双抗夹心酶联免疫吸附法检测沙门氏菌

沙门氏菌是自然界中普遍存在的一种食源性致病菌,在中国的食物中毒中沙门氏菌引起的病例占第一位。本研究以福尔马林灭活的鼠伤寒沙门氏菌免疫雌性日本大耳兔,制备抗沙门氏菌多克隆抗体。以抗沙门氏菌多克隆抗体作为捕获抗体,以抗沙门氏菌单克隆抗体C1359作为检测抗体,建立快速检测沙门氏菌双抗夹心ELISA方法。结果表明:该方法可以检测A、B、C、D、E等五种典型的沙门氏菌,对沙门氏菌纯培养液的最低检测量为1×104CFU/mL,与其他杂菌不存在交叉反应,具有较好的灵敏性和特异性。      

双抗夹心ELISA快速检测冷鲜肉中福氏志贺氏菌2a

为建立冷鲜肉中福氏志贺氏菌2a双抗夹心ELISA检测体系,采用福氏志贺氏菌2a抗原免疫获得多克隆抗体,应用杂交瘤技术进行细胞融合,筛选出1株福氏志贺氏菌2a单克隆抗体杂交瘤细胞株,命名为2C10。试验结果表明,2C10杂交瘤细胞株抗体效价为1∶10.24×104,抗体纯度为96%,免疫球蛋白亚型为IgG2a。用此单克隆抗体作为检测抗体,多克隆抗体为捕捉抗体,建立双抗夹心ELISA体系,检测限为1 000 CFU/g。用此体系检测宋内志贺氏菌、大肠杆菌O157:H7、沙门氏菌、单增李斯特菌、枯草芽孢杆菌及阪崎肠杆菌,均无交叉反应。本研究可为冷鲜肉中福氏志贺氏菌2a的检测提供一种特异性强,准确度高的ELISA检测方法。     

地西泮单克隆抗体的制备及其酶联 免疫吸附检测方法的建立

目的 制备地西泮(Diazepam DZP)单克隆抗体,并且对制备的抗体进行一系列性质鉴定。方法 利用EDC法合成免疫原和包被原,用免疫原免疫Balb/C小鼠,当效价到1:16000以后取小鼠脾脏与SP2/0进行细胞融合。然后采用竞争结合双阳性两步筛选法,筛选出能分泌特异性抗体的杂交瘤细胞,并且利用有限稀释亚克隆方法得到单株细胞,采用体内诱生法制备腹水型单克隆抗体。接着利用辛酸-饱和硫酸铵法对腹水型抗体进行纯化,利用酶联免疫吸附,SPR等方法对纯化后的抗体进行性质鉴定。结果 成功合成了地西泮免疫原和包被原,免疫Balb/C小鼠7次后效价达到1:16000,最终制备出单克隆抗体,抗体解离常数(KDs)为4.0985×10_-7M,且与大部分结构类似物没有明显的交叉反应。应用此抗体建立间接竞争ELISA法,抗体的IC₅₀=10.8ng/mL,检测范围为0.45ng/mL-862ng/mL。结论 制备出了地西泮单克隆抗体,为地西泮的免疫学检测提供了有力的支持。     

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。     

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。     

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。     

乳品中热带假丝酵母菌的双抗夹心ELISA快速检测方法

本研究用于乳品中热带假丝酵母菌的快速检测。采用热带假丝酵母菌超声破碎后的上清蛋白作为免疫原分别免疫新西兰大耳白兔和Hartley豚鼠,获得热带假丝酵母菌的多克隆抗体。以兔抗体作为捕获抗体,豚鼠抗体作为检测抗体,通过矩阵法及正交分析建立乳品中热带假丝酵母菌快速、特异的双抗夹心ELISA检测方法。该方法检出限为98 ng/m L,板内变异系数小于2%,板间变异系数小于6%,特异性及重复性良好。实际样品检测中,通过对乳制品进行滤膜集菌并选择性增菌培养,超声后提取的蛋白上清应用本研究建立的双抗夹心ELISA检测方法,100 CFU/m L热带假丝酵母菌可在22 h内准确检测出阳性反应。      

酶联免疫吸附法在沙门氏菌检测中的研究进展

酶联免疫吸附技术(ELISA)作为先进的免疫化学检测技术正越来越广泛地应用于沙门氏菌检测中.文中介绍了沙门氏菌的特性及其主要危害,阐述了ELISA的基本原理,并结合国内外研究成果,综述了目前ELISA法在沙门氏菌检测中几种常用的方法,主要有:直接ELISA法、竞争ELISA法、Dot-ELISA法、LPS-ELISA法和PCR-ELISA法,从而表明了在沙门氏菌检测中ELISA法具有良好的特异性和敏感性.     

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.     

双抗夹心酶联免疫吸附法检测荞麦过敏蛋白

提取制备荞麦过敏原蛋白(TBt)并免疫动物,分别制备鼠多克隆抗体和兔多克隆抗体,建立了双抗夹心酶联免疫吸附检测法(ELISA)用于检测食品中的荞麦过敏原。SDS-PAGE结果表明,纯化的TBt纯度达到98%以上,鼠抗血清效价为1∶6400。建立的双抗夹心ELISA法对荞麦过敏原蛋白的最低检测限为0.16μg/m L,线性范围为0.16~16μg/m L。并采用建立的双抗夹心ELISA法对市场出售的15种食品中荞麦过敏成分进行了检测。结果显示,该方法对绝大多数食品中的荞麦过敏原都有很好的特异性及灵敏性,说明该法可用于食物过敏原的诊断及食品中微量荞麦过敏成分的检测。     

牛初乳中免疫球蛋白G双抗夹心ELISA检测方法的建立

将牛免疫球蛋白G（immunoglobulin G,IgG）作为免疫原免疫BALB/c小鼠,通过细胞融合、筛选获得分泌抗牛IgG单克隆抗体的细胞株。制备小鼠腹水抗体,进一步纯化获得抗牛IgG单克隆抗体。建立双抗夹心酶联免疫吸附法检测牛初乳中IgG质量浓度,该方法在7.8～1 000 ng/mL范围内有良好的线性关系,最低检出限为7.06 ng/mL,批内变异系数为4.52%,批间变异系数为4.94%,回收率为91.85%～102.45%。此法操作简便、准确度高、稳定性好,可用于实际牛初乳样品的快速检测。     

抗副溶血弧菌OMPK单克隆抗体的制备及其ELISA双抗体夹心检测方法建立

制备全长和截短的弧菌外膜蛋白K（outer membrane protein K,OMPK）,纯化后的OMPK免疫6～8 周雌性BALB/c小鼠,间接夹心酶联免疫吸附测定（enzyme linked immunosorbent assay,ELISA）法检测小鼠的血清效价,结果表明全长OMPK重组蛋白制备的血清效价是截短OMPK1重组蛋白制备血清效价的128 倍。通过全长的OMPK重组蛋白制备多株用于检测副溶血弧菌的单克隆抗体,用生物素（biotin）和辣根过氧化物酶对单克隆抗体进行标记。间接夹心ELISA实验结果表明：只有VP3-biotin和VP16-biotin与其他单克隆抗体配对检测不同的副溶血弧菌菌株表现出较高的灵敏度及良好的特异性,并且在检测其他种属20 株菌时无交叉反应。VP16(Fab)-biotin/VP3检测副溶血弧菌时较VP16-biotin/VP3具有更高的灵敏度,该方法用于检测海鲜样品时,VP16-biotin/VP3检测三文鱼（5～10 CFU/25 g）和血蚶样品为副溶血弧菌阳性。本研究开发了一种快速、灵敏、简便的副溶血弧菌间接夹心ELISA检测方法。     

Sensitive Monoclonal Antibody-based Sandwich ELISA for the Detection of Porcine Skeletal Muscle in Meat and Feed Products

A monoclonal antibody‐based sandwich enzyme‐linked immunosorbent assay (ELISA) was developed for the sensitive detection of porcine skeletal muscle in raw and heat‐processed meat and feed products. Heat treatment of meat samples up to 132 °C for 2 h did not affect the assay performance. The assay uses a pair of monoclonal antibodies (MAbs 8F10 and 5H9) specific to skeletal muscle troponin I (TnI). MAb 8F10, reacting to mammalian TnI, is the capture antibody and the biotin‐conjugated MAb 5H9, specific to porcine TnI, the detection antibody. The sandwich ELISA is able to detect 0.05% (w/w) of laboratory‐adulterated pork in chicken, 0.1% (w/w) pork in beef mixtures, 0.05% (w/w) pork meal in soy‐based feed, and 1% commercial meat and bone meal (MBM), containing an unknown amount of pork, in soy‐based feed. This new assay provides a rapid and reliable means to detect the contamination of meat and feed products with trace amounts of porcine muscle tissue to ensure product quality and safety.     

Monoclonal Antibody-Based Sandwich ELISA for Reliable Identification of Imported Pangasius Catfish

ABSTRACT:  Pangasius catfish, primarily tra  (Pangasius hypophthalmus ) and basa ( Pangasius bocourti ), are farm-raised catfish imported from Asia and have become a common substitute for domestic catfish, grouper, and other high-valued fish in restaurant-served dishes in the United States. This article reports on the development of a monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for the identification of cooked Pangasius fish, basa, and tra. The assay uses a pair of monoclonal antibodies (MAbs F7B8 and T7E10) specific to a heat-stable 36-kDa protein present in a saline extraction of the fish muscle; MAb F7B8, which cross-reacts to all fish species, is the capture antibody and the biotin-conjugated MAb T7E10, which is specific to Pangasius fish, is the detection antibody. This sandwich ELISA reliably identified fully cooked basa and tra from more than 70 common finfish, shellfish, land animal species, and other protein materials tested. It can also sensitively detect 0.5% of adulterated basa or 0.1% tra in a mixed crabmeat products with low intra-assay (%CV: 2.59 to 4.14) and inter-assay (%CV: 3.36 to 3.71) variabilities. The new assay provides a rapid and reliable means of distinguishing fish in the Pangasiidae family from other common food fish and nonfish species and will greatly assist efforts to discourage the illegal practice of substituting high-value popular fish species by the cheaper farm-raised imported Pangasius fish at the retail and restaurant levels.     

ELISA方法与国标法在检测鲜奶中沙门氏菌的比较研究

应用直接ＥＬＩＳＡ方法对３５０份奶样进行沙门氏菌的检测，其阳性检出率为１．４％；同时采用常规方法检测，其阳性检测率为１．２％。与常规方法相比，该法的敏感性和特异性分别为１００％和９９．７％（３５０份奶样）。结果表明，该法具有快速、准确等特点，２￣３天即可完成样品筛选。     

Detection of cow milk adulteration in yak milk by ELISA

In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10–8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk.