串联两种免疫亲和柱同时检测粮食中赭曲霉毒素A和玉米赤霉烯酮

建立了一种通过串联两种免疫亲和柱的方式同时检测粮食中赭曲霉毒素A和玉米赤霉烯酮的方法。样品经乙腈∶水(90∶10)提取后,通过串联的两种免疫亲和柱净化,以高效液相色谱荧光检测器检测。结果表明,赭曲霉毒素A和玉米赤霉烯酮线性范围分别为1~30μg/mL和10~300μg/mL,相关系数R2分别为0.999 02和0.995 72,不同基质样品的回收率分别为75.25%~98.37%和83.41%~117.09%,精密度分别为1.28%~8.16%和1.23%~9.45%,最低检出限分别为0.4μg/kg和3.9μg/kg。本方法具有操作简便、快捷和准确性高等特点,适用于粮食中赭曲霉毒素A和玉米赤霉烯酮的同时检测。     

玉米赤霉烯酮化学发光免疫分析检测系统设计

为了保障粮食安全,该研究根据玉米赤霉烯酮抗原抗体反应,以及辣根过氧化物酶催化鲁米诺过氧化氢反应产生化学发光,设计一款应用于粮食行业的玉米赤霉烯酮检测系统。采用侧窗型高精度光电倍增管MD983以及16位AD转换芯片,实现化学发光强度信号的准确测量;步进电机驱动旋转精密转台,通过优化步进电机的S型脉冲驱动控制曲线参数,完成转台的高精度定位控制,实现光电倍增管的测试窗口和化学发光孔精确对准;通过精密直线导轨滑台驱动加样器的进给,实现反应液微米量级的准确微量加样。利用竞争性免疫分析方法,使得赤霉烯酮毒素为0μg/kg情况下,化学发光反应具有最大发光量,解决真菌毒素低浓度情况下的检测精度难题。经过试验验证,系统检出限为0.1μg/kg,样品加标回收率在90%以上,标准曲线决定系数为0.996 5,系统检测玉米赤霉烯酮的线性范围为0～60μg/kg。研究表明建立的玉米赤霉烯酮检测系统满足国家粮食行业对于粮食中玉米赤霉烯酮含量检测要求,为真菌毒素检测仪器的国产化提供参考。     

解淀粉芽孢杆菌NS2降解玉米赤霉烯酮的研究

为了丰富玉米赤霉烯酮(ZEN)解毒菌种库,通过采集湖南省邵阳市等7个省市粮食样品,筛选获得1株能够高效降解ZEN的解淀粉芽孢杆菌NS2。采用高效液相色谱法检测降解前后ZEN的含量发现,液态发酵72 h,该菌对5 mg·L^-1 ZEN标准品的降解率高达96.0%,固态发酵72 h,菌株对玉米粉中2.5 mg·L^-1 ZEN标准品的降解率高达88.2%。采用高效液相色谱与四级杆飞行时间质谱联用技术检测该菌处理后的ZEN降解产物,未检测出具有毒性作用的α-玉米赤霉烯醇(α-ZOL)、β-玉米赤霉烯醇(β-ZOL)等ZEN的类似物。此外,抗菌活性试验与木聚糖酶、纤维素酶和蛋白酶活性试验显示降解菌NS2还能通过产生具有抗菌作用的次生代谢产物,抑制病原微生物大肠杆菌和鼠伤寒沙门氏菌的生长。上述结果表明,解淀粉芽孢杆菌NS2可以高效降解ZEN,具有潜在应用前景,为ZEN解毒产品的开发与应用奠定了材料基础。     

玉米赤霉烯酮降解酶基因zlhy-6在枯草芽孢杆菌中的表达

玉米赤霉烯酮(ZEN)是具有雌激素作用的次生代谢产物,可以被来源于Gliocladium roseum的内酯水解酶降解为无毒物质。为了实现玉米赤霉烯酮降解酶基因zlhy-6在枯草芽孢杆菌中的表达,获得不含抗生素抗性基因的食品级重组枯草芽孢杆菌,本研究采用一步克隆及重叠延伸PCR的方法构建了单启动子和包含Hpa Ⅱ和P43双启动子的表达质粒,将质粒转化到枯草芽孢杆菌中,获得重组菌株168/pMA5-zlhy-6和168/pMA5-P43-zlhy。然后构建了重组整合载体amy-p43-zlhy,将zlhy-6基因整合到枯草芽孢杆菌168的基因组中。通过Cre/lox系统敲除抗生素抗性基因,获得整合了P43-zlhy表达盒的食品级重组枯草芽孢杆菌BZ-zlhy。将构建的3个重组菌株在37℃、pH值7.5条件下培养36 h,结果显示,双启动子表达载体重组菌株的最高酶活性为2.2 U·mL^-1,是单启动子菌株的1.2倍。双启动子重组菌株表达的降解酶对ZEN(4 μg·mL ^-1,30 min)的降解率为65.1%。重组菌株枯草芽孢杆菌BZ-zlhy表达水平最低(0.4 U·mL^-1)。本研究实现了玉米赤霉烯酮降解酶在枯草芽孢杆菌中表达,同时构建了不含抗生素抗性基因的食品级重组枯草芽孢杆菌,为玉米赤霉烯酮降解酶的工业化生产奠定了基础,也为解决粮食储藏和饲料生产中的ZEN污染提供了思路。     

玉米赤霉烯酮降解酶基因（ZEN-jjm）的克隆、表达及活性分析

玉米赤霉烯酮(zearalenone,ZEN)是世界上污染范围最广泛的一种镰刀霉毒素,能导致人或动物不孕、流产等症状,损害内脏器官并促进癌细胞的生长.本文通过设计1对特异性引物,从粉红螺旋聚孢霉(Gliocladium roseum)中克隆获得大小为795 bp的DNA片段(ZEN-jjm),通过构建E. coil原核表达载体进行表达.经HPLC检测证实,IPTG诱导后的细胞裂解上清能在3 h内完全降解液体中1μg/mL的ZEN.序列分析结果表明ZEN-jjm与zhd101存在9个碱基的差异,ZEN-jjm编码的ZEN降解酶与文献报道的乳糖水解酶存在3个氨基酸的差异,但ZEN毒素降解实验证实二者活性相似.     

一株高效降解玉米赤霉烯酮的耐酸耐高温枯草芽孢杆菌的研究

为寻求更高效、实用的生物脱毒制剂,提高其应用范围,通过采集酸性、高温的温泉环境样品,筛选获得1株高效降解玉米赤霉烯酮(ZEN)的细菌菌株Y-33,并对该菌株生长和降解特性进行分析。结果表明,菌株Y-33在50℃、pH值5.0条件下孵育36 h,菌液对2 μg·mL^-1 ZEN的降解率高达93.79%,对20 μg·mL^-1 ZEN的降解率也达82.40%。从形态、生理生化特性及16S rDNA序列对菌株Y-33进行分析,初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。该菌株发酵上清液在28℃条件下对ZEN降解率为62.02%。金属离子Mn²⁺能明显增强菌株Y-33发酵上清液对ZEN的降解能力,降解率达81.17%,而Zn²⁺严重抑制了Y-33发酵上清液对ZEN的降解能力,降解率仅为5.42%。本研究结果为ZEN解毒菌剂的开发与应用奠定了理论基础。     

抗苯巴比妥单克隆抗体杂交瘤细胞株的筛选及ciELISA试剂盒的研制

用BSA-pAPB免疫Balb/c小鼠，用细胞融合技术制备并用间接ELISA和阻断ELISA筛选抗苯巴比妥单克隆抗体(PB mAb)杂交瘤细胞株，体内诱生腹水法生产PB mAb，应用PB mAb研制PB残留竞争ELISA(ciELISA)快速检测试剂盒(PB-Kit)，并测定其性能。结果表明，筛选出3株杂交瘤细胞，最好的3F6-C4株的PB mAb间接ELISA效价为1∶6.4×105，亲和常数(Ka)为1.96×1010 L/moL，半数抑制浓度(IC50)为5.7 μg/L，与巴比妥的交叉反应率(CR%)12.4%，与其它化合物无CR；PB-Kit的线性检测范围1.0～81 μg/L，灵敏度0.75 μg/L，检测限1 μg/L；饲料样和猪尿样的平均添加回收率85.8%和91.3%，平均批内和批间变异系数均<15%。PB-Kit具有快速、敏感、特异、简便等特点，适合PB残留快速检测的推广应用。     

