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.     

ELISA detection of hazelnut proteins: effect of protein glycation in the presence or absence of wheat proteins

Hazelnuts are widely used in the food industry, especially confectionary foods. Nevertheless, these nuts contain several allergenic proteins that may be unexpectedly present as contaminants in various foods and may pose a serious threat to allergic consumers. The enzyme-linked immunosorbent assay (ELISA) is the preferred method to assess the level of hazelnut protein contamination. It is commonly used by both the food industry and enforcement agencies. Several ELISA kits are commercially available. However, protein detectability by ELISA may be affected by severe changes that proteins undergo during processing. The aim of this study is therefore to investigate the impact of processing on the ability to detect hazelnut protein by four commercial ELISA kits. Hazelnut proteins in the presence or absence of soluble wheat proteins were modified with glucose via the Maillard reaction. Changes in hazelnut proteins, such as the formation of protein-bound carbonyls, losses of reactive lysine residues and free amino groups, and severe aggregation dramatically affected the hazelnut protein detection by the commercial kits. The observed impact was highly dependent on the type of ELISA kit used.     

ELISA-Based Detection of Soybean Proteins: A Comparative Study Using Antibodies Against Modified and Native Proteins

Soybean, the world’s primary provider of protein and oil, is widely used in foodstuffs which might pose a serious threat to allergic consumers due to the presence of some allergenic proteins. Enzyme-linked immunosorbent assay (ELISA) is the preferred method for the determination of allergen contamination in foodstuffs. Due to food processing, the antibody–antigen interaction in these routinely used methods are disrupted, therefore leading to erroneous results. A comparison between an ELISA using antibodies against modified soybean proteins and against the Kunitz trypsin inhibitor (KTI) is described. Limits of detection and quantification of 115.6 ng soybean protein/mL and 346.8 ng/mL were obtained for soybean-ELISA and 117.3 ng KTI/mL and 351.9 ng/mL for KTI-ELISA, respectively. Minimal cross-reactivity with other legumes and food ingredients were obtained. The applicability of these ELISAs was evaluated to detect the presence of soybean proteins in cookies. Both matrix interferences and the baking process affected analytical recovery. However, the recoveries were found to be much higher using the soybean-ELISA. The low recovery obtained using the KTI-ELISA might be due to the inability of the antibodies used to recognize the modified proteins. These results indicate that using antibodies developed towards allergens modified through food processing simulating reactions is a better approach to be used in food allergen detection.     

Effect of proteolysis during Cheddar cheese aging on the detection of milk protein residues by ELISA

Cow milk is a common allergenic food, and cow milk-derived cheese retains an appreciable level of allergenicity. The specific and sensitive detection of milk protein residues in foods is needed to protect milk-allergic consumers from exposure to undeclared milk protein residues contained in foods made with milk or milk-derived ingredients or made on shared equipment or in shared facilities with milk or milk-derived ingredients. However, during cheese ripening, milk proteins are degraded by chymosin and milk-derived and bacterial proteases. Commercial allergen-detection methods are not validated for the detection of residues in fermented or hydrolyzed products. The objective of this research was to evaluate commercially available milk ELISA kits for their capability to detect milk protein residues in aged Cheddar cheese. Cheddar cheese was manufactured at a local dairy plant and was aged at 5°C for 24 mo, with samples removed at various time points throughout aging. Milk protein residues and protein profiles were measured using 4 commercial milk ELISA kits and sodium dodecyl sulfate-PAGE. The ELISA data revealed a 90% loss of milk protein residue signal between the youngest and oldest Cheddar cheese samples (0.5 and 24 mo, respectively). Sodium dodecyl sulfate-PAGE analysis showed protein degradation throughout aging, with the highest level of proteolysis observed at 24 mo. Results suggest that current commercial milk ELISA methods can detect milk protein residues in young Cheddar cheese, but the detection signal dramatically decreases during aging. The 4 evaluated ELISA kits were not capable of detecting trace levels of milk protein residues in aged cheese. Reliable detection of allergen residues in fermented food products is critical for upholding allergen-control programs, maintaining product safety, and protecting allergic consumers. Furthermore, this research suggests a novel use of ELISA kits to monitor protein degradation as an indication of cheese ripening.     

