Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA

Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.     

Effect of protein glycation in the presence or absence of wheat proteins on detection of soybean proteins by commercial ELISA

Soybean (Glycine max) is the world's primary provider of protein and oil and is widely used in foodstuffs. However, the use of soybean in foodstuffs might pose a serious threat to allergic consumers since some proteins can cause allergic reactions. To date mostly ELISA methods are used for testing contamination of foodstuffs with soybean. In view of the complexity regarding allergen detection in foodstuffs and appropriate food product labelling, the aim of this study was to investigate the impact of the Maillard reaction on the detectability of soybean proteins using commercial ELISA kits. Accumulation of protein-bound carbonyls, modification of reactive lysine residues and severe aggregation as a result of incubation with glucose, in the presence or absence of soluble wheat proteins, were recorded. Moreover, detection of soybean proteins by means of three commercial ELISA kits was strongly altered and was highly dependent on the type of kit used.     

Detection of the allergenic celery protein component (Api g 1.01) in foods by immunoassay

Among food allergens, celery is a frequent cause for adverse food reactions in allergic patients. In this study, the celery allergen protein Api g 1.01 RNA was amplified by RT-PCR and cloned into the pET-32a expression vector. The recombinant plasmid was transformed into E.coli BL21(DE3) pLys for the expression of protein Api g 1.01. Monoclonal antibodies were prepared against the expressed purified Api g 1.01 protein. A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of celery soluble proteins in processed foods was developed using the prepared monoclonal antibodies. The developed ELISA had a high specificity, although it showed slight cross-reactivity to carrot. The limit of quantification (LOQ) was 0.28 μg/mL (equivalent to 5.6 μg whole celery protein/g food sample). The recovery ranged from 83 to 115%, whereas coefficients of variation were 6.7–8.9%.     

Development of monoclonal antibodies and a competitive ELISA detection method for glycinin,an allergen in soybean

Soybean glycinin is a major food allergen causing anaphylaxis. A sensitive detection method for glycinin is needed to evaluate soybean allergies in food and feed products. In the present study, monoclonal antibodies (Mabs) against glycinin were prepared using purified glycinin as the immunogen. The generated Mabs, named 3B2 and 4B2, were identified as being IgG_2b and IgG_2a iso-types respectively, and exhibited high specificity to glycinin. Then we developed a competitive ELISA based on Mab 4B2 to measure glycinin which showed an IC₅₀ value of 1.7 ng/mL with a detection limit of 0.3 ng/mL, and the linear portion of the curve was 0.3–11.2 ng/mL. Recovery tests indicated that the competitive ELISA based on Mab 4B2 gave reliable reproducibility. The produced Mab 4B2 and the developed ELISA could provide a valuable tool for sensitive determination of glycinin and for future studies conducted to reveal the mechanism of how glycinin functions in anaphylaxis.     

A Sensitive Sandwich ELISA for the Rapid Detection of Mung Bean Protein: Development and Evaluation of the Effect of Thermal Processing on Detection

The mung bean allergen has not yet attracted attention on a global scale for the potential hazards it poses to allergen-sensitive individuals. In the current study, a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of trace amounts of mung bean proteins was established. Mung bean protein-specific antibodies produced in rabbits and mice immunized with protein extracted from mung bean flour were used as the capture and detection antibodies in the ELISA. The ELISA had a limit of detection of 4.99 ng/mL and did not show any cross-reactivity with nine different foods, which potentially coexisted in foodstuffs. Accuracy and repeatability were validated by spiking and recovery of mung bean protein in oat meal, milk, and soybean milk. The suitability of the ELISA for the detection of mung bean protein in thermally processed samples was established by subjecting mung bean protein, mung bean powder, and defatted mung bean powder to dry or moist heating. The results demonstrated that the accurate quantitation of mung bean protein can be established for samples processed by dry heating at ≤150 °C. Lower detection results could not be avoided for mung bean proteins subjected to either dry heating at temperatures >150 °C or with moist heat, most probably because of changes in protein solubility and structure of the mung bean protein.     

