玉米赤霉醇酶标抗原的制备及其直接和间接竞争ELISA检测方法的建立与比较

采用活性酯法将半抗原玉米赤霉醇-16-羧丙基醚与辣根过氧化物酶连接,制备了三种结合比的酶标抗原。通过紫外吸收法和直接非竞争ELISA法对酶标抗原的偶联结果和抗原性进行鉴定。最终选择结合比为1.3∶1的酶标抗原建立直接竞争ELISA检测方法。并对建立的直接竞争ELISA(DC-ELISA)与间接竞争ELISA(IC-ELISA)方法在检出限、检测线性范围、检测时间和二抗的使用方面进行了比较。     

硫代磷酸二乙酯类农药半抗原设计及抗体识别特性

通过分析硫代磷酸二乙酯类农药的结构特点, 设计并合成了系列半抗原; 采用活泼酯法将半抗原分别与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联制备了系列免疫原和包被原; 通过免疫新西兰大白兔获得了相应抗硫代磷酸二乙酯类农药的类特异性抗体. 建立检测硫代磷酸二乙酯类农药的间接竞争酶联免疫分析(ELISA)方法, 分析探讨了免疫半抗原结构对抗体特性的影响, 并阐述了包被半抗原结构对ELISA灵敏度的影响规律. 结果表明, 手臂取代位置在苯环对位且手臂较短的免疫原具有较好的免疫效果, 同时异源包被可以显著提高ELISA方法的灵敏度. 由抗体PAb-H1和包被原H6-OVA建立的间接竞争ELISA方法可以同时检测7个广泛使用的有机磷农药, 其半抑制浓度(IC50)分别为蝇毒磷(0.013 mg/L)、对硫磷(0.348 mg/L)、喹硫磷(0.022 mg/L)、三唑磷(0.035 mg/L)、甲拌磷(0.751 mg/L)、除线磷(0.850 mg/L)及辛硫磷(1.301 mg/L), 最低检测限符合国内外相关有机磷药物最大允许残留限量标准(MRLS)的检测要求.     

基于高特异性单克隆抗体的间接竞争ELISA检测食品中胭脂红的研究

该研究设计合成了一种胭脂红半抗原,分别采用重氮化法和戊二醛法将半抗原与载体蛋白偶联制备人工抗原,通过免疫Bal b/c小鼠及杂交瘤技术成功筛选制备了胭脂红高特异性单克隆抗体,与苋菜红、柠檬黄等结构类似物无交叉反应.基于该抗体建立了间接竞争酶联免疫分析方法用于检测食品中胭脂红残留.该方法对胭脂红的半抑制浓度(IC50)和...     

膜基质免疫分析法同时检测玉米中伏马菌素B1和呕吐毒素

建立了基于聚偏氟乙烯膜(Polyvinylidene fluoride,PVDF)基质的直接竞争免疫分析法,同时检测玉米中的伏马菌素B1(Fumonisin B1,FB1)及呕吐毒素(Deoxynivalenol,DON)。PVDF膜用甲醇浸湿、激活,用移液器将FB1及DON全抗原点阵于相应的膜反应区,同时采用三聚氰氯法和高碘酸钠法分别制备抗FB1、抗DON的辣根过氧化酶(Horseradish peroxidase,HRP)标抗体(monoclonal antibody,McAb),以直接竞争免疫检测方法的模式实现同时检测玉米中的FB1及DON。该方法对于FB1和DON的检出限分别为2.5和50μg/L,样品前处理简单,检测时间15 min,可肉眼辨别结果,随机检测了30份市售玉米样品,并与市售ELISA试剂盒进行方法学比较,结果无明显差异。     

