 50. Geng, T.; Liu, K.; Frazier, R.; Shi, L. F.; Bell, E.; Glenn, K.; Ward, J. M. Development of a Sandwich ELISA for Quantification of Gly m 4, a Soybean Allergen. J. Agric. Food Chem. 2015, 63, 4947–4953. 51. Hill, R. C.; Oman, T. J.; Wang, X.; Shan, G.; Schafer, B.; Herman, R. A.; Tobias, R.; Shippar, J.; Malayappan, B.; Sheng, L.; Xu, A.; Bradshaw, J. Development, Validation, and Interlaboratory Evaluation of a Quantitative Multiplexing Method to Assess Levels of Ten Endogenous Allergens in Soybean Seed and Its Application to Field Trials Spanning Three Growing Seasons. J. Agric. Food Chem. 2017, 65, 5531–5544. 52. Gendel, S. M. Comparison of International Food Allergen Labeling Regulations. Regul. Toxicol. Pharmacol. 2012, 63, 279–85. 53. Allen, K. J.; Turner, P. J.; Pawankar, R.; Taylor, S.; Sicherer, S.; Lack, G.; Rosario, N.; Ebisawa, M.; Wong, G.; Mills, E. N.; Beyer, K.; Fiocchi, A.; Sampson, H. A. Precautionary Labelling of Foods for Allergen Content: Are We Ready for a Global Framework? The World Allergy Organization Journal 2014, 7, 10.
91
54. Kirsch, S.; Fourdrilis, S.; Dobson, R.; Scippo, M. L.; Maghuin-Rogister, G.; De Pauw, E. Quantitative Methods for Food Allergens: A Review. Anal. Bioanal. Chem. 2009, 395, 57–67. 55. Sancho, A. I.; Mills, E. N. Proteomic Approaches for Qualitative and Quantitative Characterisation of Food Allergens. Regul. Toxicol. Pharmacol. 2010, 58, S42–S46. 56. Burks, A.; Fuchs, R. L. Assessment of the Endogenous Allergens in Glyphosate tolerant and Commercial Soybean Varieties. J. Allergy Clin. Immunol. 1995, 96, 1008–1010. 57. Batista, R.; Oliveira, M. Plant Natural Variability May Affect Safety Assessment Data. Regul. Toxicol. Pharmacol. 2010, 58, S8–S12. 58. Rouquie, D.; Capt, A.; Eby, W. H.; Sekar, V.; Herouet-Guicheney, C. Investigation of Endogenous Soybean Food Allergens by Using a 2-Dimensional Gel Electrophoresis Approach. Regul. Toxicol. Pharmacol. 2010, 58, S47–S53. 59. Ruebelt, M. C.; Leimgruber, N. K.; Lipp, M.; Reynolds, T. L.; Nemeth, M. A.; Astwood, J. D.; Engel, K. H.; Jany, K. D. Application of Two-Dimensional Gel Electrophoresis to Interrogate Alterations in the Proteome of Genetically Modified Crops. 1. Assessing Analytical Validation. J. Agric. Food Chem. 2006, 54, 2154–2161. 60. Nair, B.; Wheeler, J. C.; Sykes, D. E.; Brown, P.; Reynolds, J. L.; Aalinkeel, R.; Mahajan, S. D.; Schwartz, S. A. Proteomic Approach to Evaluate Mechanisms that Contribute to Food Allergenicity: Comparative 2D-DIGE Analysis of Radioallergosorbent Test Positive and Negative Patients. Int. J. Proteomics 2011, 2011, 673618. 61. Choe, L. H.; Lee, K. H. Quantitative and Qualitative Measure of Intralaboratory Two-Dimensional Protein Gel Reproducibility and the Effects of Sample Preparation, Sample Load, and Image Analysis. Electrophoresis 2003, 24, 3500–3507. 62. Voss, T.; Haberl, P. Observations on the Reproducibility and Matching Efficiency of Two-Dimensional Electrophoresis Gels: Consequences for Comprehensive Data Analysis. Electrophoresis 2000, 21 (16), 3345–3350. 63. Satoh, R.; Teshima, R.; Kitta, K.; Lang, G. H.; Schegg, K.; Blumenthal, K.; Hicks, L.; Labory-Carcenac, B.; Rouquié, D.; Herman, R. A.; HerouetGuicheney, C.; Ladics, G. S.; McClain, S.; Poulsen, L. K.; Privalle, L.; Ward, J. M.; Doerrer, N.; Rascle, J. B. Inter-Laboratory Optimization of Protein Extraction, Separation, and Fluorescent Detection of Endogenous Rice Allergens. Biosci., Biotechnol., Biochem. 2016, 80, 2198–2207. 64. Bland, A. M.; D’Eugenio, L. R.; Dugan, M. A.; Janech, M. G.; Almeida, J. S.; Zile, M. R.; Arthur, J. M. Comparison of Variability Associated with Sample Preparation in Two-Dimensional Gel Electrophoresis of Cardiac Tissue. J. Biomol. Tech. 2006, 17, 195–199. 65. Bland, A. M.; Janech, M. G.; Almeida, J. S.; Arthur, J. M. Sources of Variability Among Replicate Samples Separated by Two-Dimensional Gel Electrophoresis. J. Biomol. Tech. 2010, 21, 3–8. 66. Terry, D. E.; Desiderio, D. M. Between-Gel Reproducibility of the Human Cerebrospinal Fluid Proteome. Proteomics 2003, 3, 1962–1979. 92
