动物源性食品中氯霉素残留的间接竞争ELISA检测方法的建立

发布时间：2018-07-08 15:24

  本文选题：氯霉素 + 兽药残留　； 参考：《河南科技大学》2017年硕士论文

【摘要】：氯霉素(chloramphenicol,ACP)作为动物源性食品中残留问题较为突出的药物,建立严格的检测方法已势在必行。酶联免疫分析法用于检测兽药残留具有简便快速,高效稳定等优点,适合动物性食品残留的现场检测。本课题以合成抗原的为基础,经过细胞融合后,制备单克隆抗体,并建立间接竞争ELISA的检测方法。利用混合酸酐法使氯霉素半抗原(CAP-HS)与牛血清白蛋白(BSA)偶联,获得免疫原CAP-HS-BSA;与鸡卵清蛋白(OVA)偶联,制备检测抗原CAP-OVA,利用紫外扫描(UV)和SDS一聚丙烯酞胺凝胶电泳(SDS-PAGE)鉴定CAP-HS-BSA:CAP-HS-BSA紫外扫描图的最大吸收峰发生偏移,CAPBSA的泳动率小于BSA,判定CAP-BSA成功偶联,并推算出CAP-HS与BSA分子结合比为12.1∶1。CAP-HS-BSA免疫3只BALB/c小鼠,4次免疫后用间接ELISA检测CAP鼠源多克隆抗体(CAP pAb),其效价均大于1∶6.4×103,用间接竞争ELISA(ci-ELISA)检测CAP pAb的半数抑制浓度(IC50),其中测定结果最好的3号小鼠的IC50为16.4μg/L,CAP pAb与CAP的结构类似物及常用抗生素没有交叉反应,获得高效价,特异性强的抗CAP多克隆抗体。ELISA筛选细胞融合鼠,融合小鼠脾细胞与SPNS0瘤细胞,得到杂交瘤细胞株,体内诱生腹水法生产CAP单克隆抗体(CAP mAb),选取4株敏感度和选择性均好的抗CAP杂交瘤细胞:1B8 CAP mAb、2C4 CAP mAb、4A1 CAP mAb,其细胞上清效价:1∶2.4×102、1∶6.4×102、1∶1.2×102,腹水效价:1∶2.0×105、1∶4.8×105、1∶1.2×105。ci-ELISA测定亲和力最高的2C4株的IC50为0.53μg/L,与其类似物没有交叉反应。以CAP mAb为基础,优化了ci-ELISA最佳实验条件:CAP-HS-OVA的包被浓度:0.4μg/mL,CAP mAb的工作浓度:1:6.4×104;羊抗鼠酶标二抗(GaMIgG-HRP)的稀释浓度为1:1000;包被条件:包被原CAP-OVA的包被浓度0.4μg/mL,37℃,孵育2h,5%的猪血清封闭,37℃,孵育1h;室温条件下,显色液作用时间为9min。绘制出的CAP残留的ci-ELISA的标准曲线显示为典型的S型,与4参数logit拟合曲线相吻合,IC50为0.53 ng/m L,检测限为0.6 ng/mL;添加回收实验,阴性的鱼肉、牛奶的平均回收率分别为97.3%和97.5%,平均变异系数为4.9%、5.5%,CV均小于10%。HPLC对比实验:CiELISA测定结果与样品值的差异是3.30%~4.40%,Ci-ELISA与HPLC的测定值差异是1.13%~4.85%;实际样品的测定:河虾样品中,Ci-ELISA对HPLC的差异为4.43%。
[Abstract]:Chloramphenicolium (ACP) is an important drug in animal food. It is imperative to establish a strict method for detection of chloramphenicolium. Enzyme-linked immunosorbent assay (Elisa) has the advantages of simplicity, rapidity, high efficiency and stability, and is suitable for field detection of animal food residues. Based on synthetic antigen, monoclonal antibody was prepared after cell fusion, and indirect competitive Elisa method was established. Chloramphenicol hapten (CAP-HS) was coupled with bovine serum albumin (BSA) by mixed anhydride method to obtain the immunogen CAP-HS-BSAand chicken ovalbumin (OVA). The detection antigen CAP-OVA was prepared. The UV scanning and SDS-PAGE were used to identify the maximum absorption peak of CAP-HS-BSA-HS-BSA. The swimming mobility of CAP-OVA was less than that of BSA-BSA, and the successful coupling of CAP-BSA was determined. The binding ratio of CAP-HS to BSA was calculated to be 12.1: 1. 1. CAP-HS-BSA was used to immunize three BALB / c mice. After 4 times immunization, the titers of CAP pAb), were detected by indirect Elisa. The titers of CAP pAb), were all greater than 1: 6.4 脳 103, and the half inhibitory concentration of CAP was detected by indirect competitive Elisa (ci-ELISA). (IC50), in which the IC50 of mice with the best results was 16.4 渭 g / L, and there was no cross-reaction between the structural analogues of CAP and CAP and common antibiotics. High titer and strong specificity of anti-CAP polyclonal antibody. Elisa was used to screen the fusion cells of mouse spleen cells and SPNS0 tumor cells, and the hybridoma cell lines were obtained. In vivo induced ascites production of CAP monoclonal antibody (CAP mAb), selection of four anti-CAP hybridoma cell lines with good sensitivity and selectivity: 1B8 CAP mAbmAb2CAP mAb2CAP 4A1 CAP mAb. the supernatant titer of the cell supernatant was 12.4 脳 102: 1: 1. 4 脳 10 2: 1. 2 脳 10 2% 1.2 脳 10 2). Ascites titer was 1: 2.0 脳 10 5% 1: 4.8 脳 10 5% 1: 1.2 脳 105.ci-ELISA. The IC50 of 2C4 strain with the highest affinity was 0.53 渭 g / L, and there was no cross reaction with its analogues. On the basis of CAP mAb, the optimal conditions for ci-ELISA were optimized. The optimal conditions for ci-ELISA were as follows: the encapsulation concentration of: CAP-HS-OVA was: 1: 0.4 渭 g / mL / min, the working concentration of CAP mAb was 1: 1: 6.4 脳 10 ~ (4), the dilution concentration of goat anti-mouse enzyme-labeled second antibody (GaMIgG-HRP) was 11000: the encapsulation conditions were as follows: the encapsulation concentration of the original CAP-OVA was 0.4 渭 g / mL ~ (-1) at 37 鈩，

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