基于二聚人工抗原的β-兴奋剂多残留免疫检测技术研究

发布时间：2018-04-28 04:45

本文选题：克伦特罗 + 莱克多巴胺　；参考：《中国计量大学》2016年硕士论文

【摘要】：β-受体激动剂分为两大类:苯胺型类和苯酚型类。其中克伦特罗(Clenbuterol,CLE),属于苯胺型类β-受体激动剂;而莱克多巴胺(ractopamine,RAC),属于苯酚型类β-受体激动剂。将其作为动物的饲料添加剂,能显著提高饲料的转化率和瘦肉转化率。但其在动物体内的残留,会通过食物链进入到人体内,从而危害人类健康。它们也因此在全球范围内被禁用。本论文利用酶联免疫吸附法(ELISA)和胶体金免疫层析法(CGIA)建立了对β-受体激动剂多残留免疫检测的技术。获得的结果如下:1、以对羟基苯甲醛与硝基甲烷为起始物,并结合兰尼镍还原法,从头合成莱克多巴胺氨基衍生物,即RAC-NH2。以此生成物为起始物,与克伦特罗一样进行重氮化反应,分别合成出RAC-HSA、RAC-BSA、CLE-HSA和CLE-BSA。在此基础上,将RAC-NH2和CLE按照一定摩尔比混合,进行重氮化反应,通过紫外和SDS-PAGE鉴定,分别合成出免疫原CLE-HSA-RAC和包被原CLE-BSA-RAC。2、以合成出的CLE-HSA-RAC做为免疫原,采用常规免疫方法进行免疫小鼠,综合效价和灵敏度两个方面,选择2号小鼠进行冲击免疫,进行细胞融合试验。经过三轮亚克隆后,得到3A2和4D8两株稳定分泌抗CLE-RAC的细胞株,其中4D8这株细胞株分泌的单抗,对CLE-RAC的灵敏度最好,IC50值为0.325±0.021ng/mL,且有64000的效价。因此选择4D8细胞株注入小鼠体内,进行诱生腹水。3、以CLE-BSA-RAC人工抗原和CLE-RAC单克隆抗体(4D8)建立了检测CLE和RAC的间接竞争ELISA方法。此方法检测CLE和RAC时的IC50值为0.77ng/mL,最低检测限IC10为0.14 ng/mL。对克伦特罗、莱克多巴胺、马布特罗的交叉反应率很高(CR96%)。同时对西布特罗、溴布特罗、马贲特罗、西玛特罗、班布特罗和克伦普罗也有一定的特异性(17%CR85%),实现了多残留检测。对其余的几种受体激动剂,没有交叉反应(CR0.1%)。在实际样品检测时,猪尿、猪肉、猪肝和饲料的最低检测限分别是0.302、0.603、0.804和12.42ng/mL。而最低定量限则分别为:0.518、0.858、1.152和20.583 ng/mL。板内回收率为78%-118.00%,以及板间回收率为84.00%-114.00%。板内变异系数小于14.04%,板间变异系数小于7.69%。利用LC-MS/MS法验证,此方法是可靠的。4、采用柠檬酸钠还原法,制备了CLE-RAC胶体金标记抗体,同时成功制备出胶体金试纸条。肉眼对CLE和RAC等9种β-兴奋剂类药物的检测限可以达到5 ng/mL的水平,与前面结果icELISA结果一致,对β-兴奋剂类药物特异性较高,实现了多残留检测。同时检测时间较短,适合现场检测。本论文采用杂交瘤细胞技术,制备出CLE-RAC单抗,并建立了icELISA法和金标试纸条检测技术,为CLE和RAC等β-受体激动剂多残留提供了可靠的检测方法。
[Abstract]:尾-receptor agonists are divided into two categories: aniline type and phenol type. Clenbuterolium clenbuterolium is aniline-type 尾 -receptor agonist, while ractopamine ractopamine ractopamine belongs to phenol-type 尾 -receptor agonist. As an animal feed additive, the feed conversion rate and lean meat conversion rate can be significantly increased. But its residue in the animal body, will enter the human body through the food chain, thereby endangering human health. They are therefore banned globally. In this paper, the technique of multiresidue immunoassay for 尾 -receptor agonists was established by Elisa and CGIA-gel gold immunochromatographic assay. The results obtained are as follows: 1. The ractopamine amino derivative, RAC-NH2, was synthesized from p-hydroxybenzaldehyde and nitromethane at the ab initio using Raney nickel reduction method. RAC-HSA-RAC-BSA-CLE-HSA and CLE-BSA-CLE-HSA were synthesized by diazotization with clenbuterol. On this basis, RAC-NH2 and CLE were mixed at a certain molar ratio, diazotization reaction was carried out, the immunogen CLE-HSA-RAC and the coated CLE-BSA-RAC.2 were synthesized by UV and SDS-PAGE, respectively. The synthesized CLE-HSA-RAC was used as immunogen. Mice were immunized with routine immunization method. The mice were immunized with impact and cell fusion test in two aspects: titer and sensitivity. After three rounds of subcloning, two stable anti-CLE-RAC cell lines, 3A2 and 4D8, were obtained. The sensitivity of the McAb secreted by 4D8 cell line to CLE-RAC was 0.325 卤0.021 ng / mL, with a titer of 64000. Therefore, 4D8 cell lines were selected to be injected into mice to induce ascites. The indirect competitive ELISA method for detection of CLE and RAC was established with CLE-BSA-RAC artificial antigen and CLE-RAC monoclonal antibody (4D8). The IC50 value of CLE and RAC is 0.77 ng / mL, and the lowest detection limit IC10 is 0.14 ng / mL. Cross-reaction rates for clenbuterol, ractopamine, and Mabbuterol were high. At the same time, sibuterol, brombuterol, Mapentrol, Simatro, Bambuterol and clenpulo also have a certain specificity of 17 CR85C. For the other several receptor agonists, there was no cross-reaction between CR0. 1 and CR0. 1. The minimum detection limits for urine, pork, liver and feed were 0.302ng / mL and 12.42ng / mL, respectively. The minimum quantification limits are: 0. 518, 0. 858, 1. 152 and 20.583 ng / mL, respectively. The intraplate recovery was 78-118.00 and the inter-plate recovery was 84. 00-114. 00. Intra-plate coefficient of variation was less than 14.04 and inter-plate coefficient of variation was less than 7.69. The LC-MS/MS method was used to verify the reliability of this method. The colloidal gold labeled antibody was prepared by sodium citrate reduction method and the colloidal gold strip was successfully prepared. The detection limit of 9 尾 -stimulant drugs such as CLE and RAC by naked eye can reach 5 ng/mL level, which is consistent with the previous results of icELISA, and the specificity of 尾 -stimulant drugs is higher, and multi-residue detection is realized. At the same time, the detection time is short, suitable for field testing. In this paper, CLE-RAC monoclonal antibody was prepared by hybridoma cell technique, and icELISA method and gold-labeled strip method were established, which provided a reliable method for the detection of multi-residue of 尾 -agonist such as CLE and RAC.
【学位授予单位】：中国计量大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S859.84

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