噻虫嗪单克隆抗体制备及酶联免疫吸附分析方法的建立

通过化学修饰合成了噻虫嗪人工半抗原,采用碳二亚胺法将该半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,成功制备了分子结合比合理的免疫原和包被原。经过免疫原免疫6周龄Balb/c小鼠、PEG介导免疫鼠脾细胞和骨髓瘤细胞的融合和阳性杂交瘤细胞的筛选和克隆化,获得了效价高达1∶6.4×105的抗噻虫嗪单克隆抗体,抗体亚类为IgG1型。优化了ELISA实验条件,建立了基于单克隆抗体技术的噻虫嗪残留间接竞争ELISA方法。本方法的抑制中浓度(IC50)为0.0255mg/L,检测灵敏度(IC20)为0.0022mg/L,检出限(IC10)为0.001mg/L。除噻虫胺外,该抗体与其它噻虫嗪结构类似物无交叉反应。以自来水为基质的噻虫嗪添加回收实验显示,0.01,0.5和10.0mg/L添加水平的回收率均大于75%,且各添加水平重复测定8次的相对标准偏差均小于8%,说明所建立的间接竞争ELISA准确度高,重复性好,适合水中噻虫嗪残留的检测。     

硝酸甲酯分子间相互作用的DFT和ab initio比较

用密度泛函理论(DFT)和从头算(ab initio)方法,分别在B3LYP/6 31G和HF/6 31G水平上求得硝酸甲酯三种二聚体的全优化几何构型和电子结构,并用6 311G和6 311＋＋G基组进行总能量计算.对HF/6 31G计算结果进行MP4SDTQ电子相关校正.在各基组下均进行基组叠加误差(BSSE)和零点能(ZPE)校正求得结合能.对6 31G优化构型作振动分析并基于统计热力学求得200～600 K温度下单体和二聚体的热力学性质.详细比较两种方法的相应计算结果,发现DFT求得的分子间距离较短,分子内键长较长,所得结合能均小于相应ab initio计算值.     

分子印迹膜的制备研究进展

将分子印迹技术与膜分离技术相结合的分子印迹膜,由于其高选择性,近年来受到了国内外研究者的广泛关注.本文初步探讨了分子印迹膜(MIM) 的两种分离机理以及目前主要的制备方法:同步法和复合法.     

新型游离三碘甲腺原氨酸人工抗原的合成与表征

利用快速、温和的“Click Chemistry(点击化学)”反应,在游离三碘甲腺原氨酸(FT3)的羧基上引入一个带有三唑环的羧基“间接臂”,生成半抗原(FT3C)。 再用N-羟基琥珀酰亚胺活性酯法,将半抗原(FT3C)分别与牛血清白蛋白(BSA)和钥孔戚血蓝素(KLH)偶联,合成了免疫抗原(FT3C-BSA) 和包被抗原(FT3C-KLH)。 免疫抗原(FT3C-BSA)的紫外吸收光谱相对于FT3C和BSA有明显差异,表明成功实现了偶联,经计算得到偶联比为7∶1;用FT3C-BSA免疫Balb/c小鼠,获得高效价的抗FT3血清。 采用间接ELISA法检测抗体的IC50值为0.37 mg/L,抗血清效价可达6.24×104,是制备FT3人工抗原的一种新方法。     

分子电性距离矢量用于多溴联苯醚分子结构表达及色谱保留时间预测

应用分子电性距离矢量(MEDV)对多溴联苯醚(PBDEs)的209种同系物进行结构表征.通过多元线性回归的方法,建立了PBDEs定量结构-色谱保留(QSRR)关系的6个变量和5个变量的两种模型.两种模型的建模计算值复相关系数R均为0.995;用留一法(LOO)进行了交互检验,其复相关系数(R2cv)分别为0.987和0...     

二氢吡啶类化合物的三维定量构效关系

通过分子力学和量子化学计算,得出两种二氢吡啶衍生物的低能构象,再应用比较分子力场分析方法(CoMFA)和比较分子相似性指数分析方法(CoMSIA)分别对两种构象的43个二氢吡啶衍生物进行3D-QSAR研究. 计算结果表明,用两种方法建立的两种构象的构效关系模型均有较好的预测能力.通过分析CoMFA和CoMSIA的系数等势图,直观地了解二氢吡啶衍生物的结构对生物活性的影响,为进一步设计高活性的二氢吡啶衍生物提供一定的理论依据.     

农产品中脱氧雪腐镰刀菌烯醇的表面增强拉曼检测

利用便携式拉曼光谱仪建立了一个快速筛查与检测谷物中真菌毒素脱氧雪腐镰刀菌烯醇(DON)的表面增强拉曼散射(SERS)方法。首先利用实验室前期开发的方法制备了具有高活性的水凝胶SERS芯片。该SERS芯片是将预先制备的高SERS活性的单层碳基点(CDs)包裹的银纳米颗粒团聚体(a-AgNPs/CDs)与聚乙烯醇(PVA)水溶液混合均匀后,再利用循环冷冻-解冻的物理交联法制备而成的。实验优化了影响水凝胶SERS芯片对DON的SERS响应的实验条件,包括溶剂、浸泡温度和浸泡时间。在最佳的SERS检测条件下(溶剂为水-乙醇(1:1, v/v),浸泡温度为40 ℃,浸泡时间为5 min), DON的线性响应范围为1~10000 μg/kg(相关系数(R²)=0.9967),检出限(LOD)为0.14 μg/kg,表明该SERS基底具有较高的灵敏度。得益于水凝胶特殊的孔径结构,实际样品基质中常见的糖、蛋白质、油脂、色素等干扰物质都被阻隔在水凝胶外。因此,在复杂样品检测中仅需要简单的提取,而不需要复杂的分离处理。将该方法用于小麦粉中DON的检测,所得回收率为97.3%~103%,相对标准偏差为4.2%~5.0%。实验结果表明所建立的检测DON的SERS方法具有响应范围宽、灵敏度高、重复性好、响应迅速、操作简单、抗干扰能力强等优点,这说明本实验室所构建的水凝胶SERS芯片在粮食中生物毒素的快速筛查与检测方面具有良好的应用潜力。     

Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC₅₀) values of the SPR assay were 28.8 and 14.9 ng mL⁻¹ for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg⁻¹ for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg⁻¹ for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.     

Purity assessment of commercially available crystalline deoxynivalenol

Deoxynivalenol (DON) obtained from 2 commercial sources was characterized, and its purity was determined. The structural identity of DON was confirmed by 1H and 13C-nuclear magnetic resonance (NMR) spectroscopy, gas chromatography with mass spectrometric (GC/MS) detection, and infrared/attenuated total reflectance (IR/ATR) spectroscopy. NMR spectra showed shifts that varied from previously published data. However, we established a complete, unambiguous assignment for all signals. Chromatograms obtained by GC/MS were almost identical for both investigated samples and confirmed the structure of DON. Likewise, IR/ATR spectra verified the identity of DON. The degree of purity was determined by liquid chromatography (LC) with a variable wavelength detector, LC/MS/MS, GC with electron-capture detection (GC-ECD), and ultraviolet (UV) spectrophotometry. The purity check using LC showed a single peak in both chromatograms. With LC/MS/MS measurements, we could detect small amounts of impurities in the crystalline DON from both sources. In data obtained by GC-ECD, no differences in purity were observed. The UV measurements showed an absorption maximum at 217 nm. The mean epsilon(m) of the extinction coefficients was calculated as 6727 (L/cm/mol) for DON (Sigma) and 6825 (L/cm/mol) for DON (Biopure). Finally, the purity of DON from the 2 commercial sources was calculated as >96 and >98%, respectively. Although the DON produced by both providers can be considered sufficiently pure for routine analysis of trichothecenes in food and feed, this work again demonstrated that the impurity of the solid mycotoxin constitutes the greatest contribution to the overall uncertainty of a mycotoxin calibrant.     

Comparison of slurry mixing and dry milling in laboratory sample preparation for determination of ochratoxin A and deoxynivalenol in wheat

The significance of laboratory sample preparation for the determination of two important mycotoxins, ochratoxin A (OTA) and deoxynivalenol (DON), in wheat was investigated by comparing water-slurry mixing and dry-milling procedures. The distribution of OTA and DON in 10 kg samples of naturally contaminated wheat was established by analyzing one hundred 100 g subsamples of each sample. A normal distribution and a good repeatability of DON measurements was observed for both water-slurry mixing (mean 2290 microg/kg, CV 4.6%, median 2290 microg/kg) and dry milling (mean 2310 microg/kg, CV 6.4%, median 2290 microg/kg) procedures. For OTA determinations, reliable results could be obtained only by slurry mixing sample preparation (mean 2.62 microg/kg, CV 4.0%, median 2.62 microg/kg), whereas dry-milling comminution resulted in an inhomogeneous distribution with a high variability (mean 0.83 microg/kg, CV 75.2%, median 0.60 microg/kg) and a positive skewness (2.12). Ad hoc experiments were performed on different size portions of the same sample (10 kg) to assess accuracy and precision of the comminution/homogenization procedures (slurry mixing and dry milling). Very good results were obtained for DON determination with both procedures in terms of accuracy (>98.7% of the "weighted value") and precision (CV <3%). For OTA determination good results were only obtained by slurry mixing (99.4% of the "weighted value," CV 10%), whereas dry milling provided results with low accuracy (43.2% of the "weighted value") and high variability (CV 110%). This study clearly demonstrated that sample preparation by slurry mixing is strictly necessary to obtain reliable laboratory samples for OTA determination in wheat to minimize misclassification of acceptable/rejectable lots, mainly within official control.     

The use of supercritical fluid extraction for the determination of 4-deoxynivalenol in grains: The effect of the sample clean-up and analytical methods on quantitative results

Summary Deoxynivalenol (DON) is one of the trichothecene mycotoxins produced byFusarium molds in grains. Polar cosolvents in supercritical carbon dioxide (SC-CO₂) are needed to extract and isolate the polar DON moiety. This unfortunately results in the extraction of many interfering compounds from the grains into the extracts obtained by supercritical fluid extraction (SFE). Analysis of DON by high performance liquid chromatography (HPLC) using ultraviolet detection (UV) does not provide a specific detection method, although specific detection of DON can be enhanced by using purification steps after SFE. Alternatively, combining SFE with an immunoaffinity method can improve detection specificity and sample cleanup. In this study, SFE was employed to determine DON in grains and cereal products. The effectiveness of the SFE method was compared with two different solvent extraction methods. The extracted DON was quantitatively determined by HPLC-UV using external standardization or competitive enzymelinked immunosorbent assay (ELISA). In some cases, extracts were purified prior to quantitative analysis of the DON by using solvent partitioning, and/or solid phase extraction, or immunoaffinity columns. Therefore, this paper describes the analysis of DON in cereals using different extraction, cleanup and analysis methods. Names are necessary to report factually on available data: however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the products to the exclusion of others that may also be suitable.     

Distribution of deoxynivalenol in wheat,wheat flour,bran, and gluten,and variability associated with the test procedure

Analytical data obtained on deoxynivalenol (DON) concentration in naturally contaminated wheat during processing in an industrial mill were statistically analyzed, and the distribution functions of DON concentration in lots of wheat, bran, wheat flour, and gluten were estimated. The analytical method had acceptable precision (HORRAT 0.25-0.32) for each test sample. The total variance combined sampling, sample preparation, and analytical variances were 0.188, 0.033, 0.42, and 0.0014 ppm2 for wheat, 1.93; flour, 0.99; bran, 4.68; and gluten, 0.29, respectively. The distribution function of DON contamination presented an asymmetric tail for high values of concentration in wheat grains and wheat flour; in bran it seemed to be bimodal with 2 separated peaks of different concentrations; in gluten the normal distribution function gave a reasonably good fit to empirical data. The function eta(c) = -In(-Inp), where p (c) is the cumulative distribution function was linear with c in the so-called extreme-value type I distribution and could be fitted by a cubic polynomial in c in the distributions determined for all the products. This variability and distributional information contributes to the design of better sampling plans in order to reduce the total variability and to estimate errors in the evaluation of DON concentration in lots of wheat and wheat products.     