67. Holzhauser, T.; R. van Ree; Poulsen, L. K.; Bannon, G. A. Analytical Criteria for Performance Characteristics of IgE Binding Methods for Evaluating Safety of Buitech Food Products. Food Chem. Toxicol. 2008, 46, S15–S19. 68. EFSA Panel on Genetically Modified Organisms (GMO Panel). Scientific Opinion on the Assessment of Allergenicity of GM Plants and Microorganisms and Derived Food and Feed. EFSA Journal 2010, 8, 1700. 69. Poms, R. E.; Klein, C. L.; Anklam, E. Methods for Allergen Analysis in Food: A Review. Food Addit. Contam. 2004, 21, 1–31. 70. Kirsch, S.; Fourdrilis, S.; Dobson, R.; Scippo, M.-L.; Maghuin-Rogister, G.; De Pauw, E. Quantitative Methods for Food Allergens: A Review. Anal. Bioanal. Chem. 2009, 395, 57–67. 71. Schubert-Ullrich, P.; Rudolf, J.; Ansari, P.; Galler, B.; Fuehrer, M.; Molinelli, A.; Baumgartner, S. Commercialized Rapid Immunoanalytical Tests for Determination of Allergenic Food Proteins: An Overview. Anal. Bioanal. Chem. 2009, 395, 69–81. 72. Gonzalez, R.; Duffort, O.; Calabozo, B.; Barber, D.; Carreira, J.; Polo, F. Monoclonal Antibody-Based Method to Quantify Gly m 1. Its Application to Assess Environmental Exposure to Soybean Dust. Allergy 2000, 55, 59–64. 73. Amnuaycheewa, P.; de Mejia, E. G. Purification, Characterisation, and Quantification of the Soy Allergen Profilin (Gly m 3) in Soy Products. Food Chem. 2010, 119, 1671–1680. 74. Liu, B.; Teng, D.; Wang, X. M.; Yang, Y. L.; Wang, J. H. Expression of the Soybean Allergenic Protein P34 in Escherichia coli and Its Indirect ELISA Detection Method. Appl. Microbiol. Biot. 2012, 94, 1337–1345. 75. Tsuji, H.; Okada, N.; Yamanishi, R.; Bando, N.; Kimoto, M.; Ogawa, T. Measurement of Gly-M-Bd-30k, a Major Soybean Allergen, in Soybean Products by a Sandwich Enzyme-Linked-Immunosorbent-Assay. Biosci. Biotech. Bioch. 1995, 59, 150–151. 76. Liu, B.; Teng, D.; Wang, X. M.; Wang, J. H. Detection of the Soybean Allergenic Protein Gly m Bd 28K by an Indirect Enzyme-Linked Immunosorbent Assay. J. Agric. Food Chem. 2013, 61, 822–828. 77. Bando, N.; Tsuji, H.; Hiemori, M.; Yoshizumi, K.; Yamanishi, R.; Kimoto, M.; Ogawa, T. Quantitative Analysis of Gly m Bd 28K in Soybean Products by a Sandwich Enzyme-Linked Immunosorbent Assay. J. Nutr. Sci. Vitaminol. 1998, 44, 655–664. 78. Ma, X.; Sun, P.; He, P. L.; Han, P. F.; Wang, J. J.; Qiao, S. Y.; Li, D. F. Development of Monoclonal Antibodies and a Competitive ELISA Detection Method for Glycinin, an Allergen in Soybean. Food Chem. 2010, 121, 546–551. 79. Liu, B.; Teng, D.; Yang, Y. L.; Wang, X. M.; Wang, J. H. Development of a Competitive ELISA for the Detection of Soybean Alpha Subunit of BetaConglycinin. Process Biochem. 2012, 47, 280–287. 80. You, J. M.; Li, D.; Qiao, S. Y.; Wang, Z. R.; He, P. L.; Ou, D. Y.; Dong, B. Development of a Monoclonal Antibody-Based Competitive ELISA for Detection of Beta-Conglycinin, an Allergen from Soybean. Food Chem. 2008, 106, 352–360. 93
81. Brandon, D. L.; Bates, A. H.; Friedman, M. ELISA Analysis of Soybean Trypsin Inhibitors in Processed Foods. Adv. Exp. Med. Biol. 1991, 289, 321–337. 82. Hill, R. C.; Oman, T. J.; Shan, G.; Schafer, B.; Eble, J.; Chen, C. Development and Validation of a Multiplexed Protein Quantitation Assay for the Determination of Three Recombinant Proteins in Soybean Tissues by Liquid Chromatography with Tandem Mass Spectrometry. J. Agric. Food Chem. 2015, 63, 7450–7461. 83. Stevenson, S. E.; McClain, S.; Thelen, J. J. Development of an Isoform-Specific Tandem Mass Spectrometry Assay for Absolute Quantitation of Maize Lipid Transfer Proteins. J. Agric. Food. Chem. 2015, 63, 821–828. 84. Lee, D.-G.; Houston, N. L.; Stevenson, S. E.; Ladics, G. S.; McClain, S.; Privalle, L.; Thelen, J. J. Mass Spectrometry Analysis of Soybean Seed Proteins: Optimization of Gel-Free Quantitative Workflow. Anal. Methods. 2010, 2, 1577–1583. 85. Houston, N. L.; Lee, D. G.; Stevenson, S. E.; Ladics, G. S.; Bannon, G. A.; McClain, S.; Privalle, L.; Stagg, N.; Herouet-Guicheney, C.; MacIntosh, S. C.; Thelen, J. J. Quantitation of Soybean Allergens Using Tandem Mass Spectrometry. J. Proteome Res. 2011, 10, 763–773. 86. James, C. Global Status of Commercialized Biotech/GM Crops: 2017. ISAAA Brief. 2017, 52, 303.
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