Development of a monoclonal antibody-based competitive ELISA for detection of β-conglycinin,an allergen from soybean

Soybean β-conglycinin is one of the major food allergies for children and young animals. In order to detect immunoreactive β-conglycinin from soybean and soybean products, monoclonal antibodies against β-conglycinin were prepared using a conjugated chicken ovalbumin with a synthetic peptide that corresponded to one epitope sequence of β-conglycinin as the immunogen. The generated monoclonal antibodies, named as 6G₄, 3B₇, and 5F₁₀, were identified as being of IgG₁ isotype and exhibited high specificity to β-conglycinin. A competitive enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody 6G₄ was established to determine β-conglycinin and showed an IC₅₀ value of 4.7 ng/mL with a detection limit of 2.0 ng/mL. The recovery tests of β-conglycinin indicated that the monoclonal antibody 6G₄-based competitive ELISA gave reliable reproducibility. Therefore, the produced monoclonal antibody 6G₄ and the developed competitive ELISA could provide a valuable tool for sensitive determination of β-conglycinin and for future studies on food allergies related to soybean β-conglycinin.     

Quantification of Human IgE Immunoreactive Soybean Proteins in Commercial Soy Ingredients and Products

ABSTRACT:  The objective of this study was the detection and quantification of human IgE immunoreactive soybean proteins in commercially available soy ingredients and products. Optimum dilutions of primary antibody and antigens as well as detection sensitivity were determined for the implementation of a sandwich ELISA method using plasma from soy sensitive subjects (IgE ranging from 0.35 to 98.7 IU/mL). Human IgE immunoreactivity of commercial soybean ingredients showed that the plasma of subjects with strong allergic reaction to soybean presented proportionally higher immunoreactive response. Soy protein isolate and soy protein concentrate contained less immunoreactive proteins than soy flour and grits. As expected, a hypoallergenic soybean product presented the lowest IgE immunoreactivity. Hydrolyzed and fermented soy ingredients showed negligible human IgE immunoreactivity when proteins and peptides were < 20 kDa. The IgE immunoreactivity of soymilk samples ranged from 3.4 to 68.9 ng IgE/mg extracted protein. Tofu contained about 20-fold higher IgE immunoreactivity than soymilk products (median 171 ng IgE/mg extracted protein). Furthermore, soy cheese products presented twice the IgE immunoreactivity than tofu products (median 359 ng IgE/mg protein). Meat analogues presented considerably high extracted protein concentration (median 67.9 mg/g product). The findings of the current investigation demonstrate sandwich ELISA as a reliable immunochemical method with good repeatability, sensitivity, and low detection limit to quantify IgE immunoreactive proteins in soy ingredients and products. Quantitative measurement of specific IgE is likely to become an increasingly valuable tool for soybean industry to comply with food labeling for manufacturers, thus protecting soy-sensitive consumers.     

Immunoassays for Bowman-Birk and Kunitz Soybean Trypsin Inhibitors in Infant Formula

ABSTRACT: Because the consumption of soybean inhibitors of digestive enzymes in processed foods may have both beneficial and adverse health-related effects, reliable and rapid analytical methods for these inhibitors are needed. Monoclonal antibody-based sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the 2 major soybean protease inhibitors, the Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) of trypsin and chymotrypsin. The ELISAs had limits of quantification of approximately 1 and 3 ng/mL for BBI and KTI, respectively, and were used to measure active inhibitors in soy infant formulas. Results were compared with enzymatic analyses and demonstrated that most of the trypsin- and chymotrypsin-inhibitory activities of infant formula were due to constituents other than KTI and BBI. The sandwich ELISA for BBI was also effective in detecting soybean germplasm with atypically low levels of BBI.     

