基于纳米金颗粒和乳胶微球的卡那霉素免疫分析方法的建立及初步应用

发布时间：2018-01-24 02:17

本文关键词： 卡那霉素单克隆抗体 ELISA 纳米金颗粒乳胶微球　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：卡那霉素是一种氨基糖苷类抗生素,因其具有良好的抑制细菌生长等作用而被广泛应用于医药、农业、畜牧和水产养殖业。卡那霉素的不合理使用,一方面会引起卡那霉素在动物体内的大量残留,随着食物链的传递作用,最终进入人体,危害人体健康;另一方面未被吸收利用的卡那霉素会以原型或其代谢产物的形式排放到自然水体或土壤中,导致严重的环境污染。因此,建立灵敏度高、特异性强、稳定性好、简单快捷的分析方法来检测食品和环境中的卡那霉素含量很有必要。本研究制备了高特异性的卡那霉素单克隆抗体,结合纳米金颗粒建立了卡那霉素直接竞争ELISA分析方法,并尝试建立基于乳胶微球的免疫新方法。本文采用EDC法成功制备了免疫原(Kana-BSA)和包被原(Kana-OVA)。经动物免疫步骤免疫小鼠后,有3只小鼠体内产生的卡那霉素抗体达到实验要求,从中挑选出一只血清效价高且抑制效果好的小鼠,获取它的脾细胞与SP2/0细胞进行细胞融合。融合后经筛选,共得到10株能够稳定分泌卡那霉素单克隆抗体的细胞株。对10株杂交瘤细胞株分泌的单抗进行类与亚型的鉴定,结果显示这些单克隆抗体均为IgG1型且轻链都为?型。后续实验选取A10E5杂交瘤细胞株分泌的单抗进行卡那霉素免疫检测方法学研究。制得Kana-HRP,建立了基于HRP信号分子的卡那霉素直接竞争ELISA方法,此方法的IC50为2.15 ng/mL,LOD为0.13 ng/m L,线性区间在0.40~9.67 ng/m L;合成Au-HRP-Kana,建立了基于Au-HRP双标记信号放大的ELISA方法,该方法的IC50为1.05 ng/mL,LOD为0.16 ng/mL,线性区间为0.31~2.8 ng/m L。A10E5单克隆抗体与妥布霉素交叉反应率为99.07%,与其他结构类似物的交叉反应率均在0.3%以下。实验测得加标回收率在88.6%~132.8%,变异系数小于15.8%。对环境样品中卡那霉素残留情况进行分析测定,结果发现江苏大学环境水样中卡那霉素含量在0.45~2.78 ng/m L,牛奶样品中卡那霉素的残留量为0.35~0.55 ng/m L,而土壤中卡那霉素的含量未检出。实验采用EDC/NHS法成功合成了乳胶微球-PEG-GAM,建立了基于乳胶微球的免疫新方法,讨论了乳胶微球的非特异性结合情况,并对实验条件进行了初步的优化。基于乳胶微球的离心定量法,其IC50为4.66 ng/m L,LOD为0.25 ng/m L,线性区间在0.73~19.07ng/mL。基于乳胶微球的过滤定性法,其最低检测限为1 ng/m L,实验表明可将该方法初步应用于对卡那霉素的定性分析。最后比较了四种检测卡那霉素的分析方法,直接竞争ELISA方法灵敏度较高,可应用于实际环境样品中卡那霉素的分析检测。而基于乳胶微球的免疫分析新方法检测灵敏度虽没有ELISA方法高,但方法简单、便捷,可应用于对实际样品的初筛。
[Abstract]:Kanamycin is an aminoglycoside antibiotic, which is widely used in medicine, agriculture, animal husbandry and aquaculture. On the one hand, it will cause a large number of kanamycin residues in the animal body, along with the transmission of the food chain, eventually into the human body, harmful to human health; On the other hand, the unabsorbed kanamycin can be discharged into natural water or soil in the form of prototype or its metabolites, resulting in serious environmental pollution. Therefore, the establishment of high sensitivity, specificity and stability. It is necessary to detect kanamycin in food and environment by a simple and rapid analytical method. In this study, a highly specific monoclonal antibody against kanamycin was prepared. A direct competitive ELISA analysis method for kanamycin was established in combination with gold nanoparticles. A new immunization method based on latex microspheres was established. In this paper, the immunogen Kana-BSA and the coated proto-Kana-OVA were successfully prepared by EDC method. The mice were immunized by animal immunization. Three mice produced kanamycin antibody to meet the experimental requirements, and a mouse with high serum titer and good inhibitory effect was selected. The spleen cells were fused with SP2/0 cells. A total of 10 cell lines were obtained to stably secrete monoclonal antibodies against kanamycin. The monoclonal antibodies secreted by 10 hybridoma cell lines were identified by type and subtype identification. The results showed that these monoclonal antibodies were IgG1 type and light chain. Kana-HRP was prepared by using monoclonal antibody secreted by A10E5 hybridoma cell line for kanamycin immunoassay. A ELISA method for direct competition of kanamycin based on HRP signaling molecules was established. The IC50 of this method was 2.15 ng / mLL = 0.13 ng/mL. The linear range is 0. 40 ~ 9. 67 ng/m / L; Au-HRP-Kana was synthesized, and the ELISA method based on Au-HRP double label signal amplification was established. The IC50 of this method was 1.05 ng/mL. LOD was 0.16 ng / mL, and the linear range was 0.31 ~ 2.8 ng/m L. A10E5. The cross reaction rate between monoclonal antibody and tobramycin was 99.07%. The cross reaction rates with other structural analogues were all below 0.3%, and the recoveries were 88.6% and 132.8%, respectively. The coefficient of variation was less than 15.8.The results showed that the content of kanamycin in environmental water sample of Jiangsu University was 0.45 ~ 2.78 ng/m / L. The residual amount of kanamycin in milk samples was 0. 35 and 0. 55 ng/m / L. However, the content of kanamycin in soil was not detected. Latex microspheres PEG-GAM were successfully synthesized by EDC/NHS method, and a new immune method based on latex microspheres was established. The nonspecific binding of latex microspheres was discussed, and the experimental conditions were preliminarily optimized. The IC50 was 4.66 ng/m / L based on the centrifugation quantitative method of latex microspheres. The LOD was 0.25 ng/mL and the linear range was 0.73 ~ 19.07 ng 路mL ~ (-1). The minimum detection limit of the method was 1 ng/mL based on latex microspheres. The results show that this method can be applied to the qualitative analysis of kanamycin. Finally, four analytical methods for the detection of kanamycin are compared, and the sensitivity of the direct competition ELISA method is high. It can be applied to the analysis and detection of kanamycin in practical environmental samples, but the new method based on latex microspheres is not as sensitive as ELISA method, but the method is simple and convenient. It can be used to screen the actual sample.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X830

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