基于金纳米花标记的免疫层析法检测牛奶中黄曲霉毒素M1

研究金纳米花作为信号放大用于黄曲霉毒素M_1高灵敏检测。用粒径为(91.8±0.8)nm、分支数多的金纳米花(gold nanoflowers,AuNFs)标记黄曲霉毒素M_1(aflatoxin M_1,AFM_1)单克隆抗体(monoclonal antibody,Mab),制备用于检测牛奶中AFM_1的金纳米花免疫层析检测卡(aflatoxin M_1-gold nanoflower immunochromatographic test card,AFM_1-GFICT)。纳米金免疫分析读数仪被用于读取AFM_1系列浓度(20 pg/mL～1 000 pg/mL)的信号值。以AFM_1浓度为横坐标,信号值为纵坐标绘制非线性四参数拟合曲线。非线性四参数拟合曲线相关系数R~2=0.999 3,检测限为12.3 pg/mL。检测在空白牛奶样品加入AFM_1标准品浓度为0.1、0.3、0.5μg/kg的回收率为80.4%～118.2%,变异系数(n=10)为4.27%～9.43%。15份牛奶样品的AFM_1-GFICT和商业化AFM_1-ELISA检测结果相比,线性拟合相关系数R~2=0.976 8。     

金标免疫层析法检测黄曲霉毒素B1的方法

应用金标免疫层析法（GICA）研制的金标免疫试纸条对食品中黄曲霉毒素B1的检测，来确立该方法的各项技术指标。结果表明：方法的最低检测限：2．5ng／mL；金标免疫试纸条在4℃环境下可稳定10个月以上；与类似毒素AFB2、AFG2的交叉反应率分别是7，14％、6，25％；检测时间小于15min；GICA与ELISA方法的符合率达90％以上。该方法简便、快速，有较高的灵敏度，重复性好，特异性强，能定性或半定量检测食品中的黄曲霉毒素B1含量。     

胶体金免疫层析法检测酱油中黄曲霉毒素B1

采用胶体金免疫层析法检测酱油中的黄曲霉毒素B1。加标的酱油样品经提取后,以胶体金免疫层析法对其进行黄曲霉毒素B1测定,并与酶联免疫吸附法进行比较。结果表明,当酱油中黄曲霉毒素含量超过国家限量标准(5μg/kg),胶体金免疫层析法检测结果为阳性,说明该方法能够满足酱油样品中黄曲霉毒素B1监控的要求。     

牛奶及奶粉中黄曲霉毒素M1的快速测定

采用免疫亲和柱－荧光光度法快速测定了牛乳及乳制品中黄曲霉毒素M1。试样经过离心、脱脂、过滤后滤液经过键合有黄曲霉毒素M1特殊抗体的免疫亲和柱净化，此抗体对黄曲霉毒素M1具有专一的识别能力，黄曲霉毒素M1键合在分离柱中的抗体上，用甲醇与水之比为10：90的混合液将免疫亲和柱上杂质除去；以甲醇与水之比为80：20的混合液通过分离柱洗脱；加入溴溶液衍生，以提高测定灵敏度，衍生化后的洗脱液于荧光光度计中测定黄曲霉毒素M1，检测低限为0．1μg/kg；在0．1－1．0μg/kg范围内，回收率为89．6％－96．1％，变异系数为0．52％－5．8％，分析一个样品的时间小于30min，分析过程中不使用黄曲霉毒素M1标准物质，结果表明，本方法具有准确、简单、快速、安全等优点，可以满足少量和批量样品的检测需要。     

胶体金免疫层析法快速检测黄曲霉毒素B1的研究

本文应用胶体金免疫层析技术,建立了一种快速检测食品中黄曲霉毒素B1的方法。采用柠檬酸三钠还原法制备胶体金颗粒,标记抗黄曲霉毒素B1单克隆抗体并喷于玻璃纤维上,黄曲霉毒素B1偶联抗原和二抗鼠抗驴分别结合于硝酸纤维膜上,依次将样本垫、胶金垫、硝酸纤维膜和吸水纸组装切割成胶体金试纸条并装入检测卡中。测试结果表明黄曲霉毒素B1快速检测试纸条的灵敏度为5ng/ml,检测时间为10min,批内和批间重复性为100%,假阳性率和假阴性率均为0。使用简单方便,非常适合现场快速检测黄曲霉毒素B1。     

抗黄曲霉毒素M1免疫亲和柱的制备

制备黄曲霉毒素M1污染样品的净化前处理免疫亲和柱.利用无血清培养基制备的高纯度抗黄曲霉毒素M1单克隆抗体和溴化氰活化的Sepharose 4B进行偶联、装柱后通过IC-ELSIA对免疫亲和柱的性能评价确立了最佳反应条件.免疫亲和柱的有效柱容量为500ng,经添加回收试验测定的平均回收率为92.46%0,变异系数2.3%...     

