【作者】 李岩松；

【导师】 柳增善；

【作者基本信息】 吉林大学 ， 预防兽医学， 2011， 博士

【摘要】 伏马菌素(Fumonisins)主要是由串珠镰刀菌、多育镰刀菌等产生的一类真菌毒素,至今已经发现的伏马菌素及其衍生物已达20余种,其中最为主要的是B族伏马菌素中的伏马菌素B1(FB1)、伏马菌素B2(FB2)和伏马菌素B3(FB3)。FB1主要污染的食品(或饲料)是玉米及其制品,且广泛发生于世界各地。伏马菌素属于2B类致癌物质,很多动物实验已经证明其具有致癌性,有调查显示伏马菌素与人类的食管癌具有相关性。由于伏马菌素对人和动物健康的严重危害性,其在食品卫生领域中越来越受到人们的重视。目前,伏马菌素的检测方法主要有液相色谱法、液相色谱-质谱联用法、免疫学检测法等。由于仪器分析方法样品处理相对复杂、成本高,限制了应用的推广。而免疫学方法具有灵敏度高、特异性好、样品处理简单、成本较低等优点,尤其适合大量样品的快速筛检。本实验在制备伏马菌素完全抗原及单克隆抗体的基础上,建立了玉米样品中伏马菌素的间接竞争ELISA(ic-ELISA)、胶体金免疫层析(GICA)和间接竞争化学发光酶免疫测定(ic-CLEIA)快速免疫学筛检方法,以期为进一步研究伏马菌素快速检测试剂产品奠定基础,满足实际检测的需求。本文采用戊二醛法将小分子半抗原FB1分别与卵清蛋白(OVA)、牛血清蛋白(BSA)偶联制备检测抗原和免疫抗原,SDS-PAGE电泳及紫外扫描法鉴定偶联成功。常规免疫和快速免疫两种方式免疫BALB/c鼠,常规方法融合并筛选杂交瘤细胞。获得3株稳定分泌伏马菌素单克隆抗体的杂交瘤细胞株,并优选了其中的4B3细胞株,小鼠腹水诱生法制备单克隆抗体。该单克隆抗体为IgG1亚类,辛酸-硫酸铵法纯化后单克隆抗体浓度为2.19 mg/mL,纯度为85.95%。抗体亲和常数为5.21×109 L/mol、效价为1:2.56×106。4B3抗体与FB2、FB3的交叉反应率分别为198%和59%;与脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮、T2毒素、桔青霉素、赭曲霉素A、黄曲霉素B1的交叉反应率均小于10%。利用该单克隆抗体建立了伏马菌素间接竞争ELISA检测方法,优化后的检测条件为:抗原包被浓度为0.5μg/mL,包被条件为37℃2 h;抗体工作浓度为1:30 000; 37℃竞争反应45 min;酶标二抗工作浓度1:5 000,作用时间37℃60 min;底物作用时间37℃避光25 min;间接竞争ELISA方法的线性回归方程为:y = -41.499x + 89.715,相关系数(R2)为0.981,线性范围为1.56 ng/mL～50 ng/mL,最低检出限为1.30 ng/mL;玉米加标回收实验结果表明,该方法总的平均回收率为(94.73±4.54)%、批内变异系数为8.31%、批间变异系数为8.54%;对14个玉米实际样品进行了检测,5个样品未检出FB1、6个样品中FB1的含量小于1 mg/kg、3个样品中FB1的含量在1 mg/kg～2 mg/kg之间。柠檬酸三钠还原法制备20 nm的胶体金溶液,与伏马菌素单克隆抗体合成金标探针,组装了简易胶体金免疫层析试纸条,优化了试纸条各项参数,试纸条裸眼可视检出限为2.5 ng/mL、最佳判定浓度为30 ng/mL,试纸条稳定性可达6个月以上。加标样品测定结果显示,甲醇:水(70:30,v/v)提取的玉米样品,可直接稀释5倍后上样检测,检测结果与标准品曲线相符。14个玉米样品测定结果显示,3个样品中伏马菌素浓度不低于0.75 mg/kg;10个样品中伏马菌素含量小于0.25 mg/kg;1个样品中伏马菌素浓度在0.25 mg/kg～0.75 mg/kg之间。利用该单克隆抗体建立了伏马菌素间接竞争化学发光酶免疫检测法,优化后的检测条件为:包被抗原25 ng/mL,4℃过夜;抗体使用浓度为1:100 000,37℃45 min;酶标二抗工作浓度1:10 000,37℃60 min;现配化学发光底物溶液,室温避光10 min。标准曲线方程为:logit (y) = 1.079 5 - 2.299 8 log (x),相关系数(r)为-0.998 2,线性范围为0.32 ng/mL～25 ng/mL,最低检出限为0.32 ng/mL。玉米加标回收实验表明,该方法总的平均回收率为(100.15±9.55)%、批内变异系数为7.90%、批间变异系数为8.12%。对14个玉米实际样品进行了检测,其中5个样品未检出FB1、6个样品中FB1的含量小于1 mg/kg、3个样品中FB1的含量在1 mg/kg～2 mg/kg之间。利用LC-MS/MS检测方法,乙腈/水提取、伏马菌素TC-F120专用净化柱净化处理样品,对玉米样品中的伏马菌素进行了测定并与前述建立的免疫学方法进行了比较分析。LC-MS/MS方法验证结果表明,免疫学检测方法分析结果与LC-MS/MS分析结果相符。所建立的胶体金免疫层析法、间接竞争ELISA检测方法和化学发光酶免疫检测法可以用于玉米样品中伏马菌素的快速筛检。更多还原