60Co-γ辐照模式下玉米中赤霉烯酮和品质的变化及对绵羊定时人工授精的影响

为研究经赤霉烯酮(ZEN)自然污染与⁶⁰Co-γ辐照处理后的玉米对绵羊定时人工授精中澳洲白种公羊、湖羊母羊的生殖性能的影响,本试验选取含ZEN玉米样本,分别用0、3、5、8、10 kGy剂量的⁶⁰Co-γ辐照,并测定辐照后玉米主要营养成分变化。然后分别将未污染玉米(Ⅰ对照组)、经10 kGy ⁶⁰Co-γ辐照的污染玉米(试验Ⅱ组)、污染玉米(试验Ⅲ组)饲喂定时人工授精的绵羊,检测澳洲白种公羊的精液质量,测定湖羊母羊的繁殖性能指标。结果表明,未经⁶⁰Co-γ辐照的玉米含ZEN 2 153 μg·kg^-1,在10 kGy ⁶⁰Co-γ辐照剂量下,玉米中ZEN降解率为83.7%;当⁶⁰Co-γ辐照剂量低于10 kGy时,玉米淀粉表面结构无变化,主要营养成分无显著变化。试验Ⅱ组澳洲白种公羊的采精量、精子活力、精子密度与试验Ⅲ组相比显著升高(P<0.05),与Ⅰ对照组相比无显著差异(P>0.05);试验Ⅲ组澳洲白种公羊第30天精子畸形率与试验Ⅱ组、Ⅰ对照组相比均显著升高(P<0.05),显微镜下观察可见双尾精子、双头精子、无尾精子等现象,其中双尾精子头部、尾部不完整;试验Ⅱ组湖羊母羊同期发情率、受胎率、产羔率和羔羊始重等指标与试验Ⅲ组相比显著升高(P<0.05),与Ⅰ对照组无显著差异(P>0.05)。综上,使用经10 kGy ⁶⁰Co-γ辐照后ZEN含量低于2 153 μg·kg^-1的玉米饲喂繁育绵羊,对绵羊定时人工授精无不利影响。本研究结果为⁶⁰Co-γ辐照模式下玉米中赤霉烯酮和品质的变化对绵羊定时人工授精的影响研究提供了理论依据。     

二氟沙星残留检测直接竞争ELISA方法的建立

应用分泌抗二氟沙星(DIF)单克隆抗体(DIFmAb)的杂交瘤细胞株建立直接竞争ELISA分析方法.特异性实验结果表明,与达氟沙星的交叉反应率为0.32%,与其它氟喹诺酮类药物和磺胺类药物无交叉反应,该方法对DIF的检测限可达到0.1μg/L,半数抑制浓度IC(50)为2.2 μg/L,线性范围0.1～128.0μg/L,平均批内和批间变异系数小于15%.对牛奶和鸡肉分别添加0.4、2.0、10.0和50.0μg/L DIF标准品.平均回收率分别为89.3%和83.9%.直接竞争ELISA准确度高、重复性好、特异性强,适用于DIF残留快速检测的推广应用.     

磺胺甲噁唑单克隆抗体的研制及免疫金标试纸检测方法的建立

用磺胺甲噁唑人工免疫原（BSA-SMZ）免疫BALB/C小鼠（Mus muscalus）,细胞融合技术筛选抗磺胺甲噁唑单克隆抗体（SMZmAb）杂交瘤细胞,体内诱生腹水法制备SMZ mAb,并鉴定其免疫学特性;应用SMZmAb研制SMZ残留快速检测金标试纸（SMZ-strip）,并测定其性能。结果表明,筛选出3株杂交瘤细胞,其中最好的3G6株间接ELISA效价细胞培养上清为1∶8.1×102,腹水为1∶6.4×105,亲和常数（Ka）为3.07×109 L/mol,对SMZ的半数抑制浓度（IC50）为12.4 μg/L,与磺胺嘧啶的交叉反应率（CR%）为2.84%,与其它磺胺类药物无交叉反应;SMZ-strip目测检测限为6 μg/L;其敏感性与竞争ELISA试剂盒相当,符合率为100%;SMZ-strip具有快速、敏感、特异、简便等特点,适合于SMZ残留快速检测的推广应用。     

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.     

