氟喹诺酮类药物多残留酶联免疫检测方法的建立  

Development of an ELISA Method for Multi-Residue Detecting of Fluoroquinolones

作　　者：李新朋[1,2] 姜金庆[2] 钱爱东[1] 王自良[2] 范国英[2] 单晓峰[1] 康元环[1] 李艺[2] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]河南科技学院动物科学学院,河南新乡453003

出　　处：《中国农业科学》2014年第23期4726-4735,共10页Scientia Agricultura Sinica

基　　金：国家十二五科技支撑计划重大项目(2011BAK10B01);国家自然科学基金(U1204310)

摘　　要：【目的】氟喹诺酮类药物(FQs)在畜牧业中广泛用于预防和治疗细菌性疾病,不合理使用导致在动物性食品中残留,严重危害消费者健康,其检测受到世界各国重视。制备抗多种FQs单克隆抗体(m Abs),建立间接竞争ELISA(ic ELISA)检测方法,为动物性食品中FQs多残留检测奠定基础。【方法】环丙沙星(CPFX)羧基上引入氨基丁酸后为半抗原(CPFX-A),质谱鉴定;分别用碳二亚胺法(DDC)和混合酸干法将半抗原与牛血清白蛋白(BSA)和卵清蛋白(OVA)偶联合成免疫原(CPFX-A-BSA)和包被原(CPFX-A-OVA),紫外和红外光谱鉴定是否偶联成功;CPFX-A-BSA免疫Balb/c小鼠,间接ELISA和ic ELISA方法测定多抗血清效价和敏感性,选取抗血清效价高和敏感性好的鼠用于细胞融合;NS0细胞和脾细胞按1﹕5比例在PEG-1500作用下融合,筛选抗FQs杂交瘤细胞株,并进行效价、亚型、敏感性和交叉反应率鉴定;体内诱生腹水法制备m Ab,液体石蜡作为诱导剂,每只鼠注射108个杂交瘤细胞;选敏感性和广谱特异性好的腹水m Ab,建立ic ELISA标准曲线,对ic ELISA检测方法优化,在鸡肉中添加10种FQs进行测定,将测定结果与HPLC法比较,用SPSS 17.0软件进行显著差异性分析。【结果】半抗原改造和人工抗原合成成功;3只鼠血清效价均达到1﹕1.28×104以上,2号鼠血清效价最高(1﹕2.56×104),且IC50值最低(12.92 ng·m L-1),选择2号鼠作为细胞融合用鼠;4次亚克隆筛选并鉴定后获得3株敏感广谱特异的杂交瘤细胞,分别命名为2H5、3D11、4F4,细胞上清效价分别为1﹕1 600、1﹕1 600和1﹕800,腹水效价分别为1﹕1.6×106、1﹕8.2×105和1﹕8.2×105;ic ELISA方法的包被原浓度为1μg·m L-1,5%猪阴性血清4℃包被过夜,单抗1﹕40 000稀释,山羊抗鼠酶标二抗(Ga MIg G-HRP)1﹕8 000稀释;反应温度均为37℃,标准品与单抗同时加入反应15 min,洗板后加二抗反应25 min;显色10 min,2H5细胞株腹水敏感性和广谱特异性【 Objective 】 Fluoroquinolones（FQs） are widely used in veterinary medicine for the treatment and prevention of bacterial infection. With the increasing use, FQs residues in animal edible tissues have caused serious public health problems and attracted serious attention by research scholars all over the world. The objective of this study was to produce class-specific monoclonal antibodies（m Abs） against fluoroquinolones（FQs）, establish competitive indirect enzyme linked immunesorbent assay（ic ELISA）, and in order to lay a foundation for detection of multi-residue FQs in animal foods.【Method】The aminobutyric acid was introduced to carboxyl of ciprofloxacin as hapten（CPFX-A） and was proved by（＋） ESI-MS spectrum, which was conjugated to bovine serum albumin（BSA） as immunogen（CPFX-A-BSA） by the N,N＇-Dicyclohexylcarbodiimide（DDC） method, and to ovalbumin（OVA） as coating antigen（CPFX-A-OVA） by mixed anhydride method, respectively, which were then identified by infrared ray（IR） and ultraviolet（UV）. Balb/c mice immunized by CPFX-A-BSA were selected for cell fusion, which was identified by ELISA and ic ELISA. Under the effect of PEG-1500, NS0 cells and spleen cells were fused at the ratio of 1﹕5. Hybridoma lines that secrete m Ab against FQs were selected and their immunological traits were characterized by titer, subtype, sensitivity and cross reaction rate, which ascites were carried out by injecting 108 hybridoma cells in vivo, and ic ELISA standard curve was established and optimized. High titer, class-specific monoclonal antibody was used to detect 10 FQs in chicken for calculating recovery rate and variation coefficient. The data were also compared with that of HPLC, and SPSS 17.0 software was used to conduct the significant difference analysis.【Result】 The hapten and artificial antigen were synthesized successfully and antiserum titers of three mice were higher than 1﹕1.28×104, in which the titer and IC50 of No.2 mouse were 1﹕2.56×104 and 12

关 键 词：环丙沙星 氟喹诺酮类药物 杂交瘤细胞株 广谱特异性单克隆抗体 间接竞争ELISA 

分 类 号：S859.84[农业科学—临床兽医学]