【Abstract】 Fumonisins (FBs), as a group of mycotoxins, are mainly produced by Fusarium moniliforme, Fusarium proliferatum, etc. There are more than 20 kinds of derivatives of mycotoxins. FB1, FB2 and FB3, are the most toxic and prevalent. FB1 usually be found in maize and maize-based food or animal feed. FBs are one kind of the possible carcinogen. The carcinogenicity of FBs have been proved in many animals, and some surveys have showed that the FBs might relate to esophageal cancer in humans. Because the serious threat of FBs to human and animal, More attention has been paid in the field of food hygiene. Currently, several analytical methods have been reported for FBs determination in food samples, including high performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC-MS) and immunological methods, etc. For the need of expensive equipment and complex sample preparation, the application of these methods by using large instrument were limited. Because the immunological methods were sensitive, specific, cheap and simple (sample processing), they were fit for the rapid screening of large numbers of samples. In this study, a indirect competitive ELISA (ic-ELISA), colloidal gold immunochromatographic assay (GICA) and chemiluminescent enzyme-linked immunosorbent assay (CLEIA) were developed to analyze FB1. These results would be contributed to the progress of rapid screening methods for FBs.In this study, the glutaraldhyde was used to conjugate FB1 to ovalbumin for the detection antigen and bovine serum albumin for the immunogen. The conjugates FB1-OVA and FB1-BSA were analyzed by SDS-polyacrylamide gelelectrophoresis and UV scanning method. Routine immunization protocol and quickly immunization protocol were adopted to immunize BALB/c mice, the hybridomas were screened after the cell fusion. Three hybridomas which stable secrete the antibodies against FBs have been selected and the hybridoma named 4B3. The subclass of the McAb was IgG1, the concentration was 2.19 mg/mL, the ELISA titers was 1: 2.56×106, and the affinity constant was 5.21×109 L/mol of the McAb. The cross-reactivities with FB2 and FB3 was 198% and 59%, respectively, and all cross-reactivities (CRs) with DON, ZEN, T-2, CIT, Och-A and AFB1 were lower than 10%.An ic-ELISA method for detection FB1 using the McAb have been established and optimized. The optimized concentration of antigen added to microtiter plates was 0.5μg /mL at 37℃for 2 h. The working concentration of the McAb in the competition condition was 1:30 000 at 37℃for 45 min. The reaction time of HRP secondary antibody and OPD substrate solution were at 37℃for 1 h and 25 min, respectively. The linear regression equation of the ic-ELISA was y = -41.499 x + 89.715, with the correlation coefficient (R2) of 0.981. The linear range was 1.56 ng/mL - 50 ng/mL with the lowest detection limit of 1.30 ng/mL. Recovery experiment of spiked corn samples showed that the total average recovery, coefficient of variation, and inter-assay coefficient of the method were (94.73±4.54)%, 8.31% and 8.54%, respectively. 14 corn samples was tested for FB1 using the ic-ELISA. In these samples, the FB1 was not detected in 5 samples, less than 1 mg/kg in 6 samples, between 1 mg/kg and 2 mg/kg in 3 samples.The nanoparticle-monoclonal antibody probes for FB1 have been synthesized using the 20 nm colloidal gold particles prepared by sodium citrate reduction and the simple colloidal gold immunochromatographic test strip have been assembled. The visible detection limit of the test strip was 2.5 ng/mL and optimal decidable concentration was 30 ng/mL (the test line was disappeared). The stability of the test strip was up to 6 months. When the spiked corn samples were extracted by methanol: water (70:30, v/v) and diluted for 5 times, the results were consistent with the standard curve. The corn samples were detected with the test strip. The results show, the concentration of FB1 was higher than 0.75 mg/kg in 3 samples, was less than 0.25 mg/kg in 10 samples and was between 0.25 mg/kg - 0.75 mg/kg in 1 sample.An ic-CLEIA method for detection FB1 using the McAb have been established and optimized. The optimized concentration of antigen added to polystyrene white flat bottom micro-plate was 25 ng /mL at 4℃overnight. The concentration of the McAb in ic-CLEIA was 1:100 000 at 37℃for 45 min. The working concentration and time of goat anti-mouse IgG-HRP were 1:10 000, at 37℃for 60 min. The chemiluminescent substrate solution was at room temperature away from light for 10 min. The standard curve equation of the ic-CLEIA was logit (y) = 1.079 5 - 2.299 8 log (x) with the correlation coefficient (r) of -0.998 2. The linear range of 0.32 ng/mL - 25 ng/mL and the lowest detection limit of 0.32 ng/mL. The total average recovery, coefficient of variation, and inter-assay coefficient of the method were (100.15±9.55)%, 7.90% and 8.12% in detection of spiked corn samples, respectively. Of the 14 corn samples, the FB1 was not detected in 5 samples, was less than 1 mg/kg in 6 samples and was between 1 mg/kg and 2 mg/kg in 3 samples.After extracted with acetonitrile/water and purified by TC-F120 SPE columns, the FB1 was detected by LC-MS/MS method in corn samples. The results from the immunoassays were compared with those of LC-MS/MS method, which indicated that the results between the immunological detection methods and LC-MS/MS method were consistent. The developed three immunological detection methods including GICA, ic-ELISA and ic-CLEIA can be used for rapid screening FBs in corn.更多还原