Development of an enzyme-linked immunosorbent assay based a monoclonal antibody for the detection of pyrethroids with phenoxybenzene multiresidue in river water

A sensitive and broad class selective direct competitive enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (McAb) has been described for the detection of pyrethroids with phenoxybenzene group. One monoclonal antibody, 2G(2)E(7), was obtained and characterized after fusion of myeloma cells with spleen cells isolated from BALB/c mice. The assay with the most selectivity for the family pyrethroids with phenoxybenzene group was optimized. The IC(50) values of the optimized immunoassay were 1.8 μg L(-1) for deltamethrin, 1.5 μg L(-1) for cypermethrin, 2.0 μg L(-1) for fluvalinate and fenvalerate, 2.2 μg L(-1) for phenothrin, 2.4 μg L(-1) for flucythrinate, 3.0 μg L(-1) for fenpropathrin, and 5.0 μg L(-1) for permethrin. River water samples fortified with pyrethroids were analyzed with the ELISA to evaluate the accuracy of the assay. The recoveries of pyrethroids in spiked water samples ranged from 74 to 108%. The results indicate that the ELISA developed can accurately simultaneously determine pyrethroids with phenoxybenzene group in water samples.     

结合条件对羊抗兔抗体连接到细菌磁小体的影响

用羊抗兔抗体进行抗体连接到细菌磁小体(BMPs)实验，以探讨结合条件对抗体连接效率(以每毫克BMPs上连接抗体的微克数表示，单位为μg/mg)的影响。实验表明，冷冻干燥后的BMPs的抗体连接效率降低36%~55%；在液氮中(-196 ℃)预处理的抗体连接效率显著高于在-70 ℃和-20 ℃预处理的。室温(15 ℃)下反应18 h的抗体连接效率最高，为64.06 μg/mg，明显高于其它温度和连接时间的。反应体系中，BMPs用量由5 mg降到0.1 mg，抗体用量由10 μg增加到300 μg时，抗体连接效率逐渐上升，当BMPs用量为0.1 mg，抗体用量为300 μg时，抗体连接效率达762.37 μg/mg。PBS、HEPES、Tris-HCl、MOPS、VBS以及磷酸盐等溶液可用于抗体连接到BMPs体系中，在通常使用的缓冲液pH和浓度下，几种溶液的抗体连接效率变化范围为46.28±0.58 ~ 90.83±1.64 μg/mg；并且不同的溶液有相应的使抗体连接效率达到最高时的pH和溶液浓度，当HEPES的pH为3.5，浓度为20 mmol / L；Na3PO4的pH为5.6，浓度为5 mmol / L时，抗体连接效率分别为108.01和76.78 μg/mg。对连接到BMPs上的抗体进行SDS-PAGE定性检测结果表明，抗体连接到了BMPs的膜上。     

Monoclonal antibody-based ELISA and colloidal gold immunoassay for detecting 19-nortestosterone residue in animal tissues

This article presents the generation of monoclonal antibodies (mAbs) with high specificity against 19-nortestosterone (NT) through cell fusion techniques and the development of a mAb-based indirect competitive ELISA (icELISA) method and colloidal gold-based immuno-chromatographic assay to detect NT residues in beef and pork samples. A modified carbodiimide method was employed to synthesize the artificial antigen, and BALB/c mice were used to produce anti-NT mAbs. On the basis of the checkerboard titration, an indirect competitive ELISA standard curve was established. This assay was sensitive and had a linear range from 0.03 to 38 ng/mL in phosphate buffered saline (PBS), with IC(50) and LOD values of 0.52 ng/mL and 0.01 ng/mL, respectively. Of all the competitive analogues, the produced mAb exhibited a high cross-reactivity to 17α-nortestosterone (83.6%), the main metabolite of NT in animal tissues. Except for moderate cross-reactivities with trenbolone (22.6%) and β-boldenone (13.8%), the other interference to the assay was negligible (<0.05%). In contrast, the strip test had a visual detection limit of 1 ng/mL in PBS, 2 μg/kg in beef, and 2 μg/kg in pork, respectively, and the results can be judged within 10 min. The ELISA and GC-MS results showed close correlation in beef (R2=0.9945) and in pork (R2=0.9977). Therefore, the combination of two immunoassays provides a useful screening method for quantitative or qualitative detection of NT residues in animal-origin products.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for the screening of tylosin and tilmicosin in muscle, liver, milk, honey and eggs