胶体金免疫层析法快速检测食用油中黄曲霉毒素残留

为建立用于快速检测粮油样品中黄曲霉毒素药物残留含量的胶体金免疫层析检测试剂,我们采用免疫竞争法,将抗黄曲霉毒素单克隆抗体-胶体金复合物包被在微孔中,并将人工合成的黄曲霉毒素抗原包被在硝酸纤维素膜(NC膜)表面作为检测线(T 线)。待测样品中的黄曲霉毒素残留物将与NC膜上的黄曲霉毒素抗原竞争结合胶体金标记的抗黄曲霉毒素单克隆抗体,并以颜色直观显示检测结果。检测食用油样品时,灵敏度可达到0.5μg/L,仅需20 min。实验证明该检测试剂具有较高的灵敏度及很好的特异性,操作便捷,稳定可靠,可作为黄曲霉毒素残留现场监控的有效快速筛检手段。     

利用荧光免疫层析卡测定黄曲霉毒素M1的研究

将量子点与抗黄曲霉毒素M1(AFM1)的单克隆抗体偶联,以AFM1-BSA偶联物包被于NC膜作为检测线,山羊抗鼠二抗包被于NC膜上作为质控线,构建了直接竞争检测AFM1的荧光定量免疫层析检测卡体系,在0.156μg/L~1.25μg/L范围内能够准确定量黄曲霉毒素B1,灵敏度达到0.156μg/L。     

牛奶中黄曲霉毒素的来源与控制

本文概述了牛奶中黄曲霉毒素的存在种类,来源及其性质.对近年来国内外对牛奶中黄曲霉毒素毒素的检测方法、控制和消除方法进行了研究.     

牛奶中黄曲霉毒素M1测定方法的比较

本试验采用薄层层析法、黄曲霉毒素M1试剂盒快速检测法、酶联免疫法,测定了牛奶中黄曲霉毒素的含量并比较了三种测定方法的异同。三种检测方法中,薄层层析法试验成本底,但样品前处理繁琐,最低检测限高,不能检测出低含量的黄曲霉毒素;黄曲霉毒素M1试剂盒快速检测法属于半定量检测法,方法简单快速,但偶尔出现假阳性,适于原奶的筛选。酶联免疫法方法为定量检测法,相对准确,但成本高,适于对可疑牛奶的仲裁实验。     

Comparison of immunochromatographic assays based on fluorescent microsphere and quantum-dot submicrobead for quantitative detection of aflatoxin M1 in milk

The performance of fluorescent microsphere immunochromatographic assay (FM-ICA) and quantum-dot submicrobead immunochromatographic assay (QB-ICA) was systematically and comprehensively compared in quantitative detection of aflatoxin M₁ in milk. Under optimum conditions, the advantages of FM-ICA include lower limit of detection of 42.3 pg/mL with better accuracy, precision, reliability, and practicability. The advantages of QB-ICA include shorter detection time and lower monoclonal antibody consumption. The 2 ICA were consistent with liquid chromatography-tandem mass spectrometry. This study serves as a reference for selecting appropriate fluorescent labels for the immunochromatographic assay of aflatoxin M₁.     

黄曲霉毒素M1 ELISA试剂盒的检测效果研究

目的为验证本公司生产的黄曲霉毒素M1酶联免疫法(ELISA)检测试剂盒的检测效果。方法用ELISA检测试剂盒对牛奶、酸奶、奶粉和干酪等4种样品中黄曲霉毒素M_1的残留量进行检测,并与高效液相色谱荧光检测法进行对照。结果该试剂盒对4种样品的最低检测限分别为0.039、0.192、0.306、0.199μg/kg(L),试剂盒板内板间变异系数均小于5%;对4种样品做加标回收试验,其回收率均在93%~120%之间,变异系数均小于10%;该方法与高效液相色谱法检测实际样品的阴、阳性判断结果一致。结论该方法稳定、可靠、灵敏,可满足食品中黄曲霉毒素M_1残留快速检测的需要。     

酶联免疫法测定婴幼儿配方乳粉中黄曲霉毒素M1的方法研究

采用酶联免疫法建立婴幼儿配方奶粉中黄曲霉毒素M1的测定方法。样品经冷冻离心、脱脂棉去脂等简便的前处理操作,采用黄曲霉毒素M1的酶联免疫试剂盒进行分析。结果显示,黄曲霉毒素M1在0～0.08 μg/L范围内线性关系良好,方法检出限为0.05 μg/kg,回收率为90.0%～105.0%,精密度相对标准偏差（RSD）为8.1%。该方法快速、简便、特异、可靠,适于现代批量检测的需求,为保证乳制品的质量安全提供了一可靠的筛选方式,将更好地配合液相色谱-质谱法作定性和定量分析。     

Determination of aflatoxins by enzyme-linked immunosorbent assay with special reference to aflatoxin M1 in raw milk

There is an increasing requirement for the screening of animal feedstuffs, plant products and edible animal tissues for human consumption, for the presence of contamination by aflatoxins. An enzyme-linked immunosorbent assay is described which may be used to determine aflatoxins in raw milk at 0.1μg litre^?1 and in extracted feedstuffs at the level of 5μgkg^?1. The method includes a novel conjugation procedure allowing heterologous conjugates to be prepared resulting in increased sensitivity without interference by sample matrix effects. Suitably cross-reactive antisera were produced using the cyclopentanone ring of the aflatoxin molecule as the principal immunogenic determinant.     