Natural occurrence of deoxynivalenol (DON) in wheat based noodles consumed in Malaysia

In this first study performed in noodle samples consumed in Malaysia for the presence of deoxynivalenol (DON), a total of 135 sample of noodles, comprised of instant noodle, yellow alkaline noodle and white salted noodle, were randomly collected from food stores and analyzed for DON using high performance liquid chromatography (HPLC) with a PDA (photodiode array) detector at 218 nm. The objective of this study was to investigate the DON contamination levels in different types of wheat-based noodle consumed in Peninsular Malaysia. An acetonitrile:water (17:83 v/v) mixture was used as a mobile phase and clean-up was accomplished with a Mycosep 225 column. There was a high variation in the DON concentrations in all types of noodle group as well as between brands. Only one sample of instant and yellow alkaline noodle each were contaminated with DON, at concentrations of 1.003 and 1.243 ng/g, respectively. The minimum detectable concentration for the DON was 0.627 ng/g in instant noodle. In the case of white salted noodle, none of the samples contained any detectable amount of DON. The results indicate a low occurrence of DON mycotoxins in commercial noodle products in Malaysia.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

Stable isotopic labelling-assisted untargeted metabolic profiling reveals novel conjugates of the mycotoxin deoxynivalenol in wheat

An untargeted screening strategy for the detection of biotransformation products of xenobiotics using stable isotopic labelling (SIL) and liquid chromatography–high resolution mass spectrometry (LC-HRMS) is reported. The organism of interest is treated with a mixture of labelled and non-labelled precursor and samples are analysed by LC-HRMS. Raw data are processed with the recently developed MetExtract software for the automated extraction of corresponding peak pairs. The SIL-assisted approach is exemplified by the metabolisation of the Fusarium mycotoxin deoxynivalenol (DON) in planta. Flowering ears were inoculated with 100 μg of a 1?+?1 (v/v) mixture of non-labelled and fully labelled DON. Subsequent sample preparation, LC-HRMS measurements and data processing revealed a total of 57 corresponding peak pairs, which originated from ten metabolites. Besides the known DON and DON-3-glucoside, which were confirmed by measurement of authentic standards, eight further DON-biotransformation products were found by the untargeted screening approach. Based on a mass deviation of less than ±5 ppm and MS/MS measurements, one of these products was annotated as DON-glutathione (GSH) conjugate, which is described here for the first time for wheat. Our data further suggest that two DON-GSH-related metabolites, the processing products DON-S-cysteine and DON-S-cysteinyl-glycine and five unknown DON conjugates were formed in planta. Future MS/MS measurements shall reveal the molecular structures of the detected conjugates in more detail.     

Deoxynivalenol and its conjugates in beer: a critical assessment of data obtained by enzyme-linked immunosorbent assay and liquid chromatography coupled to tandem mass spectrometry

Enzyme-linked immunosorbent assays (ELISAs) are often employed for the control of deoxynivalenol (DON) in barley and other intermediates involved in beer production chain. Because of the occurrence of high levels of DON-3-glucoside (DON-3-Glc) in malt and beer that have been reported for the first time in our earlier study, research focused on the accuracy of DON determination by immunoassays in cereal-based matrices has been initiated. DON-3-Glc was strongly cross-reacting in all examined commercial ELISA test kits (Ridascreen^® DON (R-Biopharm), Veratox 5/5 DON^® (Neogen Corporation), Deoxynivalenol EIA (Euro-Diagnostica), and AgraQuant^® DON Assay 0.25/5.0 Test Kit (Romer Labs). The highest overestimation in beer analysis, up to 1000%, when taking the DON content determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as a reference method, was obtained by AgraQuant assay. Besides of DON-3-Glc and 3- and 15-acetyldeoxynivalenol (ADONs), also other, not known yet, matrix components contributed to false positive results. Similar phenomenon, although in a lesser extent due to lower content of these substances, was observed for using ELISA in the analysis of wheat. The relationship between a way of sample handling and DON overestimation was demonstrated; higher ELISA response was measured in an aqueous extract compared to that prepared by acetonitrile-water (84:16, v/v). Most of cross-reacting co-extracts were removed by MycoSep™# 226 cartridge, what leads us to the hypothesis on the presence of currently unknown cross-reactive species.