Incorrect use of tylosin and tilmicosin could result in allergy and select resistance. To monitor the illegal use of these antibiotics in animals, a monoclonal-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been established. Several haptens were synthesized and conjugated to carrier protein. Female Balb/c mice were inoculated with the four different conjugates to produce monoclonal antibodies according to the schemes of immunization. Aftercell fusion and culture several times, nine hybridoma cell lines were isolated. Only one, 3C4 that has isotype IgG2a, was selected for detailed study. The cross-reactivity of the monoclonal antibody 3C4 to tylosin and tilmicosin was 100% and 51% respectively. The standard curves based on the tylosin and tilmicosin matrix calibration ranged from 2.5 to 40 μg L(-1), with an IC(50) value of 6.1 μg L(-1) and 12.1 μg L(-1), respectively. The limits of detection of the ic-ELISA ranged from 5.1 μg kg(-1) to 13.8 μg kg(-1) in edible animal tissues. The recoveries were 74.1% to 120.7% with less than 18.6% of the coefficient of variation when tylosin and tilmicosin were spiked in various biological matrices with the concentrations of 25.0-200.0 μg kg(-1). Good correlations between the results of the ic-ELISA and high performance liquid chromatography were observed in the incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for the screening of the residues of tylosin and tilmicosin in muscle, liver, milk, honey and eggs.     

Mercury Levels of Soils,Sediments and Fish in French Guiana,South America

This study examines the level of mercury contamination for different components of the biosphere in French Guiana. The spatial variability of the contamination of the waters, soils and sediments in the catchment area of the Sinnamary River is studied. The contamination of the trophic chain isdiscussed by analysis of the flesh of fish for the principal species represented in nine Guianese rivers, especially species consumed by human populations. The mercury contamination of sediments and soils, is extremely heterogeneous and that of fish is generalised. For carnivorous fish, the average mercury content is 0.48 μg g^-1 (±0.28) (fresh weight) and for non-carnivorous species, 0.05 μg g^-1 (±0.07). The concentrationsmeasured are greater than the European standards forconsumption for 4.7% of the carnivorous individuals.     

Development and application of an indirect competitive enzyme-linked immunoassay for aflatoxin m(1) in milk and milk-based confectionery

High-titer rabbit polyclonal antibodies to aflatoxin M(1) (AFM1) were produced by utilizing AFM1-bovine serum albumin (BSA) conjugate as an immunogen. An indirect competitive enzyme-linked immunosorbent assay was standardized for estimating AFM1 in milk and milk products. To avoid the influence of interfering substances present in the milk samples, it was necessary to prepare AFM1 standards in methanol extracts of certified reference material (CRM) not containing detectable AFM1 (< 0.05 ng/g). The reliability of the procedure was assessed by using CRM with AFM1 concentrations of < 0.5 and 0.76 ng/g. Also, assays of milk samples mixed with AFM1 ranging in concentration between 0.5 and 50 ng/L gave recoveries of > 93%. The relative cross-reactivity with aflatoxins (AF) and ochratoxin A, assessed as the amount of AFM1 necessary to cause 50% inhibition of binding, was 5% for AFB1 and much less for AFB2, AFG1, and AFG2; there was no reaction with ochratoxin A. AFM1 contamination was measured in retail milk and milk products collected from rural and periurban areas in Andhra Pradesh, India. Of 280 milk samples tested, 146 were found to contain < 0.5 ng/mL of AFM1; in 80 samples it varied from 0.6 to 15 ng/mL, in 42 samples from 16 to 30 ng/mL, and in 12 samples from 31 to 48 ng/mL. Most of the milk samples that contained high AFM1 concentrations were obtained from periurban locations. The results revealed a significant exposure of humans to AFM1 levels in India and thus highlight the need for awareness of risk among milk producers and consumers.     

Solvent Extraction of Zein from Dry‐Milled Corn

Batch extraction of zein from dry‐milled whole corn with ethanol was optimum with 70% ethanol in water, an extraction time of 30–40 min, and temperature of 50°C. High yields (60% of the zein in corn) and high zein contents in the extracted solids (50%) were obtained at a solvent‐to‐solids ratio of 8 mL of 70% ethanol/g of corn. However, zein concentration in the extract was higher at lower ratios. Multiple extraction of the same corn with fresh ethanol resulted in a yield of 85% after four extractions, whereas multiple extractions of fresh corn with the same ethanol resulted in high (15 g/L) zein concentration in the extract. Optimum conditions for batch extraction of zein were 45°C, with 68% ethanol at a solvent‐to‐solids ratio of 7.8 mL/g for an extraction time of 55 min. Column extractions were also best at 50°C and 70% ethanol; a solvent ratio of 1 mL/g resulted in high zein concentrations in the extract (17 g/L) but yields were low (20%).