Development of a sandwich enzyme-linked immunosorbent assay for the detection of egg residues in processed foodst.

Chicken eggs are used extensively as an excellent source of dietary proteins. These proteins have many functional properties, making them valuable food ingredients. However, eggs are a frequent cause of food hypersensitivity, especially in children. Of major concern to food processors is the inadvertent cross-contact of food products with allergenic residues, which could result in potentially life-threatening reactions in those with a food allergy. The aim of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of undeclared egg residues in foods. Commercially purified ovalbumin (OVA) and dehydrated egg white solids were used as antigens to induce antibodies in rabbits and goats. Reference pasta standards and various food samples were extracted, then clarified by centrifugation. Goat anti-egg white antibodies were used as the capture reagent, nonspecific sites were blocked with gelatin, then standard and sample extracts were added. Rabbit anti-OVA antibodies were used as detector antibodies, followed by addition of commercial goat anti-rabbit IgG antibody labeled with alkaline phosphatase and subsequent substrate addition. Twenty brands of egg-free pasta (two lots each) were analyzed using the ELISA. Fourteen common pasta ingredients were also evaluated for cross-reactivity problems in the method. The detection limit of the assay was 1 ppm spray-dried whole egg. Fifty-five percent (22 samples) of the egg-free pasta samples tested positive for the presence of undeclared egg residues, with values ranging from 1 to >100,000 ppm. Minimal cross-reactivity was encountered in general, but portobello mushrooms and basil caused some minor matrix effects. This sandwich-type ELISA method can be used to detect undeclared egg residues in processed foods and to evaluate industrial clean-up operations.     

Challenging the Limit of Detection for Egg Allergen Detection in Red Wines by Surface Plasmon Resonance Biosensor

The improvement of a surface plasmon resonance (SPR)-based immunoassay for the detection of traces of egg-based fining agents in red wines is herein described. The latter represents an extension of a previously developed direct assay targeted to the detection of ovalbumin (OVA) as marker of the presence of egg white powder residues, a typical fining agent utilized by the winery industry. In this paper, a suitable pre-treatment procedure was optimized for the sensitive detection of OVA at sub-ppm levels in red wines fortified with egg-white powder, by using an immunoassay proved to be reliable for both white and roseé wines. A red wine from Chianti grapes selected as reference matrix was artificially contaminated with egg-white powder before undergoing different sample purification. Several purification strategies were investigated and tested in order to challenge the limit of detection (LOD) obtained with the methods currently in use for egg allergen detection. Finally, the optimized two-step pre-treatment, combining polyvinylpolypyrrolidone-based purification and size exclusion chromatography, enabled to achieve an LOD in red wine as low as 0.2 μg/mL. The optimized SPR-based method met the method performance criteria issued by the International Organization of Vine and Wine (OIV) concerning the minimum sensitivity required for the analyses of potentially allergenic fining agent proteins in wines, confirming the biosensor as promising tool to monitor the residual contamination level of fined red wines.     

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.     

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay for the Determination of Thiacloprid in Agricultural Samples

An enzyme-linked immunosorbent assay (ELISA) was developed based on a polyclonal antibody for the analysis of thiacloprid in agricultural samples. Thiacloprid hapten was synthesized and conjugated to bovine serum albumin to produce an immunogen and ovalbumin to produce a coating antigen. Polyclonal antibodies were obtained from immunized New Zealand white rabbits. Under optimal conditions (5 % methanol, 0.1 mol/L Na⁺, pH 5.5), the ELISA showed a 50 % inhibitory concentration (IC₅₀) value of 0.01 mg/L and a limit of detection (IC₁₀) of 0.47 μg/L. No obvious cross-reaction with the other structural analogs of neonicotinoid insecticides showed that the polyclonal antibodies had a high specificity for thiacloprid. The average recoveries from spiked water, soil, pear, and tomato were in the range of 80 to 119 %. The results of the ELISA were confirmed by high-performance liquid chromatography, and the correlation of the results from the two methods had a high correlation coefficient of 0.99 (n?=?3) in spiked samples (soil, pear, and tomato). The proposed ELISA could successfully be applied to the determination of thiacloprid residues in agricultural samples.     