Texturisation and modification of vegetable proteins for food applications using microbial transglutaminase

Microbial transglutaminase (MTG) isolated from Streptomyces mobaraensis has been available on a commercial scale for several years. MTG generates inter- and intramolecular cross-links between γ-carboxylamide groups of glutamine residues and ɛ-amino groups of lysine residues in proteins. Due to its great potential to improve various functional properties of proteins, MTG is mainly used to enhance texture, stability, and water binding. Application of MTG for the production of plant protein-based foodstuffs such as tofu, noodles, bread and bakery products, is still limited to raw materials from soybean and wheat. However, with the increasing demand for vegetarian foods, the utilisation of novel proteins as functional ingredients, e.g. from peas, lupins, sesame, and sunflower, seems promising. To open new horizons for MTG application, this review aims at demonstrating the actual potential of MTG in processing foodstuffs based on vegetable proteins. Particular focus was laid on novel plant protein sources suitable for cross-linking with MTG. Furthermore, strategies for improving texture and nutritive value of the proteins are discussed.     

Easy determination of the addition of soybean proteins to heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins

The addition of non-meat proteins to processed meat products is limited by regulations. Therefore, this work has investigated the determination of added soybean proteins in commercial heat-processed meat products prepared with turkey meat or pork-turkey meat blends that could also contain milk proteins. The method consisted of extracting proteins from the meat products in a Tris-HCl buffer (pH 8) and analysing the extract by high-performance liquid chromatography with a linear gradient water-acetonitrile containing 0.05% (v/v) TFA. This method enabled the detection and quantitation of up to 0.08 and 0.28% (w/w), respectively, of soybean proteins (related to 6 g initial product) in these products. Satisfactory precision and recovery data were established. Accuracy was evaluated by a comparison of soybean protein contents determined by the proposed method and the existing AOAC official method based on an enzyme-linked immunosorbent assay (ELISA) from which no statistically significant differences were observed.     

Towards a Comprehensive Validation of ELISA Kits for Food Allergens: Case 1—Egg

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose.     

Towards a Comprehensive Validation of ELISA Kits for Food Allergens. Case 2—Milk

There are several enzyme-linked immunosorbent assay (ELISA) kits in the market that have been proven to be useful for the determination of egg in foods. However, inconsistent results that are obtained when different kits are used make the selection of one kit over another very difficult. Two different approaches were used to help understand why results vary among kits. Different kits were used to analyze spiked egg material [NIST reference material (RM) 8445] in wheat flour (raw ingredients) and cookies containing egg as an ingredient baked for different periods of time (processed food). These results were compared with immunoblotting using conjugated antibodies from the commercial kits to determine the antibody specificity and sample extraction efficiency. ELISA results can be difficult to compare because reporting units differ among kits. Results from both ELISA and immunoblotting are in agreement regarding the decreased detection of proteins in baked cookie extracts. Moreover, immunoblotting showed that this reduction is due to reduced protein content in these extracts. However, a properly selected extraction solution may help improve the solubility of certain egg proteins in processed foods. Harmonization of the reporting unit system along with the use of a common reference material is recommended as the path forward in the standardization of detection methods for food allergens. This would assist the end user in making an informed decision regarding the selection of the most appropriate kit for his or her purpose.     

Comparison of commercially available ELISA kits with human sera-based detection methods for peanut allergens in foods

Undeclared peanut allergens as contaminants in foodstuffs represent a major health problem for sensitized persons. Various immunochemical techniques are employed to detect and quantify peanut allergens. There is an urgent need to compare and standardize those test systems to enable comparable allergen analyses of foodstuffs, comparable studies, and consequent and consistent measures against the presence of hidden peanut allergens. The present study compared commercially available peanut ELISA kits with human sera-based immunoassay techniques (dot blotting and Western blotting), enabling semiquantitative and quantitative detection, and identification of peanut contaminants in foodstuffs. Additionally, the effect of conventional roasting conditions on the detection and quantification of peanut with the selected methods was investigated.     

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.     

Evaluation of three commercial ELISA kits for rapid screening of deoxynivalenol in unpolished wheat

Evaluation of commercial ELISA kits for the screening of deoxynivalenol (DON) was carried out. Three kinds of commercial kits supplied by different companies were used. Three lots of naturally contaminated wheat and DON-free wheat (<0.05 microg/g) were used as samples. The values obtained from ELISA were compared with those of the HPLC-UV method. In the results obtained from 14 institutions using ELISA, the CV values were less than 17.6% for all the commercial kits. The DON-free sample was not detected as positive with any commercial kits. Also there was no negative finding among the naturally contaminated samples used in this experiment. Coefficients of determination in ELISA and HPLC analysis were 0.979-0.999. These results indicate that ELISA using any of the three commercial kits is efficient for the screening of DON in wheat.     

