动物性食品中玉米赤霉醇ELISA快速检测方法的研究
发布时间：2018-09-19 07:34
【摘要】：近年来,真菌毒素对食品的污染越来越严重,其中因玉米赤霉醇(ZER)所造成的污染已不容忽视。玉米赤霉醇及其代谢产物具有一定的雌激素作用,它随食物链进入机体后,会造成机体生长发育障碍,对机体第二性征产生影响。我国农业部早在2002年就明确禁止玉米赤霉醇作为增重剂用于畜禽养殖。目前,用于食品及饲料中ZER的检测方法主要有薄层色谱法、高效液相色谱法和免疫分析技术。薄层色谱法只能用于定性分析;高效液相色谱法的前处理过程复杂,需要专业人员操作,应用大量的有毒有机溶剂,使其应用受到一定的限制。而ELISA免疫分析技术具有快速、灵敏、简便、特异等优点,正好可以弥补上述方法的不足,已被成功运用于食品和饲料中真菌毒素的快速检测。1、玉米赤霉醇人工抗原的合成及多抗血清的制备针对ZER第十六位上的羟基进行改造,设计并合成半抗原ZER-16-羧丙基丁醚。分别采用混合酸酐法和碳二亚胺(EDC)法将改造后的半抗原与载体蛋白BSA和OVA偶联,制备出人工免疫抗原ZER-BSA和人工检测抗原ZER-OVA。经SDS-PAGE电泳鉴定,偶联蛋白在凝胶上的迁移距离比载体蛋白小,故偶联成功。之后免疫小鼠并成功制备出效价达1:2.56×104、IC50为15.77ng/m L的鼠源多抗血清。2、玉米赤霉醇单克隆抗体的制备及其特性鉴定成功获得高效ZER多抗血清后,采用单克隆抗体技术和细胞融合技术将有效小鼠的脾细胞与SP/20骨髓瘤细胞进行融合,多次亚克隆后筛选出了三株能够稳定分泌ZERm Ab的杂交瘤细胞株,分别为6B2E6、6B2E11和12B10A7,其细胞上清效价可达1:2.56×104、1:5.12×104和1:2.56×104。将敏感性最好的12B10A7细胞株注射到处理过的小鼠腹腔内,收集到的腹水效价为1:6.4×105。所得单抗经鉴定为Ig G1型,亲和常数Ka为4.7×1011L/mol,回归方程为y=-0.3796x+0.7746,相关系数R2=0.9885,IC50=0.529ng/m L。ZERm Ab与黄曲霉毒素B1(AFB1)、伏马菌素B1(FB1)等非同类毒素的交叉反应率低,均小于0.5%。该细胞株不论是体外传代还是冻存复苏后都能稳定地分泌抗体。3、ZER间接竞争ELISA检测分析法的建立及在动物性食品中ZER残留检测的应用通过反复的ELISA检测试验,确定了抗原最佳包被浓度为1:2000倍稀释,抗体最佳工作浓度在1:10000倍稀释,建立了ZER间接竞争ELISA检测方法,用此方法对动物性食品中ZER残留进行检测,回归方程为y=-0.3582x+0.5949,相关系数R2=0.9925,IC50=0.184ng/m L。回收率在85%以上,变异系数小于10%,试验结果表明本试验建立的ZER间接竞争ELISA检测分析法灵敏性强,准确率高,能够有效地检测食品中ZER的残留。
[Abstract]:In recent years, the contamination of food caused by mycotoxins is becoming more and more serious, and the pollution caused by gibberellin (ZER) can not be ignored. Corn gibberellin and its metabolites have some estrogenic effects. When the food chain enters the body, it will cause the growth and development of the body and affect the secondary sexual characteristics of the body. As early as 2002, China's Ministry of Agriculture specifically banned gibberellin as a weight gain agent for livestock and poultry breeding. At present, the detection methods of ZER in food and feed mainly include thin layer chromatography, high performance liquid chromatography and immunoassay. TLC can only be used for qualitative analysis. The pretreatment process of HPLC is complex and requires professional operation. A large number of toxic organic solvents are used, so its application is limited to a certain extent. ELISA immunoassay has the advantages of rapidity, sensitivity, simplicity and specificity, which can make up for the shortcomings of the above methods. It has been successfully used in the rapid detection of mycotoxins in food and feed. The synthesis of artificial antigen of Zea gibberellin and the preparation of polyantiserum were modified to modify the hydroxyl group at the sixteenth position of ZER and to design and synthesize hapten ZER-16- carboxypropyl butyl ether. The modified hapten was coupled with the carrier proteins BSA and OVA by mixed anhydride method and carbodiimide (EDC) method, respectively. The artificial immune antigen (ZER-BSA) and the artificial detection antigen (ZER-OVA.) were prepared. SDS-PAGE electrophoresis showed that the transfer distance of the conjugated protein on the gel was smaller than that of the carrier protein, so the coupling was successful. Then mice were immunized and the mouse polyclonal antisera with the titer of 1: 2.56 脳 104 IC50 as 15.77ng/m L were successfully prepared. After the preparation and characterization of monoclonal antibody to Zea gibberellin, the highly effective ZER polyclonal antibody was obtained. The effective mouse spleen cells were fused with SP/20 myeloma cells by monoclonal antibody technique and cell fusion technique. After several subclones, three hybridoma cell lines were selected to secrete ZERm Ab stably. The supernatant titers of 6B2E6B2E11 and 12B10A7 were 1: 2.56 脳 10 ~ 4 ~ 1: 5.12 脳 10 ~ 4 and 1: 2.56 脳 10 ~ 4 脳 10 ~ 4 respectively. The most sensitive 12B10A7 cell line was injected into the peritoneal cavity of the treated mice. The titer of ascites collected was 1: 6.4 脳 105. The McAbs were identified as Ig G1 type, the affinity constant Ka was 4.7 脳 1011L / mol, the regression equation was y1-0.3796x 0.7746, and the correlation coefficient was R2O0.9885C500.29ngr / m L.ZERm Ab with aflatoxin B1 (AFB1), fumonisin B1 (FB1) and other non-congener toxins, all of which were less than 0.5mg / m. This cell line can secrete antibody. 3ZER indirectly competitive ELISA assay and its application in animal food by repeated ELISA detection test, no matter whether it is passaged in vitro or after cryopreservation and resuscitation. The best coating concentration of antigen was 1: 2000 times dilution and the best working concentration of antibody was 1: 10 000. An indirect competitive ELISA method for the detection of ZER residues in animal food was established by using this method. The regression equation was yr 0.3582x 0.5949, and the correlation coefficient was R2O 0.9925IC500.184ng/ mL. The recovery was more than 85%, and the coefficient of variation was less than 10. The results showed that the indirect competitive ELISA method established in this study was sensitive and accurate, and could be used to detect ZER residues in food effectively.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TS207.5;S816.17