Impact of wine manufacturing practice on the occurrence of fining agents with allergenic potential

Proteinogenic wine fining agents are hidden allergens and could present a risk for consumers with allergies. Therefore, the European Parliament adopted Directive 2003/89/EC amending Directive 2000/13/EC to declare ingredients, contaminations and processing aids that are known to trigger allergic reactions. The Amendment Regulation (EU) 1266/2010 excluded the labelling of wines which are processed with hen’s egg and products thereof until 30 June 2012 to get more scientific findings. After 1 July 2012 wine fining agents have to be declared if above 0.25 mg l^–1 (Regulation (EU) 579/2012 in conjunction with article 120 g of Regulation (EU) 1234/2007). The Organisation International de la Vigne et du Vin (OIV) advises this limit of detection (LOD) for potential allergenic residues of proteins. Wine fining agents are processing aids and according to the wine producer’s knowledge will be removed after coagulation by filtration or other production steps. Due to lack of scientific data, residues of fining agents in the final product could not be excluded. In this risk assessment, highly sensitive ELISA methods for ovalbumin of known origin for wine have been developed. The objective was to investigate the presence of allergen residues in wine after certain technological treatments were applied to remove the wine fining agents. For all developed ELISA methods the LODs are in the low µg l^–1 range between 5 and 10 µg l^–1 fining agent, whereas the LOQ varies between 5 and 80 µg l^–1 fining agent. The results of the investigation of well-known wines and fining agents demonstrate that white wines fined with white or ovalbumin from hen’s egg could retain allergens. The use of certain technological procedures during wine processing leads to different results. In white wine, bentonite or sheet filtration followed by sterile filtration lead to wines containing no detectable amounts of ovalbumin. In red wine, especially the final sterile filtration removes the fining agents.     

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.     

Impact of irradiation and thermal processing on the immunochemical detection of milk and egg allergens in foods

There is little information available on the antigenicity of allergens in foods subjected to gamma irradiation and thermal processes. The objective of the study was to evaluate the effects of gamma irradiation and thermal processing on the recovery of milk and egg allergens in foods prepared using irradiated wheat flour incurred with these allergens. Bread, boiled pasta and extruded cereal were selected as food matrices for the study.Allergen detection was performed using Surface Plasmon Resonance (SPR) and commercially available enzyme linked immunosorbent assay (ELISA) kits. Protein structure was characterized using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and circular dichroism (CD) spectroscopy. The ELISA kits performed well in detecting allergens in the unprocessed flours with recoveries ranging from 63–120%. The results for dough and uncooked pasta were similar to those of unprocessed flours. Recoveries for boiled pasta were significantly (p ≤ 0.05) reduced. The lowest allergen recoveries were obtained in bread samples. Allergen recoveries in gamma irradiated samples depended on the irradiation dose, the type of ELISA kit used, the tested matrix, and the processing method. FTIR and CD results showed that among the allergens casein was the most thermostable followed by ovomucoid. β-Lg and ovalbumin showed thermal sensitivity. In conclusion, gamma irradiation may affect the antigenicity of allergenic food residues at low levels depending on the food matrices and the processing methods.     

Immunochemical detection of ovalbumin in dairy-based foods

Commercially available antibodies were used for the detection and quantitation of ovalbumin in ice cream and cheese. Electrophoretic separation of protein components, followed by blotting and immunochemical recognition of ovalbumin allowed the detection of the addition of 50 g kg^-1 egg white to processed cheese and allowed detection of egg in ice cream. For quantitative purposes a competitive ELISA method was set up. Calibration curves for ovalbumin in various products were obtained. A marked matrix effect was evident in ice cream, cheese and yoghurt. Different types of cheese gave nearly identical matrix effects. Analytical application of the method should therefore be possible, at least within a given class of products. A study on the influence of thermal treatments on the ELISA response showed that melting of cheese at various temperatures modifies only slightly the ELISA response for ovalbumin.     

Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle,chicken muscle,egg, fish,milk and kidney

A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 μg/kg in kidney and 20 μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 μg/kg, and the detection limits were 1.5 μg/kg in pig muscle and 6 μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r²) which was greater than 0.9.     

Validated sandwich enzyme-linked immunosorbent assay for casein and its application to retail and milk-allergic complaint foods

Cows' milk is a commonly allergenic food. Cross-contamination of milk proteins into nondairy, kosher-pareve foods prepared on shared processing equipment can cause severe, life-threatening reactions in milk-allergic individuals. A sandwich-type enzyme-linked immunosorbent assay (ELISA; 96-well plate format) was developed for the detection of undeclared casein in foods. Rabbit anti-casein antibodies were used as the capture reagent. Food samples and standards were ground, extracted in 0.01 M phosphate-buffered saline, clarified by centrifugation, and added to the wells. Goat anti-casein antibodies were employed as the detector antibody, and the amount of antibody bound was determined with a commercial rabbit anti-goat immunoglobulin conjugated to alkaline phosphatase, with subsequent substrate reaction. Antibodies developed were specific to casein, with no cross-reaction observed with 30 foods and food ingredients. Non-milk-containing products such as fruit juices, fruit juice bars, sorbets, and dark and pareve-labeled chocolate were purchased from June 2002 through June 2003. In addition, samples allegedly causing eight milk-allergic consumer complaints were analyzed. The ELISA had a detection limit of less than 0.5 ppm of casein. The casein content in the analyzed foods ranged from less than 0.5 ppm to more than 40,000 ppm casein; undeclared casein residues were found in all of the samples implicated in allergic reactions. The levels of milk contamination in some of the other surveyed products could also be hazardous for milk-allergic consumers. This ELISA method provides a useful quality control tool for the food industry and could also be used as a validation of kosher-pareve status.     