Characterization,antigenicity and detection of fish gelatine and isinglass used as processing aids in wines

Fish gelatine (FG) and isinglass (IG) are widely used in the pharmaceutical industry and as ingredients or processing aids in food production. Both products are the focus of interest since several countries, particularly the member states of the EU and Japan, USA, Australia and New Zealand, have introduced special labelling regulations for allergenic foodstuffs, such as fish and products thereof. Thus, there is a demand for a reliable and sensitive method for the detection of FG and IG in foodstuffs. In this study, the characterization of various FGs and IGs, polyclonal antibodies raised against fish collagen and the development of a sensitive indirect ELISA for the detection of FG and IG is described. The ELISA method detected ≤0.11 µg ml^?1 from all FGs and IGs tested. The indirect ELISA was applied to various experimental wines where FG and IG had been used as processing aids and to several commercial wines. No residues of FG and IG were detected in the experimental wines additionally treated with bentonite, a strong adsorbent for proteins, and successive filtration. Additionally, no residues were found in the commercial wines. However, amounts of up to 0.33 µg ml^?1 were found in some experimental wines not treated with bentonite and successive filtration. Therefore, wines may contain traces of FG and IG that were used as processing aids during wine production. However, treatment with bentonite in combination with additional filtration had a clear impact on these residues.     

Influence of baking time and matrix effects on the detection of milk allergens in cookie model food system by ELISA

Milk allergens are common allergens occurring in foods, therefore raising concern in allergic consumers. Enzyme-linked immunosorbent assay (ELISA) is, to date, the method of choice for the detection of food allergens by the food industry although, the performance of ELISA might be compromised when severe food processing techniques are applied to allergen-containing foods. In this paper we investigated the influence of baking time on the detection of milk allergens by using commercial ELISA kits. Baked cookies were chosen as a model food system and experiments were set up to study the impact of spiking a matrix food either before, or after the baking process. Results revealed clear analytical differences between both spiking methods, which stress the importance of choosing appropriate spiking methodologies for method validation purposes. Finally, since the narrow dynamic range of quantification of ELISA implies that dilution of samples is required, the impact of sample dilution on the quantitative results was investigated. All parameters investigated were shown to impact milk allergen detection by means of ELISA.     

Commercial Milk Enzyme‐Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk‐Derived Ingredients

Numerous commercial enzyme‐linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α‐, β‐, or κ‐casein) and whey proteins (α‐lactalbumin or β‐lactoglobulin). Nine commercially‐available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk‐derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk‐derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions.     

Modern immunoassays in meat-product analysis

The increased regulation of foodstuffs in modern society requires analytical methods which are easy to perform, sensitive, specific and relatively inexpensive. The basic antigen-antibody reaction provides means for very specific analytical procedures. Immunoassays are powerful analytical tools that permit the specific and rapid detection or measurement of antigens and haptens to which antibodies can be produced. Sensitive recognition of the interaction is made possible by labelling the analyte or antibody, mainly with radioisotope (RIA) and enzyme (ELISA). Wide applications of these modern immunoassays to food analysis began about 1980. The paper reviews investigations, where various types of RIA and ELISA were developed for the use in meat product analysis. Detection and determination of various meat species, non-meat proteins, microorganisms and bacterial toxins, drugs, anabolic hormones, pesticides, mycotoxins, and other contaminants in meat and meat products by the means of immunoassays is described. Now, the commercial kits are available for most of these compounds. They make possible to perform analysis in different laboratories under standard conditions. The reason of an enthusiasmic acceptance of this technology is related to its inherent specificity, high sensitivity, and the facility of application. In fact, immunoassays compete with other analytical technics. They have the advantage of economy when screening large numbers of samples.     

Determination of soybean proteins in commercial heat-processed meat products prepared with chicken,beef or complex mixtures of meats from different species

The addition of foreign proteins (mainly soybean proteins and milk proteins) to heat-processed meat products is a common practice. This work approaches the determination of additions of soybean proteins in heat-processed meat products prepared with chicken meat, beef meat, and complex mixtures of meats from different species (chicken, pork, beef, and turkey) by perfusion reversed-phase high-performance liquid chromatography. The applied method was previously developed for the determination of soybean proteins in pork and turkey meat products but it has never been tested for the determination of soybean proteins in other heat-processed meat products containing other kinds of meats. This paper demonstrates the validity of this method for the detection of soybean proteins in heat-processed meat products containing different varieties of meats and even in the presence of other foreign proteins such as milk proteins. The specificity and existence of matrix interferences have been checked for these samples and accuracy has been evaluated by the comparison of the soybean protein contents determined by the proposed method and the official ELISA method.     

