Collaborative Interlaboratory Studies for the Validation of ELISA Methods for the Detection of Allergenic Fining Agents Used in Wine According to the Criteria of OIV Resolution 427–2010 Modified by OIV–Comex 502–2012

The clarification or fining of wine removes undesired substances (mainly proteins, phenols, and tannins), which would roil the wine and cause bitterness and astringency. A common fining agent, egg white, can be directly added to wine through the inlet of a circulating pump, but more typically egg white comes as commercial preparation in powdered form (commercially named egg albumin). Skimmed milk or more frequently purified caseinates are used to remove bitterness and hardness of white wine and sherry. Both egg white and caseinates are fining agents with optimal enological properties, but their residues could represent a risk for subjects suffering from food allergy. The rules for allergen labeling were detailed in Directives 2003/89/EC, and Directive 2005/26/EC established a list of food ingredients provisionally excluded from labeling, that included wine fining agents. Extended till June 2012, wine labeling exemption can be now maintained only if (1) egg and milk derivatives are not used and cross-contamination is under control; and (2) wine clarified with such products is negative for the presence of residues using techniques with detection and quantification limits of 0.25 and 0.5 ppm, respectively. Analytical requirements were defined in the OIV resolution 427–2010 (OIV 2010) modified by OIV/COMEX 502–2012 (OIV 2012). On the basis of a previous experience, an interlaboratory collaborative trial was organized to validate a commercial ELISA kit designed to measure allergenic residues in red wine fined with egg white proteins. In the meantime, the performance of the commercial caseinate ELISA kit for white wine was rechecked according to the new limit of detection and limit of quantification values, recommended by OIV in 2012. The collaborative interlaboratory studies showed that both ELISA kits had good reproducibility, repeatability, and robustness in detecting residues of allergenic fining agents in wine, in good agreement with the requirements of the OIV resolution 427–2010 modified by OIV/COMEX 502–2012.     

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.     

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.     

Detection of Traces of Ovalbumin and Casein in White and Red Wines by Quantitative Western Blotting

Fining of wine with agents containing cow's milk or hen's egg white is a common and traditional procedure. In light of increasing food allergies all over the world, the presence of fining residues has been subject of intense debate. Switzerland does not make exception, and since 2009 the Federal Department of Home Affairs has modified its food regulations stating that the labels must show if traces of fining agents are present. Nevertheless, the application of this regulation is not based on an official analytical method. In this study we show that immunoblotting is an efficient technique to detect and quantify ovalbumin and casein residues in bottled wine. We showed that final filtration is an essential step to remove finings in red wine, and that overfining of white wine may result in fining residues in finished products. Finally, for the first time in Switzerland, 22 samples were taken by food safety inspectors and officially analyzed for the regional food control authority of the Canton of Vaud. These samples were allergen free, but a larger study is currently planned in collaboration with other regional authorities of Switzerland to complete these results and make a complete picture of the Swiss wine production.     

Implementation of an Enzyme Linked Immunosorbent Assay for the Quantification of Allergenic Egg Residues in Red Wines Using Commercially Available Antibodies

Since the early 2000s, labeling of potentially allergenic food components to protect people who suffer from food allergies is compulsory in numerous industrialized countries. In Europe, milk and egg components used during the winemaking process must be indicated on the label since July 1, 2012. Several ELISA procedures have been developed to detect allergenic residues in wines. However, the complexity of the wine matrix can inhibit the immunoenzymatic reaction. The aim of this study was to implement an ELISA assay for the detection of ovalbumin in red wines using commercially available antibodies. The specificity of the acquired antibodies and the absence of cross reactivity were assessed by immunoblotting and ELISA. An ELISA assay with a LOD of 14.2 μg/L and a LOQ of 56.4 μg/L of ovalbumin in aqueous solution was obtained. Differences in ELISA signals were observed when analyzing various fining agents, although reproducible conformation of the antigen could be reached for the comparison of ovalbumin and Ovicolle. The differences between samples in terms of pH could be leveled but the inhibition of the ELISA signal, positively correlated to the tannin content of the wines, could not be suppressed. Thus, standard curves of ovalbumin in several wines were obtained by relative quantification. The control steps and the difficulties encountered presented in this study should be considered by anybody working toward the development of ELISA assays for the detection of allergenic residues in complex food matrices.     