基于Exo III信号放大的荧光适配体传感器检测Kunitz型大豆胰蛋白酶抑制因子

Kunitz型大豆胰蛋白酶抑制因子（Kunitz Soybean Trypsin Inhibitor，KTI）是一种很关键的抗营养因子，不仅对动物消化系统和生长发育有不良影响，还制约各个行业对大豆的利用率，因此快速有效的检测方法是非常必要的。该研究建立一种基于核酸外切酶III（Exonuclease III，Exo III）和碳纳米颗粒（Carbon Nanoparticles，CNPs）的信号辅助放大荧光传感体系用于KTI的检测。具体体系包括KTI适配体（Aptamer，APT）、互补链（Complementary DNA，cDNA）、信号探针（Signal Probe，SP）、Exo III和CNPs共5个部分。通过可行性分析和CNPs浓度优化试验，测得KTI在100~600 ng/mL范围内呈线性相关，检测限为12.59 ng/mL。以豆浆作为样品，采用加标回收测得回收率为97.42%~102.85%，RSD在0.61%~2.36%之间，该方法可对实际样品中的KTI进行测定。     

Purification and Quantification of Kunitz Trypsin Inhibitor in Soybean Using Two-Dimensional Liquid Chromatography

Kunitz trypsin inhibitor (KTI) is one of the major antinutritional factors in soybean and results in inhibition of digestion of dietary protein. In this study, we developed a novel strategy to purify and quantify KTI from soybean using two-dimensional liquid chromatography. Lipids from ground soybean were removed using hexane after which the ground soybean was extracted with protein extraction buffer. The crude extract was first purified by weak anion exchange chromatography, and then the fraction containing KTI was further separated by size exclusion chromatography. The fraction containing KTI was collected and analyzed by SDS-PAGE and electrospray ionization mass spectrometry. Results indicated that purified KTI has a molecular mass of 20 kDa and a purity of ~98% with inhibitory activity of 2425 TIU/mg protein. This assay was used for the quantification of KTI in soybean samples. The assay showed concentrations with a range between 7.81 and 500.00 μg/mL and a limit of detection of 0.12 mg/g. The recoveries of KTI in spiked soybean samples were between 82.19% and 86.65%, and intra- and interday precisions (% CV) were less than 7.35% and 8.42%. The developed method was used to analyze soybean samples from different sources and soybean products derived from different processing techniques, which demonstrated that the developed procedure provided an accurate and sensitive tool for separation and quantification of intact KTI in soybean.     

Detection of BT transgenic maize in foodstuffs

Different foodstuffs for humans and monogastric animals were analysed to detect CryIA(b)gene and quantify the CryIA(b) protein present in the transgenic maize used as an ingredient. Eight out of 32 foods obtained from the market showed to have been elaborated with transgenic Bt maize. Specific primers used to identify the transgenic event revealed that Mon810 was predominantly present in the foodstuffs. A commercial ELISA test allowed the quantification of the CryIA(b) protein in low processed foods, and found that 0.1 ppm was the highest value per gram of food. A Western blot carried out with immuno-purified polyclonal antibodies was capable of detecting both the intact or degraded CryIA(b) protein depending on the food assayed.     

Quantification of the thaumatin-like kiwi allergen by a monoclonal antibody-based ELISA

Thaumatin-like proteins (TLPs) have been established as a new family of fruit and pollen allergens. The aim of this study was to develop a two-site ELISA for the quantification of the thaumatin-like kiwi allergen (Act d 2) in kiwifruit extracts and kiwifruit-containing food products. Genomic DNA (gDNA) of Act d 2 was amplified and the deduced amino acid sequence was determined to obtain a primary structure. Act d 2 purified from kiwifruit extract by HPLC was identified by Edman degradation and MS. Balb/c mice were immunized with Act d 2 for the production of mAbs by hybridoma technology. The optimized ELISA measured Act d 2 concentrations ranging from 0.2 to 9.0 ng/mL, with intra- and interassay coefficients of variation of 3.65 and 10.44%, respectively. The developed ELISA is a useful method for the quantification of the thaumatin-like kiwi allergen in kiwifruit extracts as well as the allergen level in kiwifruit-containing food products. It may be a helpful analytical tool for the evaluation of the stability (integrity) of fruit allergen extracts for in vitro diagnosis.     

