磺胺氯吡嗪钠化学发光免疫分析方法的建立

发布时间：2018-01-27 08:26

本文关键词： 磺胺氯吡嗪钠单克隆抗体化学发光免疫检测磺胺噻唑　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：磺胺氯吡嗪钠(sulfachlorpyrazine sodium,SPZ)是磺胺类药物的一种,在兽医临床上用来治疗家禽球虫病。若大剂量使用或不遵守休药期规定,药物易在动物体内蓄积,威胁人类健康。免疫学检测方法作为兽药残留大规模检测的主要方法,具有特异性强,灵敏度高等特点。目前国内外关于SPZ残留的酶联免疫检测方法(ELISA)的报道较少,未见关于SPZ的化学发光免疫分析(CLIA)检测方法的报道。本文制备了SPZ的单克隆抗体,在此基础上建立CLIA方法;同时对磺胺药物多残留快速检测技术进行探索,合成了药物的母核抗原-磺胺噻唑(TS)并制备其多克隆抗体,分别用ELISA方法和CLIA方法对抗体进行鉴定。1、SPZ单克隆抗体的制备及鉴定。本实验通过重氮化方法制备了完全抗原SPZ-OVA,免疫小鼠4次后,选取血清抗体效价较好的小鼠脾细胞进行细胞融合。经过筛选并将阳性克隆按有限稀释法亚克隆3次,得到稳定分泌抗体的细胞株。将细胞株扩大培养后,腹腔注射小鼠制备腹水。将腹水单克隆抗体D9纯化后通过间接ELISA确定包被原最佳浓度为0.01μg/m L,单抗的工作浓度为1:4×105,在最佳抗原抗体工作浓度下进行ci-ELISA实验,建立标准曲线,得到曲线线性方程为y=-0.3369x+1.347,R2=0.994,计算得抗体半数抑制浓度(IC50)为326ng/m L;与其他测试药物的交叉反应小于5%,表明单抗特异性良好。2、SPZ化学发光免疫检测(CLIA)方法的建立。在单克隆抗体Mab D9的基础上建立SPZ的CLIA方法,在0.02-4μg/m L的范围内,SPZ浓度的对数值与相对发光值呈线性关系,线性方程为y=-0.3602x+1.2874,R2=0.981,IC50值为151ng/m L;标准曲线的批内变异系数为7.02%,批间变异系数为8.5%;鸡肉样品最低检测限为36.3ng/g,样品添加回收率在75%-85%之间。3、磺胺类药物族特异性抗体的制备。首先合成磺胺母核结构TS,然后用碳二亚胺(EDC)法合成免疫原TS-BSA,将其免疫小鼠后,对抗血清进行筛选,得到两只小鼠血清效价分别为1:2.56×105和1:1.28×105;选用效价高的小鼠血清进一步检测。经ci-ELISA实验和CLIA实验测得抗体对TS的IC50值分别为33ng/m L和7.9ng/m L;与供试磺胺药物中含有五元杂环的药物有较强交叉反应(CR26%);与部分六元杂环药物有一定的交叉反应(1%CR11%);与磺胺氯吡嗪钠(SPZ)和磺胺二甲嘧啶钠(SM2)无交叉反应(CR0.01%)。实验表明抗体与多种R基结构的SAs可产生交叉反应。
[Abstract]:Sulfadiazine sodium sulfadiazine is a kind of sulfonamides. Used in veterinary clinic for the treatment of coccidiosis in poultry. If the drug is used in large doses or does not comply with the withdrawal period, the drug is easily accumulated in the animal body. As the main method of large-scale detection of veterinary drug residues, immunological detection has strong specificity. The characteristics of high sensitivity. At present, there are few reports at home and abroad about the Elisa method of SPZ residue detection. There was no report on the chemiluminescence immunoassay of SPZ. The monoclonal antibody against SPZ was prepared and the CLIA method was established. At the same time, the rapid detection technique of sulfadiazine multiresidue was explored, and the mother nucleus antigen-sulfathiazolium (TS) of the drug was synthesized and its polyclonal antibody was prepared. ELISA method and CLIA method were used to identify the monoclonal antibody. The complete antigen SPZ-OVA was prepared by diazotization. After the mice were immunized for 4 times, the spleen cells with good antibody titers were selected for cell fusion, and the positive clones were subcloned by limited dilution method for 3 times. The cell lines secreting antibodies stably were obtained. Ascites were prepared by intraperitoneal injection of mice. After purification of monoclonal antibody D9 in ascites, indirect ELISA was used to determine the optimal concentration of the coating material was 0. 01 渭 g / mL, and the working concentration of the monoclonal antibody was 1: 4 脳 10 ~ 5. The ci-ELISA test was carried out at the best working concentration of antigens and antibodies, and the standard curve was established. The linear equation of the curve was yang-0.3369x 1.347R2O0.994. IC50 was calculated to be 326ng / mL; The cross-reaction with other tested drugs was less than 5%, indicating that the specificity of the McAb was good (.2%). The establishment of SPZ chemiluminescence immunoassay (CLIA). Based on the monoclonal antibody Mab D9, the CLIA method of SPZ was established in the range of 0.02-4 渭 g / mL. The logarithmic value of SPZ concentration is linearly related to the relative luminescence value. The linear equation is YCU -0.3602x 1.2874N R2O 0.981N IC50 = 151ng / mL; The coefficient of variation in the standard curve was 7.02 and the coefficient of variation between batches was 8.5. The minimum detection limit of chicken samples was 36.3 ng / g. The recovery of the samples was between 75% and 85%. The preparation of sulfonamides group specific antibodies was performed. First, the mother nuclear structure TS of sulfanilamide was synthesized. Then the immunogen TS-BSAwas synthesized by carbodiimide EDCmethod and then immunized with TS-BSA. the antiserum was screened. The titers of two mice were 1: 2.56 脳 105 and 1: 1.28 脳 105, respectively. Ci-ELISA test and CLIA test showed that the IC50 value of antibody to TS was 33ng / mL and 7.9 ng / mL, respectively. There was a strong cross reaction between CR26 and sulfamethylamine containing five heterocyclic compounds. There was a certain cross reaction with some hexagonal heterocyclic drugs. There was no cross reaction with sulfamethazine (SSP) and sulfadimethazine sodium (SM2). The results showed that the antibody could cross react with SAs with various R-group structures.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79

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