呋喃西林代谢物酶联免疫吸附测定方法的建立及性能测定

建立动物源性食品中呋喃西林代谢物酶联免疫吸附测定方法。采用自制的呋喃西林代谢物抗原、抗体为基础原料,通过优化反应条件,建立间接竞争酶联免疫吸附试验(enzyme-linked immunosorbent assays,ELISA)检测方法,并对方法的性能进行测定。结果表明:建立的检测方法在0.03μg/L^2.43μg/L范围内呈线性,检测灵敏度为0.03μg/kg,鱼肉组织样本、虾肉组织样本的检测限分别为0.236、0.229μg/kg,回收率均在75%~120%之间,批内、批间变异系数均在15%以下,通过与标准方法液相色谱-串联质谱(high performance liquid chromatography-mass spectrum/mass spectrum,LC-MS/MS)对比验证,两种方法用于鱼肉样本的检测呈现较好的相关性,相关系数R2为0.9944。建立的呋喃西林代谢物酶联免疫吸附测定方法的灵敏度、准确度和精密度等参数均符合我国动物源性食品中兽药残留检测方法的规定。     

Polymerase chain reaction (PCR) for detection of potentially allergenic hazelnut residues in complex food matrixes

Corylus avellana ) residues in complex food matrixes has been developed applying the polymerase chain reaction (PCR) with “hot start” and “time-release” features. By amplifying a 182 bp- fragment of the coding deoxyribonucleic acid (cDNA) of Cor a 1, the major hazelnut allergen, detection of even 0.001 % of hazelnut in commercial food products could be demonstrated. Results were confirmed by our previously described sandwich-type enzyme-linked immunosorbent assay (ELISA) that quantifies potentially allergenic hazelnut protein at trace levels. Received: 9 November 1999 / Revised version: 9 December 1999     

一种多菌灵酶联免疫快速检测方法的建立

利用酶联免疫吸附法原理建立了一种能够定量测定果蔬、茶叶、烟叶中多菌灵残留量的方法.通过酸碱中和化学反应,制备多菌灵半抗原,再通过免疫小鼠筛选出抗多菌灵单克隆抗体,制得酶标羊抗鼠抗抗体,并将制备出的抗多菌灵单克隆抗体和酶标羊抗鼠抗抗体用于酶联酶联免疫试剂盒的研制.结果表明,在0.1～8.1 μg/L的线性范围内,目标物多...     

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 real-time PCR assays for the detection of lupin residues in food products

Lupin is a legume from the Leguminosae family that is used, amongst other, for human nutrition. In Europe, lupin is used as a substitute for soy in bakery and dietary products and recently its consumption has increased significantly. Unfortunately lupin is known to trigger allergic reactions in sensitised individuals and therefore its use in food products requires a mandatory declaration on the label in accordance with Directive 2007/68/EC. To protect the allergic consumer the availability of detection methods for the identification of lupin in food products is required. Here we present the development of two real-time polymerase chain reaction (PCR) methods that allow the detection of lupin-specific DNA as a marker for the presence of this allergenic ingredient in food products. Genomic DNA sequences coding for conglutin genes were chosen as targets for the detection of lupin. One primer set and probe was designed for the amplification of a 153 bp fragment of α-conglutin; another primer set and probe was designed for the detection of a 150 bp δ-conglutin amplicon. Lupin at a level of 10 mg/kg food matrix could be detected in cookies baked from a lupin containing dough using the α-conglutin method. Since lupin is used in bakery products the effects exerted by heat treatments on lupin detection by real-time PCR have been investigated. Enzyme-linked immunosorbent assay (ELISA) analyses were performed in parallel to compare the detection of lupin DNA with that of lupin protein in market products. Qualitative ELISA results confirm results obtained by the real-time PCR methods targeting α- and δ-conglutin.     

Development of a chemiluminescent enzyme‐linked immunosorbent assay for five sulfonamide residues in chicken muscle and pig muscle

BACKGROUND: An enzyme‐linked immunosorbent assay (ELISA) based on polyclonal antibodies with enhanced chemiluminescent (ECL) detection of sulfonamides in food samples has been optimised and characterised. The specificity of the assay was assessed by determining cross‐reactivities with a set of 16 sulfonamides. The aim of this study was to develop a method for determining sulfonamides with high sensitivity. RESULTS: The sensitivity of the developed ECL‐ELISA was higher than that of colorimetric ELISA. The sensitivities of five of the sulfonamides (sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine and sulfapyridine) ranged from 0.73 to 2.92 µg L^?1, with limits of detection of 0.10–0.43 µg L^?1. The coefficients of variation of intra‐assay and inter‐assay studies carried out over 5 days were mostly less than 10%. Recovery studies of chicken muscle and pig muscle were performed with simple and rapid extraction. Good recoveries (62.1–110.3%) were achieved and the results correlated well with those obtained using high‐performance liquid chromatography analysis. CONCLUSION: This study has provided an effective analytical technique for the rapid and reliable determination of residues of sulfisozole, sulfathiazole, sulfameter, sulfamethoxypyridazine and sulfapyridine in food samples with high sensitivity. To the authors' knowledge, this is the first report on chemiluminescent ELISA for sulfonamide analysis. Copyright © 2008 Society of Chemical Industry     

Preparation of Artificial Antigen and Development of Indirect Competitive ELISA Based on Chicken IgY for the Detection of Acid Orange II in Food Samples

To evaluate the feasibility of specific egg yolk antibodies (IgY) technology for the detection of hazardous chemical residues in foodstuffs, IgY were generated to detect acid orange II (AO2) residues. Bovine serum albumin (BSA) was made to bind with AO2 by succinic anhydride (SA), and the conjugate was used to immunize the laying chickens. PEG-6000 precipitation was used to extract IgY antibodies. The titer of anti-AO2 IgY attained the peak (1:12,800) after fifth booster immunization. Checkerboard titration showed that 1:640 dilution of anti-AO2 IgY could approximately give an optical density (OD) 1.0 at 5 μg/mL AO2-OVA coating concentration. The anti-AO2 IgY based indirect competitive ELISA (ic-ELISA) showed that the IC₅₀ value of anti-AO2 IgY was 10.72 μg/mL and regression curve equation was y?=?24.41?×?+75.11 (R ²?=?0.946). The ic-ELISA showed less cross-reactivity in range between 0.09 and 21.07 %. Recoveries from dried bean curd, sausage, and chilli powder samples were from 78.80 to 152.90 %, with relative standard deviation lower than 19.13 %. The study suggests that generated anti-AO2 IgY antibodies could be used in routine screening analysis of AO2 residues in food samples.     

Multi-allergen screening immunoassay for the detection of protein markers of peanut and four tree nuts in chocolate

A multiresidue enzyme immunoassay was developed to check for the presence of markers of peanut, hazelnut, almond, cashew and Brazil nuts in a single run. The assay was designed under the competitive indirect format and adapted for screening purposes applied to chocolate samples. The limit of detection for this assay was below 1 µg g^-1 protein for each allergenic food. In most cases, the high specificity of the antibodies used allowed the identification of each particular allergenic food with no possible confusion. This assay was proven to be useful as part of an analytical procedure involving the identification of the unknown allergenic food among peanut and other tree nuts in recalled samples before the application of a quantitative technique to determine the level of cross-contamination.     

莱克多巴胺人工抗原及抗体的制备

莱克多巴胺是一种禁用β-兴奋剂,建立莱克多巴胺药残的快速检测方法是实现对其进行有力监控的有效途径,而莱克多巴胺抗体是快速免疫检测法的基本试剂。本实验用全新的方法研究了莱克多巴胺免疫原的合成,采用对氨基苯甲酸(ABA)和1,4-丁二醇缩水甘油醚(BDE)将莱克多巴胺分别和cBSA、cOVA偶联,合成了莱克多巴胺的免疫原和包被抗原,并对其进行了紫外分析。用免疫原免疫新西兰大白兔,获得了高灵敏度和特异性的莱克多巴胺多克隆抗体,采用间接ELISA法检测抗体的IC50值为4.34ng/ml,所得多克隆抗体效价达到102400。     

氯霉素毛细管电泳免疫分析方法研究

针对目前食品安全领域中氯霉素的检测,建立一种快速灵敏的氯霉素毛细管电泳免疫分析方法。该方法对氯霉素检测的线性范围和最低检测限分别为0.008～5μg/L 和 0.0016μg/L。与相应固相酶联免疫分析ELISA相比,对氯霉素的检测灵敏提高了约20 倍且有效缩短了分析时间。建立的氯霉素毛细管电泳免疫分析方法对动物源性食物(牛奶、鸡肉和鱼肉)进行分析,得到氯霉素在实际样品中的检测限为0.035μg/kg。