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.     

Advances of Research on Glycinin and β-Conglycinin: A Review of Two Major Soybean Allergenic Proteins

Being an important crop, soybean is widely used in the world and plays a vital role in human and animal nutrition. However, it contains several antinutritional factors (ANFs) including soybean agglutinin, soybean protease inhibitors, soybean allergenic proteins, etc., that may result in poor food utilization, decreased growth performance, and even disease. Among these ANFs, soybean allergenic proteins can lead to allergic reactions in human and animals, which has become a public problem all over the world, but our knowledge on it is still inadequate. This paper aims to provide an update on the characteristics, detection or exploration methods, and in vivo research models of soybean allergenic proteins; especially glycinin and β-conglycinin are deeply discussed. Through this review, we may have a better understanding on the advances of research on these two soybean allergenic proteins. Besides, the ingredient processing used to reduce the allergenicity of soybean is also reviewed.     

Rapid detection of the addition of soybean proteins to cheese and other dairy products by reversed-phase perfusion chromatography

The undeclared addition of soybean proteins to milk products is forbidden and a method is needed for food control and enforcement. This paper reports the development of a chromatographic method for routine analysis enabling the detection of the addition of soybean proteins to dairy products. A perfusion chromatography column and a linear binary gradient of acetonitrile-water-0.1% (v/v) trifluoroacetic acid at a temperature of 60°C were used. A very simple sample treatment consisting of mixing the sample with a suitable solvent (Milli-Q water or bicarbonate buffer (pH = 11)) and centrifuging was used. The method enabled the separation of soybean proteins from milk proteins in less than 4 min (at a flow-rate of 3 ml/min). The method has been successfully applied to the detection of soybean proteins in milk, cheese, yogurt, and enteral formula. The correct quantitation of these vegetable proteins has also been possible in milk adulterated at origin with known sources of soybean proteins. The application of the method to samples adulterated at origin also leads to interesting conclusions as to the effect of the processing conditions used for the preparation of each dairy product on the determination of soybean proteins.     

Commercial soybean lecithins: a source of hidden allergens?

 Soybeans are known to be allergenic for adults as well as for infants. Processed products derived from soybeans are used in a wide spectrum of foods, drugs and other industrial products. In particular, soybean lecithins are used as stabilizers and emulsifiers and may not be suspected as possible source of allergens. To test this hypothesis, six commercial soy lecithins were investigated for residual allergenicity and compared with extracts from raw and heat-treated soybeans. They were characterized, the protein content was determined by enzyme-linked immunosorbent assay (ELISA) and allergens were analyzed with specific IgE from patients' sera using the enzyme allergosorbent test (EAST), EAST inhibition and protein blotting followed by immunodetection. For further characterization a polyclonal antiserum directed against soybean extract and a monoclonal antibody (mAb 025) directed against the acidic subunit of the soybean storage protein glycinin were used. The EAST studies revealed that three of six sera from patients with allergy to soybeans contained IgE to four soy lecithins (Topcithin 50, Topcithin 300, Emulfluid FD 12, Epikuron 100 P), the same lecithins which were found to contain residual proteins. Two lecithins with a protein content of less than 20 ppb did not bind IgE. EAST inhibition showed that the allergens from soy lecithin were immunologically more closely related to allergens from heat-treated soybeans than to those from raw soybeans. Protein blotting and immunodetection of the protein extract from the lecithins resulted in various allergen bands between 14 kDa and 94 kDa. A heat-stable allergen of 39 kDa was recognized by the monoclonal antibody and thus identified as a subunit of glycinin. The results obtained were confirmed by a mediator release assay based on a rat basophil leukemia cell line. Lecithins that contained residual proteins caused a specific mediator release, suggesting that these products may induce allergic symptoms. Our results show that soybean lecithins are capable of introducing hidden allergens to processed foods and that the IgE binding potential corresponds to the total protein determined by ELISA. Furthermore, it appears to be possible that by monitoring the protein content soy lecithins can be applied which may be safe for the allergic consumer. Received: 22 January 1998