特布他林残留的酶联免疫检测方法的建立

采用自主制备的特布他林特异性抗体建立特布他林残留的间接竞争酶联免疫(ELISA)检测方法,对该检测体系的灵敏度、准确度、精密度、特异性进行测定,并将该检测方法与高效液相色谱(HPLC)法进行对比分析。结果表明：特布他林残留检测体系的检测范围为1～100ng/mL,灵敏度为0.47ng/mL,检测限为1ng/mL,回收率80%～99%,与盐酸克伦特罗、硫酸沙丁胺醇的交叉反应率分别为94.9%、93.9%,与盐酸莱克多巴胺及肾上腺素的交叉反应率小于0.01%。采用HPLC 法进行沙丁胺醇残留的检测时,其检测范围为10～200μg/mL,检测限为1μg/mL,回收率为79.2%～94.8%。ELISA 法与HPLC 法相比,灵敏度较高、特异性强、检测结果准确度相近,但在检测结果稳定性方面逊于HPLC 法。     

鸡蛋致敏蛋白免疫分析方法的建立

采用硫酸铵沉淀法和离子交换法分离纯化鸡蛋清得致敏蛋白后免疫兔子制备抗多克隆抗体,并建立快速、灵敏、特异性强的鸡蛋致敏蛋白酶联免疫检测方法。在优化条件下,鸡蛋致敏蛋白在1×10-4～10μg/mL质量浓度范围内与抑制率线性关系良好,其线性回归方程为y＝0.74145＋0.21692 lgC(r＝0.9971),半抑制浓度(IC50)和最低检测限(IC10)分别为为77.076、1.104ng/mL;样品加标回收率在98.29%～101.55%之间;且抗鸡蛋致敏蛋白多克隆抗体对火鸡蛋蛋白、鸭蛋蛋白、鹅蛋蛋白均具有交叉反应,与花生、小麦蛋白无交叉反应;批内和批间变异系数分别为4.63%和5.49%,且可在4℃可保存6个月以上,能够快速灵敏检测食品中鸡蛋致敏蛋白的存在。     

双酚A单克隆抗体的制备及酶联免疫分析方法的建立

采用活泼酯法将双酚A的结构类似物双酚酸与载体蛋白偶联制备人工抗原,用制备的人工抗原免疫BALB/c小鼠,采用聚乙二醇法进行细胞融合制备双酚A单克隆抗体,成功获得一株分泌抗双酚A单克隆抗体的细胞株3H1,经鉴定抗体属于IgG1亚型,轻链为κ,并建立了间接竞争酶联免疫分析法。线性范围为1～50 ng/mL,最低检测限为0.43 ng/mL,半数抑制浓度为6.56 ng/mL。回收率为82.83%～101.94%,变异系数为2.94%～12.95%。该方法具有较高的灵敏度和特异性,具有良好的应用前景。     

Effect of Processing on the Detectability of Pecan Proteins Assessed by Immunological and Proteomic Tools

The present study evaluates the effect of food processing on the antigenicity of pecan proteins as measured by enzyme-linked immunosorbent assay (ELISA). In addition, proteomic tools were used to identify potential pecan markers suitable for confirming the presence of pecan proteins in food and validating new methods developed to detect traces of the commodity. To assess the effects of processing on protein stability and antigenicity, pecan nuts were submitted to heat treatments and extracts were analysed by ELISA, sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. The ELISA method was able to detect pecan traces even after submitting the commodity to rigorous treatments, though these treatments affected the detectability to varying degrees. Proteomic assessment showed that the majority of pecan proteins were matched by homology to walnut proteins, which are more abundantly populated in the protein sequence databases. However, there were a few important exceptions: 7S vicilin, 11S legumin and putative allergen I1, unambiguously identified as pecan in origin. Interestingly, putative allergen I1 offered unique analytical advantages to be used as a pecan marker for validation and confirmation purposes.     