本文编号：2249464
[Abstract]:In recent years, the contamination of food caused by mycotoxins is becoming more and more serious, and the pollution caused by gibberellin (ZER) can not be ignored. Corn gibberellin and its metabolites have some estrogenic effects. When the food chain enters the body, it will cause the growth and development of the body and affect the secondary sexual characteristics of the body. As early as 2002, China's Ministry of Agriculture specifically banned gibberellin as a weight gain agent for livestock and poultry breeding. At present, the detection methods of ZER in food and feed mainly include thin layer chromatography, high performance liquid chromatography and immunoassay. TLC can only be used for qualitative analysis. The pretreatment process of HPLC is complex and requires professional operation. A large number of toxic organic solvents are used, so its application is limited to a certain extent. ELISA immunoassay has the advantages of rapidity, sensitivity, simplicity and specificity, which can make up for the shortcomings of the above methods. It has been successfully used in the rapid detection of mycotoxins in food and feed. The synthesis of artificial antigen of Zea gibberellin and the preparation of polyantiserum were modified to modify the hydroxyl group at the sixteenth position of ZER and to design and synthesize hapten ZER-16- carboxypropyl butyl ether. The modified hapten was coupled with the carrier proteins BSA and OVA by mixed anhydride method and carbodiimide (EDC) method, respectively. The artificial immune antigen (ZER-BSA) and the artificial detection antigen (ZER-OVA.) were prepared. SDS-PAGE electrophoresis showed that the transfer distance of the conjugated protein on the gel was smaller than that of the carrier protein, so the coupling was successful. Then mice were immunized and the mouse polyclonal antisera with the titer of 1: 2.56 脳 104 IC50 as 15.77ng/m L were successfully prepared. After the preparation and characterization of monoclonal antibody to Zea gibberellin, the highly effective ZER polyclonal antibody was obtained. The effective mouse spleen cells were fused with SP/20 myeloma cells by monoclonal antibody technique and cell fusion technique. After several subclones, three hybridoma cell lines were selected to secrete ZERm Ab stably. The supernatant titers of 6B2E6B2E11 and 12B10A7 were 1: 2.56 脳 10 ~ 4 ~ 1: 5.12 脳 10 ~ 4 and 1: 2.56 脳 10 ~ 4 脳 10 ~ 4 respectively. The most sensitive 12B10A7 cell line was injected into the peritoneal cavity of the treated mice. The titer of ascites collected was 1: 6.4 脳 105. The McAbs were identified as Ig G1 type, the affinity constant Ka was 4.7 脳 1011L / mol, the regression equation was y1-0.3796x 0.7746, and the correlation coefficient was R2O0.9885C500.29ngr / m L.ZERm Ab with aflatoxin B1 (AFB1), fumonisin B1 (FB1) and other non-congener toxins, all of which were less than 0.5mg / m. This cell line can secrete antibody. 3ZER indirectly competitive ELISA assay and its application in animal food by repeated ELISA detection test, no matter whether it is passaged in vitro or after cryopreservation and resuscitation. The best coating concentration of antigen was 1: 2000 times dilution and the best working concentration of antibody was 1: 10 000. An indirect competitive ELISA method for the detection of ZER residues in animal food was established by using this method. The regression equation was yr 0.3582x 0.5949, and the correlation coefficient was R2O 0.9925IC500.184ng/ mL. The recovery was more than 85%, and the coefficient of variation was less than 10. The results showed that the indirect competitive ELISA method established in this study was sensitive and accurate, and could be used to detect ZER residues in food effectively.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TS207.5;S816.17
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