Absence of allergenic residues in experimental and commercial wines fined with caseinates

The possible presence of allergenic residues in wines treated with one of the potassium caseinates used as fining agents has been investigated.     

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。     

Development of two immunoassay formats to detect beta-lactoglobulin: influence of heat treatment on beta-lactoglobulin immunoreactivity and assay applicability in processed food

Milk proteins are commonly used as ingredients in the food industry because of their functional properties, but they can cause severe reactions in milk-allergic individuals. In this work, two enzyme-linked immunosorbent assay (ELISA) formats were developed to detect bovine beta-lactoglobulin. The indirect competitive ELISA involved the use of anti-beta-lactoglobulin antisera, and the sandwich ELISA involved the use of specific antibodies isolated using a beta-lactoglobulin immunosorbent material. The effect of heat treatment on immunoreactivity of the protein in buffer and in milk was determined with both assays. The amount of immunoreactive protein in buffer and in milk decreased as determined by the sandwich ELISA, whereas the amount increased when measuring with the competitive ELISA. Several food products, including meat, bakery products, sauces, and snacks, were analyzed. With both assays, 10 of 11 products in which the ingredient list included the terms "powdered milk" or "milk proteins" contained beta-lactoglobulin. However, the beta-lactoglobulin concentration in these products obtained with the competitive ELISA were much higher than those obtained with the sandwich ELISA. These differences could be explained by the fact that the determination of beta-lactoglobulin concentration by immunoassay is influenced by differences in antibody recognition of the protein present in highly processed foods. Therefore, the antigen-binding properties of antibodies used in a particular immunoassay are important for a correct interpretation of results obtained in food processed at high temperature.     

双抗夹心ELISA方法检测食品中单核细胞增生李斯特氏菌

采用0.5% 福尔马林灭活的单核细胞增生李斯特氏菌作为免疫原免疫日本大耳兔,获得抗单核细胞增生李斯特氏菌多克隆抗体,并以此多克隆抗体作为捕获抗体,以抗单核细胞增生李斯特氏菌Internalin A(InlA)单克隆抗体作为检测抗体,建立快速、特异的检测该菌的双抗夹心ELISA 方法。结果表明：该方法对单核细胞增生李斯特氏菌纯培养液的最低检测量为1.7 × 105CFU/mL。在检测食品样品的实验中,食品样本对本方法干扰较小,运用选择性增菌液进行前增菌可提高该方法的准确性。     

Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk

The demand for commercially available human breast milk has significantly increased in recent years. For various reasons, a significant amount of commercially available human breast milk is being adulterated with other types of milk. This fraudulent practice poses a threat to consumers' health due to potential adulterants such as cow milk, which may put the infant at risk due to intolerance or allergy. A direct sandwich anti-bovine IgG ELISA has been developed for the sensitive and specific detection of cow milk in adulterated human breast milk. This assay uses polyclonal anti-bovine IgG antibody as a capture antibody and monoclonal anti-bovine IgG-alkaline phosphatase antibody as a detection antibody. Once optimized, the assay was found to be highly sensitive, and specific to bovine IgG. The assay had no significant cross-reaction with human breast milk, indicating that it was highly specific. The anti-bovine IgG ELISA was able to detect the presence of cow milk in adulterated human breast milk with a detection limit of 0.001% cow milk. The developed assay was highly reproducible (coefficient of variation <10%). The developed direct sandwich anti-bovine IgG ELISA is simple, reliable, and reproducible, making it an ideal test for this purpose.     

Fining of Red Wines with Pomace Cell Wall Material: Effect on Wine Phenolic Composition

Fining is a winemaking technique used to remove unwanted wine components that affect clarification, astringency, color, bitterness, and aroma. One of the objectives of fining is often to reduce the wine tannin content due to its effect on wine astringency. Proteinaceous agents are commonly used with this objective, but problems related with their possible allergenic properties or the excessive enrichment of wine with proteins, which may cause stability and turbidity problems, have led to the search for new fining agents. In this way, the cell wall material from processed grape pomace could be a good alternative since it has a high affinity for tannins. In this work, the effect of cell wall material from processed pomace from Monastrell and Cabernet Sauvignon grapes on the reduction of the phenolic content of red wines is studied and the results are compared with those obtained with commercial fining products. Also, the varietal effect due to the different composition of the cell wall material from these two varieties has been evaluated. The concentration and composition of wine anthocyanins and tannins, before and after the fining process, were analyzed by HPLC and their molecular mass distribution by SEC. The results showed that, at laboratory scale, pomace cell walls have a fining effect that exceeds that of most the protein-based fining agents, even when used at their highest recommended doses. The cell wall material significantly reduced the wine phenolic content, the reduction ranging from 48 to 68% for anthocyanins and from 44 to 64% for tannins, although varietal differences exist regarding the cell wall-binding capacity. Monastrell cell walls exhibited the highest capacity and could be used at much lower doses than those used in this study, reducing the formation of lees and the wine adsorbed on them. The results indicate that this material could be an appropriate alternative for protein-based fining agents in red wine and their use would avoid allergen-related effects.     

酶联免疫吸附技术在食品检测分析中的研究进展

酶联免疫吸附技术(ELISA)是酶免疫测定技术中应用最广泛的技术,是将已知的抗原或抗体吸附在固相载体表面,使酶标记的抗原抗体反应在固相表面进行,通过酶与底物产生颜色反应,用洗涤法将液相中的游离成分除去后再进行定量测定。主要分为夹心法、间接法和竞争法。本文简要介绍了几种常用方法及其基本作用机制,详述了ELISA检测技术在食品检测中具体应用及发展历史与现状,对其在食品中农药残留、病原微生物、生物毒素、转基因食品、重金属残留、过敏性残留物及违规添加成分检测等方面的应用情况进行系统总结,并对其发展前景进行了分析。     

Effects of fining agents on trans-resveratrol concentration in wine

Resveratrol has been identified as a wine component related to moderate wine consumption and a reduction in serum cholesterol levels. Processes such as wine fining that result in loss of resveratrol during winemaking are therefore of interest, and led to these present studies. A number of agents were compared and were found to lower resveratrol levels in all wines to some extent. Results from two studies (1996 and 1997) are reported. The standard addition method was used in combination with High Pressure Liquid Chromatography to calculate resveratrol levels. In Study 1 (1996), recommended maximum levels of all fining agents (Level 3), bentonite, egg white, gelatin + kieselsol and polyvinylpolypyrrolidone (PVPP), lowered resveratrol levels significantly compared to controls (Level 0). Nevertheless, addition of fining agents at Level 0 resulted in resveratrol levels that were significantly higher than those at Level 3, but resveratrol levels in wine from Level 0 were not significantly different from Level 1. In Study 2 (1997), carbon + egg white, and gelatin + kieselsol fining was studied, and their effects differed according to grape variety. Least removal of resveratrol by carbon fining occurred in wine from Cabernet Sauvignon (Vitis vinifera) whereas most removal occurred in wines from Cynthiana (Vitis aestivalis) and Noble (Vitis rotundifolia). Resveratrol levels of control wine were significantly higher than resveratrol levels of wine treated with recommended maximum addition of fining agent in all varieties. Taken overall, any addition of any fining agent lowered resveratrol levels in all wines to some extent, but complex interactions between fining agent and wine variety resulted in different regression trends. While recognising the constraints set by our particular data that could reflect unique circumstances, we are nevertheless able to infer from these trends that low levels of fining agents can be used without statistically significant loss of resveratrol.     

Use of patatin, a protein extracted from potato, as alternative to animal proteins in fining of red wine

The use of plant-derived proteins as wine fining agent has gained increased interest owing to the potential allergenicity of animal proteins in susceptible subjects. Patatin P is the name of a family of glycoproteins that can be recovered from potato aqueous by-product. In this study, a comparative fining trial simulating industrial procedures with 10, 20 and 30?g/hL of commercial preparations of patatin, potassium caseinate, gelatin and egg albumin on an Aglianico (Vitis vinifera L.) red wine was performed. Color indexes and phenolics were analyzed by spectrophotometric methods and HPLC. The potential astringency has been evaluated by an index based on the ability of wine to precipitate salivary proteins (SPI, Saliva Precipitation Index). Patatin is a suitable alternative to animal proteins used as fining agent because: (i) a decrease in total phenolics and tannins after the treatments with 10, 20 and 30 g/hL of commercial preparation containing P was detected; (ii) Patatin, as well as all the fining agents used in this experiment, is able to diminish astringency and the content of red wine phenolics able to react with salivary proteins. Considering all concentrations tested, the effectiveness in reducing proteins reactive towards wine polyphenols was patatin?=?gelatine > egg albumin > casein (p?<?0.05); (iii) at each concentration considered, the treatment with patatin causes no depletion of chromatic characteristics of red wine although a significant slight loss of individual anthocyanins was observed.     

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.     

Sandwich Enzyme‐Linked Immunosorbent Assay (ELISA) for the Detection of Lupine Residues in Foods

ABSTRACT: Lupine has been increasingly used in food applications due to its high nutritional value and excellent functional properties. However, lupine provokes allergic reactions in susceptible individuals. The presence of undeclared lupine residues in foods can pose a serious health risk to lupine‐allergic individuals. Therefore, the objective of this research was to develop a sandwich‐type ELISA for the detection of lupine residues in foods. Lupine flour derived from Lupinus albus was used to immunize 3 rabbits and a sheep. Pooled lupine‐specific antibodies were partially purified from the sera by ammonium sulfate precipitation. A sandwich lupine ELISA with a limit of quantification (LOQ) of 1 ppm was developed by utilizing the rabbit antisera as the capture reagent and the sheep antiserum as the detector reagent. The binding of the antigen‐antibody complex was visualized by the addition of commercial rabbit antisheep IgG antibody labeled with alkaline phosphatase with subsequent addition of p‐nitrophenyl phosphate substrate to produce a colored product for quantification. Minor cross‐reactivity was observed with soy (Glycine max) and black bean (Castanospermum australe). The performance of the lupine ELISA was evaluated in reference food standards (beef frankfurter and apple cinnamon muffin) and laboratory‐prepared cooked frankfurters and corn muffins. The mean percent recovery for lupine spiked‐frankfurters and corn muffins were 108.4%± 8.8% and 103.1%± 11.5%, respectively. The sandwich‐type lupine ELISA developed in this study provides food manufacturers and regulatory agencies with an effective analytical tool to detect and quantify lupine residues in processed foods.     

小分子化合物开放夹心免疫分析研究进展

基于抗原-抗体识别的免疫分析技术在小分子监测领域占有重要地位,已成功应用于生理活性物质、化学有害物、农兽药等的快速检测,在临床诊断、环境、食品以及卫生领域发挥重要作用。由于缺乏结合位点,小分子化合物的分析大多采用竞争模式,与传统的三明治夹心法相比,稳定性和灵敏度往往都难以满足实际检测需求。近几年来,科学家开始尝试建立针对小分子化合物的非竞争模式免疫分析方法,比如基于抗独特型抗体、基于抗体可变区片段、基于抗异型抗体及抗免疫复合物多肽。其中基于抗体重链和轻链结合的开放夹心免疫分析由于具有只需要单个抗体、快速非竞争的均相检测小分子等优点,备受研究者关注。本文主要综述基于抗体可变区片段的小分子非竞争免疫分析方法,重点介绍了建立小分子非竞争检测模式所需要抗体可变区的获得及筛选途径、几种形式信号放大载体的研究现状,最后对其在小分子物质检测领域的应用前景进行了评述,期望能为本领域研究提供借鉴。     

Performance of purified grape pomace as a fining agent to reduce the levels of some contaminants from wine

The quality of red wine depends on the absence of compounds which may affect its safety and/or stability such as ochratoxin A, biogenic amines and some metals and trace compounds. The presence of ochratoxin A in musts and wines is due to fungal contamination of the grapes and has been classified as a possible human carcinogen. Biogenic amines are formed by the microbiological decarboxylation of the corresponding amino acid precursors during the fermentation or ageing and storage, and, at high concentrations, they may induce adverse reactions in sensitive people. Trace elements may have both a nutritional and a toxic effect on health, but also can cause turbidity and stability problems. Their presence is affected mainly by natural factors such as soil mineral content and direct contact with tank surfaces and metallic tubing during winemaking. One of the best options to remove these compounds when present in excess in wine is fining. However, some fining agents commonly used may themselves present problems related with their allergenic properties or with their propensity to increase the protein content, which can cause turbidity problems. In an attempt to avoid such these problems, purified grape pomace was tested as a fining alternative since it has been seen to have a high capacity to reduce the astringency, turbidity and also the ochratoxin A content. The main aim of this work, therefore, was to study if this material can limit the presence of ochratoxin A, biogenic amines and metals and some trace elements in a Monastrell red wine, thus increasing the value and safety of this product.     

Quantitative analysis of shrimp allergen in food matrices using a protein chip based on sandwich immunoassay

Food allergy is increasingly becoming a serious concern these days. With packaged foods becoming the norm of the day, food allergy cases out of accidental consumption are becoming rampant, thereby generating great risks for the subjects involved and prompting food authorities in different countries to formulate new regulations about displaying food allergen data on food labels. Detection of food allergens is conventionally carried out by ELISA or PCR tests. These techniques are limited in that they can only detect one or few allergens at one time. Therefore, in the present study a novel sandwich protein chip assay was developed for quantitation of shrimp allergens in food matrixes. The shrimp allergen model used 3D aldehyde slides as the solid carrier, rabbit antisera as the capture reagent, and biotin-labeled monoclonal antibody as the detector reagent. Resulting antigen–antibody complexes were visualized in the presence of commercial strepavidin labeled with Cy3 to produce fluorescence for quantification. With the LOD of the protein chip being 0.054 mg tropomyosin/kg, the protein chip can quantify down to 0.096 mg tropomyosin/kg. The protein chip was not found to be sensitive to other kinds of foods but cross-reacted to some extent with allergens of some other crustaceans. The recoveries ranged from 69.2 to 99.9%, while the intra- and inter-assay coefficients of variation were <13% and <19%, respectively. It seems that the new assay is reliable enough to detect shrimp allergens in food and food products and help minimize the instances of shrimp allergy. It is also possible to use the protein chip for simultaneous detection of other food allergens.     

定量检测转基因植物蛋白Cry1Ac的双抗体夹心ELISA方法建立

以典型且研究较多的抗虫CrylAc基因表达的CrylAc蛋白为检测目标,制备其单克隆抗体及辣根过氧化物酶标记的单克隆抗体,在优化抗体纯化方案,获得纯化抗体的基础上,以CrylAc单克隆抗体为包被抗体,以辣根过氧化物酶标记的CrylAb单克隆抗体为检测抗体,建立了CrylAc蛋白的双抗体夹心酶联免疫吸附分析(ELISA)检测法,并用于玉米中CrylAc蛋白含量的检测。结果显示,所建立的双抗体夹心ELISA法稳定性较好,测定变异系数在3%以内;在10 ng/mL~200 ng/mL的浓度范围内,线性回归方程为+=0768.0x0114.0y(R2=0.9989),检测限为9.49 ng/mL;对玉米样品提取液中Cry1Ac蛋白含量的测定回收率在102.5%~103%范围内。本研究所建立的双抗体夹心ELISA法为玉米中Cry1Ac蛋白的定量检测提供了有效的手段,可作为一种潜在的检测方法用于转基因产品的检验检疫中,在出入境检验检疫工作中也有较高的应用价值。