Development and validation of a sandwich ELISA for the determination of potentially allergenic lupine in food

The paper presents a sandwich enzyme linked immunosorbent assay (ELISA) for the detection of traces of lupine proteins in foods. Anti-lupine antibodies were produced by immunising a rabbit and a hen with a protein extract from white lupine flour. IgY were used as coating and IgG as secondary antibodies. The ELISA detects proteins from white (Lupinus albus) and blue (Lupinus angustifolius) lupine and, with a lower sensitivity, proteins from yellow (Lupinus luteus) lupine. The ELISA does not show any cross-reactivity with 34 plant species potentially used in lupine containing foodstuffs. Accuracy, repeatability, limit of detection (LOD) and limit of quantification (LOQ) were determined by analysing model biscuits and noodles containing from 0 to 10,000 ppm lupine flour. Lupine flour could be detected in the unprocessed doughs as well as in the processed products down to a spiking level of 1 ppm.     

大豆过敏原夹心ELISA和竞争ELISA方法的建立及其在加工食品中应用研究

本文以大豆混合过敏原为目标,建立了快速、便捷检测大豆过敏原的夹心酶联免疫吸附方法（sandwich-enzyme linked immunosorbent assay）和间接竞争酶联免疫吸附方法（indirect competitive enzyme-linked immunosorbent assay）,通过实际加工样品的回收实验、加标食品回收实验以及对真实食物样本的检测,对这两种方法进行了比较,确定了各自的适用范围。结果表明,夹心ELISA方法标准品浓度在0.0078~30 μg/mL范围内呈现出良好的线性关系,曲线方程为y=0.2333x+0.0692,决定系数R2=0.995。竞争ELISA方法的检测范围为10~100000 ng/mL,最低检测限为10 ng/mL。对购入橙汁进行加标回收实验,夹心ELISA检测后的回收率要高于竞争ELISA检测后的回收率,达100%以上;而对成分和加工方式都比较复杂的巧克力、牛肉酱、面包或蛋糕来说,竞争ELISA检测后的回收率要高于夹心ELISA检测后的回收率。对发酵类食物进行检测,竞争ELISA方法检测到的浓度要高于夹心ELISA,而对成分比较简单的食物比如芝麻糊、豆奶等进行检测时,夹心ELISA的检测浓度要略高于竞争ELISA。综上,竞争ELISA方法更适用于食物基质复杂,经过深度加工的食品,而夹心ELISA方法更适用于食物成分简单,轻加工后的食品,两种方法在各自的适用范围内均能实现较准确的检测。     

Determination of Nε-carboxymethyllysine in heated milk products by immunochemical methods

  N ^ε-Carboxymethyllysine (CML) is an important Maillard product which is formed in vivo and during food processing and heating, and which can therefore be used as a marker for heat damage of foodstuffs. A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect CML modifications on proteins. CML protein was synthesized and anti-CML antiserum was prepared, which recognized CML modifications specifically on CML proteins and proteins which were incubated with various carbohydrates. Heated milk and milk powder samples could be directly tested by ELISA without further clean-up procedures and the CML contents were determined in relation to reaction time and heating conditions. Positive results were confirmed by SDS-PAGE/immunoblotting using the same antiserum. ELISA proved to be a fast, specific, and easy-to-handle method to evaluate CML formation in heated milk products. Additionally, SDS-PAGE/immunoblotting can be helpful to detect CML also in insoluble food proteins. Received: 10 September 1998     

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8 μg/g pre-hydrolysis and 171.5 to 250.1 μg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3 μg/g pre-hydrolysis and 9.0 to 18.7 μg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0 μg/g pre-hydrolysis and 4.0 to 13.2 μg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion.