澳洲坚果蛋白夹心ELISA方法的建立及应用

利用碱溶酸沉法提取未加工的澳洲坚果蛋白,分别制备鼠、兔多克隆抗体,并以兔多抗为捕获抗体,鼠多抗为检测抗体建立了双抗夹心酶联免疫检测方法。该方法的检出限为(0.95±0.17)ng/mL。方法与大豆、核桃、榛果、杏仁、花生、开心果、腰果和小麦蛋白无明显交叉反应,证明该方法特异性良好。焙烤的澳洲坚果和焙烤饼干中澳洲坚果蛋白的添加回收率为73.67%～77.03%,说明该方法不仅适用于未加工澳洲坚果蛋白的准确检测,也适用于热加工后以及焙烤食品中澳洲坚果蛋白的定量检测。     

检测食品中志贺氏菌的双抗夹心ELISA方法的研究

研究获得纯化抗志贺氏菌IgY,经检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320;以志贺氏菌免疫新西兰大耳白兔,获得抗志贺氏菌的兔抗体,效价可达1∶12800。以此两种抗体建立双抗夹心ELISA,正交试验分析表明,兔抗体包被条件为4℃过夜,采用不封闭,抗原与包被抗体结合条件为37℃、1h;加入检测抗体(IgY)的浓度为0.25mg/mL,其结合条件为37℃、1h。该方法对纯培养菌液检出限为105cfu/mL,具有良好的敏感性;10株其他菌株的检测结果表明,该方法只与志贺氏菌发生特异性反应,不与其他菌株的抗原发生交叉反应。染菌的食品样品经选择性增菌后进行双抗夹心ELISA检测,含0.1～1cfu/mL志贺氏菌的样品在增菌13h后可检出阳性反应,含1～10cfu/mL志贺氏菌的样品在增菌11h后可检出阳性反应。     

食品中过敏原胡桃蛋白间接竞争ELISA检测方法研究

胡桃是坚果类食品中引起过敏反应的首要食品。采用胡桃总蛋白产生的多克隆抗体,建立检测胡桃成分的间接竞争ELISA方法,并通过特异性试验和样品回收试验对该方法进行验证。结果表明:该方法对胡桃蛋白的定量检测限(LOQ,相当于IC80)为94 ng/mL;在特异性试验中,只有美洲山核桃出现交叉反应,其它植物样品均未出现交叉反应;将50~1 000 ng/mL〔相当于10~200μg/g〕的胡桃蛋白添加到小麦蛋白中,胡桃蛋白的回收率在78%~93%之间。     

双抗夹心ELISA检测食品中大肠杆菌O157:H7方法研究

研究获得纯化抗大肠杆菌O157:H7IgY抗体,经检测10mg/ml纯化IgY抗体的效价为1:320;以大肠杆菌O157:H7免疫新西兰大耳白兔,获得兔抗大肠杆菌O157:H7IgG抗体,效价达1:25600。以兔抗大肠杆菌O157:H7IgG抗体稀释3200倍作为捕获抗体,抗大肠杆菌O157:H7IgY抗体为检测抗体建立双抗夹心ELISA方法检测大肠杆菌O157:H7,正交试验分析表明,捕获抗体于37℃包被2h、不封闭、抗原与捕获抗体于37℃结合2h、检测抗体浓度为0.25mg/ml、与抗原于37℃结合1h为最优反应条件。该方法对纯培养菌液检出限为105CFU/ml,具有良好的敏感性及特异性。染菌样品经在EC增菌液中选择性培养后进行双抗夹心ELISA检测,接种量为0.1～1CFU/g(ml)的样品在培养12h后可检出阳性反应,1～10CFU/g(ml)的样品在培养8h后可检出阳性反应。     

双抗夹心ELISA检测转Bar基因抗除草剂大豆

为快捷有效地检测转Bar基因抗除草剂大豆,利用已制备的抗除草剂Bar基因编码蛋白,膦丝菌素乙酰转移酶（phosphinothricin acetyltransferase,PAT）单克隆抗体和多克隆抗体,建立PAT蛋白双抗夹心酶联免疫吸附检测方法,对转Bar基因抗除草剂大豆不同组织材料进行定量检测。结果显示,最佳检测条件为捕获抗体质量浓度0.125 μg/mL,包被酶标板,37 ℃孵育1 h后4 ℃静置过夜,检测样品37 ℃孵育1.5 h,检测抗体质量浓度6.25 μg/mL,37 ℃孵育1.5 h;PAT蛋白的最低检测限为0.04 ng/mL,大豆蛋白体系中为8 ng/mL;重复性变异系数小于3%。利用上述检测条件,对实验建立的转Bar基因抗除草剂大豆进行PAT蛋白定量检测,成功地在根、茎、花、叶、种子不同部位检测到该蛋白的表达。     

β-伴大豆球蛋白间接竞争ELISA检测方法建立

β–伴大豆球蛋白是引起过敏现象发生的主要大豆蛋白过敏原之一,建立β–伴大豆球蛋白过敏原的检测为研究大豆中过敏原降低程度奠定了良好的基础。采用自制的兔抗β–伴大豆球蛋白多克隆抗体,并以标准β–伴大豆球蛋白为抗原、辣根过氧化物酶标记的羊抗兔IgG为酶标二抗,建立了定量检测β–伴大豆球蛋白的间接竞争ELISA方法。并对该方法进行了条件优化及精密度的测定。结果为:抗原包被浓度为0.3μg/mL,一抗最佳稀释度为1∶3200,酶标二抗稀释度为1∶10000;板内误差2.19%,板间误差9.61%。回归直线方程为y=–2.439 2x–1.871 8,相关系数R2=0.995 7,其检测的线性范围为0.1²μg/mL。表明所建方法具有一定的重复性和灵敏度,可用于大豆制品过敏原的检测。     

大豆过敏原的检测方法研究进展

大豆是八大食物过敏原之一,如何快速、准确、低成本地检测大豆过敏原已成为科研、食品工业以及医药卫生等行业关注的焦点。大豆中的主要过敏原是大豆球蛋白、β-conglycinin、Gly m Bd 60K、Gly m Bd 30K和Gly mBd 28K。其中,大豆过敏原的主要检测方法有电泳法、免疫学方法、PCR法、色谱法、质谱法以及生物芯片技术。本文通过对大豆中主要过敏原的检测方法进行综述,旨在为不同食物中大豆过敏原的检测提供方向,以保障大豆过敏患者安全。     

水产品中志贺氏菌双抗夹心ELISA检测方法建立与应用

志贺氏菌(Shigella)通称痢疾杆菌,是一类具有传染性强和危害严重的革兰阴性食源性致病菌,广泛污染各类动物源性食品.该研究将志贺氏菌经0.5％福尔马林溶液灭活后,多次免疫雌性大耳兔获得抗志贺氏菌多克隆抗体,以抗志贺氏菌多克隆抗体作为捕获抗体,以辣根过氧化物酶(horseradish peroxidase,HRP)标...     

双抗体夹心ELISA法测定食物中大豆过敏原蛋白成分

通过建立双抗体夹心ELISA法测定食物中大豆过敏原蛋白成分,为进出口食品中大豆过敏原成分检测和食物过敏性疾病的预防提供技术基础.提取大豆总蛋白,免疫小鼠制备抗大豆总蛋白的多克隆抗体,用该抗体做包被抗体,并用生物素标记该多抗作为捕捉抗体,从而建立双多抗体夹心ELISA法;检测该方法的灵敏度,同时检测10种食品中是否含有大豆过敏原蛋白成分.SDS-PAGE电泳结果显示大豆过敏原总蛋白在120、60、35、30、28、18 ku处条带明显;免疫印迹显示,这些条带与制备的高效价抗大豆蛋白的多抗具有明显的阳性反应.检测食品中大豆过敏原蛋白成分双抗体夹心ELISA法最低检出限为～100 ng/mL,标准曲线在100ng/mL～12.8μg/mL范围内线性良好;10种进口食品检测结果与食物过敏原标签标注内容相符.     

双抗夹心ELISA法定量检测食品中大肠杆菌O157：H7初探

以鸡抗O157:H7特异性脂多糖(LPS)抗体(IgY)为捕获抗体,酶标抗体(HRP-IgY)为检测抗体建立双抗夹心ELISA法检测食品中大肠杆菌0157:H7的方法,确定了抗原浓度对数与OD450值的高度线性相关性,根据检测OD450值可从拟合回归曲线确定样品中的含菌量,含菌量≥104CFU/ml的食品样品可直接用双抗夹心法进行检测,含菌量≤104CFU/ml的食品样品可增菌14h后检测.     

间接竞争ELISA法检测牛乳中β-乳球蛋白含量的准确性评价

研究建立了以β-乳球蛋白标准品为包被抗原、自制抗体为一抗、辣根过氧化物酶标记的羊抗兔IgG:HRP为二抗、邻苯二胺为底物的间接竞争酶联免疫检测法(indirect competitive enzyme-linked immu nosorbent,ELISA)。同时以反相高效液相色谱法定量检测相同样品中β-乳球蛋白含量,采用t检验方法来评价间接竞争ELISA的方法定量检测β-乳球蛋白的准确性。t检验方法结果显示:在显著水平α=0.05下,该间接竞争ELISA法检测β-乳球蛋白的测量值较为准确。     

两种PCR方法检测食品中花生过敏原Arah1成分

采用套式PCR和荧光实时定量PCR两种方法检测食品中花生主要过敏原Ara h1基因成分,从而推断食品中是否含有花生过敏原成分。根据GenBank提供的花生主要过敏原基因Ara h1 DNA序列(登录号为AF432231)的中一段序列分别设计2对内外特异性引物,扩增目的基因片段来建立套式PCR方法;再用上述内引物扩增目的片段,建立SYBRGreenⅠ荧光实时定量PCR方法,绘出拷贝数-CT标准曲线;并通过这2种PCR方法检测8种食品中含有花生主要过敏原Ara h1基因成分。套式PCR方法具有较高的特异性和灵敏度,SYBR GreenⅠ荧光实时定量PCR方法的标准曲线在3×102至3×108 copies范围内线性关系良好,R2值为0.993 5,检测低限设定为3×103 copies。2种方法检测8种食品样品,结果均与食物过敏原标注内容相符。本研究建立的2种方法具有快速、灵敏等优点,可用于食品中花生主要过敏原基因Ara h1成分的检测。     

Control of Food Animals in Their Chains: Tools to Detect Unwanted Substances and Factors

Food safety asks for zoonoses/animal health, residues/ contaminants, zoonotic agents/ resistance genes, also for wellbeing of animals. Food chains with animal categories, the presence of risk factors in the region, also local keeping techniques characterize the list of risk factors to be considered. Good Practices are intended to keep the chains safe (prevention), for verification, internal or external control tests are established for the absence/ presence of unwanted factors in daily work's performance (called “meat inspection” for slaughter animals). Consequently, for meat inspection, appropriate methods from different disciplines should be provided for any of inspection targets.     

Development of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Buckwheat Residues in Food

Abstract: Buckwheat is a pseudocereal (an eudicot with seed qualities and uses similar to those of monocot cereals, family Poaceae) that is consumed in some Asian countries as a staple, and in some western countries as a health food. Allergic reactions to buckwheat are common in some countries. The objective was to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to detect traces of buckwheat that might inadvertently contaminate other foods in order to assure accurate labeling and consumer protection. Buckwheat-specific antibodies produced in 3 species of animals were tested for specificity and titer by direct ELISA and immunoblot. A sandwich ELISA was developed utilizing pooled rabbit antibuckwheat sera to capture buckwheat proteins and pooled goat antibuckwheat sera, followed by enzyme-labeled rabbit antigoat immunoglobulin G (IgG), to detect bound buckwheat proteins. The lower limit of quantification (LOQ) of the sandwich ELISA was 2 parts per million (ppm) of buckwheat in the presence of complex food matrices. The ELISA is highly specific with no cross-reactivity to any of 80 food ingredients and matrices tested. Validation studies conducted with buckwheat processed into noodles and muffins showed greater than 90% and 60% recovery, respectively. The percent recovery of buckwheat from noodles was similar to that achieved with a commercial buckwheat ELISA kit (ELISA Systems Pty. Ltd., Windsor, Queensland, Australia) at high buckwheat concentrations. However, the sensitivity of this ELISA was greater than the commercial ELISA. This newly developed ELISA is sufficiently specific and sensitive to detect buckwheat residues in processed foods to protect buckwheat-allergic subjects from potential harm. Practical Application: Buckwheat is becoming a common food ingredient in a number of processed foods due to potentially beneficial nutritional properties, without the celiac disease inducing glutenin proteins of wheat and related cereals. However, buckwheat causes allergy in some individuals and must be labeled and tested accurately to protect those with allergy to buckwheat. We describe the development of a new test assay to help food producers ensure that buckwheat is not present in foods that are not intended to contain buckwheat.     

间接竞争ELISA法检测呋喃唑酮代谢物

以人工抗原和特异性抗体为基础,建立了以2-硝基苯甲醛为衍生试剂检测呋喃唑酮代谢物3-氨基-2-恶唑烷酮(AOZ)间接竞争ELISA方法。通过方阵滴定和间接竞争法确定ELISA方法的抗原抗体的最佳工作浓度;得到标准曲线的线性范围为0.25~10ng/mL,线性关系良好;根据标准曲线计算IC50为0.5881~1.1333 ng/mL。此外,检测了与其他类似物的交叉反应率,显示了很好的特异性;并且通过对虾样品添加回收率的测定,证明了该方法的准确性。高温短时间衍生化处理可达到与37℃孵育过夜同样的衍生效果。     

Purification, Thermal Stability, and Antigenicity of the Immunodominant Soybean Allergen P34 in Soy Cultivars, Ingredients, and Products

ABSTRACT:  Protein P34 ( Gly m Bd 30K) is the immunodominant allergen in soybean ( Glycine max L.). The objectives of this study were (1) to study the effect of thermal treatment on P34 antigenicity and secondary structure after isolation and purification of P34 from soybean by chromatographic techniques; (2) to identify the variability of P34 allergen within 138 accessions from a diverse USDA soybean germplasm collection by ELISA; and (3) to quantify P34 immunoreactivity in various commercial soy ingredients and products. Thermal processing decreased P34 antigenicity. Soybean accessions with the highest P34 content were ancestral (12 mg/g defatted flour) followed by modern (10 mg/g defatted flour) and exotic (8 mg/g defatted flour). The cultivar that emerged as the lowest-expressing P34 accession was PI548657 (2.3 mg/g defatted flour). Among commercial soy ingredients, soy flour yielded the highest P34 antigenicity (32 mg/g extracted protein) followed by soy protein isolate (29 mg/g extracted protein) and soy protein concentrate (24 mg/g extracted protein). Among soy consumer products, soymilk presented the highest P34 antigenicity, ranging from 7 to 23 mg/g extracted protein, followed by tempeh (8 mg/g extracted protein), soy infant formula (3.4 mg/g extracted protein), soy powder (2 mg/g extracted protein), and soy cheese products (0.50 mg/g extracted protein). Korean miso, soy sauce, soy chili mix, soy nuts, soy cream cheese, soy meat patty, texturized soy protein, and soy cereal exhibited undetectable P34 antigenicity (detection limit = 0.45 ng). Selecting soybean varieties with low levels of this allergen, or via processing, could potentially make soybean products less antigenic.     

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.