Time-resolved fluorescent immunochromatographic assay-based on three antibody labels for the simultaneous detection of aflatoxin B1 and zearalenone in Chinese herbal medicines

ABSTRACT

A time-resolved fluorescent immunochromatographic assay (TRFICA) was successfully developed for the sensitive, simultaneous, and quantitative detection of aflatoxin B₁ (AFB1) and zearalenone (ZEN) in Chinese herbal medicines. Eu-nanospheres (EuNPs) with unique optical properties increased the stability and sensitivity of the immunochromatographic assay. To obtain stable quantitative results, we applied a three-label system in which monoclonal antibodies for AFB1 and ZEN were conjugated to the EuNPs as detection probes on the test line (T line), and EuNP-labelled chicken IgY conjugates acted as the reference on the control line (C line). The fluorescence intensities of the T and C lines were recorded, and the T/C ratio was employed as the quantitative signal for the elimination of strip variation and matrix effects. The parameters that affected the TRFICA were optimised. Under optimal conditions, the established TRFICA gave good linear ranges from 0.60 μg/kg to 3.92 μg/kg for AFB1 and from 0.40 μg/kg to 1.28 μg/kg for ZEN. The limits of detection for AFB1 and ZEN were as low as 0.60 and 0.40 μg/kg, respectively, in Chinese herbal medicines Semen coicis, Rhizoma dioscoreae, and Platycodon grandiflorus, respectively. The average recoveries of the spiked samples were 73%–95% for AFB1 and 75.83%–90% for ZEN, both with a relative standard deviation of < 9.08%. The results of 15 actual samples detected by the developed TRFICA showed a satisfactory correlation with those of ultra-performance liquid chromatography tandem mass spectrometry. Therefore, the TRFICA is a simple, rapid, and sensitive approach to quantitatively detect mycotoxins in Chinese herbal medicines.     

基于聚丙烯酰胺分子印迹包金核壳纳米粒子对食用油中黄曲霉素B1的SERS检测

在金纳米粒子周围包覆聚丙烯酰胺黄曲霉素B1分子印迹聚合物,制得以金纳米为核,分子印迹聚合物为壳的核壳结构纳米粒子。黄曲霉素B1分子可以被聚丙烯酰胺壳层空穴特异性捕获,进入金纳米粒子等离子体共振磁场有效范围而实现黄曲霉素B1的SERS检测,检测线性范围为3×10^-113×10^-4mol/L,相关系数R²为0.982,检测限达到3×10^-11mol/L。将建立的方法用于食用油样品中的黄曲霉素B1检测,黄曲霉素B1的含量在4.65×10^-94.98×10^-5mol/L之间,样品回收率为89.84%¹01.24%,相对标准偏差为4.51%¹3.11%。结果表明,本方法快速准确,操作简便,可以用于食用油中黄曲霉素B1的快速检测。      

Quantum dot bead-based competitive immunochromatographic assay for enterotoxin aureus A detection in pasteurized milk

Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.     

Occurrence of aflatoxin M1 in domestic milk in Japan during the winter season

A total of 208 samples of commercial pasteurized milk gathered from retail outlets across Japan during the winter season were analysed for aflatoxin M₁ (AFM₁). Japan was divided into 11 regions from north to south, and nine to 45 milk samples from each region were randomly purchased between December 2001 and February 2002. Each milk sample was cleaned up by an immunoaffinity column, and AFM₁ was quantified by liquid chromatography with fluorescence detection in four independent laboratories. The limit of detection of the method was 0.001 μg kg^-1. The identity of the putative AFM₁ in milk sample was confirmed by the formation of AFM₁ hemi-acetal with trifluoroacetic acid. Based on the results obtained with spiked samples (0.05 μg AFM₁ kg^-1), the mean recovery was 91.4%, the relative standard deviation for repeatability was 4.6%, and the relative standard deviation for reproducibility was 8.0% among four independent laboratories. AFM₁ was detected in 207 (99.5%) of 208 milk samples at 0.001-0.029 μg kg^-1, with a mean of 0.009 μg kg^-1 and a 90th percentile of 0.014 μg kg^-1. No significant difference of the level of AFM₁ contamination was observed among the regions.