摘要
为了建立快速、简单、方便的克伦特罗(Clenbuterol, CL)检测方法,本试验采用重氮法将CL与牛血清白蛋白(Bovine serum albumin, BSA)、卵清白蛋白(Ovalbumin, OVA)偶联,产生免疫原CL-BSA,包被抗原CL-OVA,以CL-BSA作为免疫原,用等剂量的福氏完全佐剂乳化CL-BSA,免疫6周龄BALB/C小鼠。试验目的：(1)通过制备高效价的CL多克隆抗体,构建克伦特罗酶联免疫吸附检测试剂盒；(2)运用杂交瘤技术,分离CL单克隆抗体,构建克伦特罗胶体金检测试纸条。
1、CL酶联吸附检测试剂盒
小鼠经CL-BSA多次免疫后,分离小鼠血清,得抗血清效价为128000,与莱克多巴胺(Ractopamine, Rac)、沙丁胺醇(Salbutamol, Sal)交叉反应率为0.1%,具有很强的特异性。用CL抗血清建立了CL间接竞争ELISA法,得到标准曲线的回归方程loght y=-0.8756x+1.6958,相关系数为0.995,可检测范围为1ng/mL-10μg/mL,并检测出饲喂了含CL猪的尿样中含有CL。
2、CL胶体金检测试纸条
小鼠经CL-BSA加强免疫后3天,取小鼠脾细胞,利用聚乙二醇(PEG)4000将其与骨髓瘤细胞SP2／0进行融合,制备杂交瘤细胞。经过对培养上清的筛选和杂交瘤细胞的克隆化,最终得到3株能分泌克伦特罗抗体的杂交瘤细胞株：1A1、1B5、和2H9。用间接ELISA法测得杂交瘤培养上清效价分别达到1：2000、1：1200、1：800。最后用扩大培养所得的杂交瘤细胞诱导小鼠产生产生腹水,制备单克隆抗体。
CL检测试纸条的研制：本试验利用CL单抗标记40nm胶体金,标记胶体金稳定。将标记胶体金CL-OVA和羊抗鼠免疫球蛋白G(IgG)喷涂在硝酸纤维膜(NC)膜上,CL-OVA条带作为检测线,羊抗鼠IgG条带作为质控线,制备得到可快速检测盐酸克伦特罗的胶体金检测试纸条。将待测样品滴在试纸条吸样品区,在毛细管作用下,样品沿着膜移动。根据竞争抑制免疫层析原理,如果样品中没有CL,质控线和检测线均显色；相反,检测线不显色,质控线显色。该法可在10分钟内完成对CL的检测,最小检测限为lppb。该检测方法快速、准确、灵敏、操作简便,适合非专业人员在屠宰现场检测克伦特罗。
In order to establish a rapid, simple and convenient method of detecting clenbuterol, CL-conjugated antigen were produced by coupling diazotized CL with Bovine Sera Albumin(BSA) and Ovalbumin (OVA). Thus get immunogen CL-BSA and antigen-coated CL-OVA, using CL-BSA as a immunogen with equal dose of Freund's complete adjuvant emulsified to immune 6-week-old BALB/C mice. Thus to (1) build Clenbuterol ELISA Test Kit by high titer polyclonal antibodies, (2) build Clenbuterol colloidal gold test dipstick by monoclonal antibody CL
1.CL ELISA Assay Kit
Separated anti-Clenbuterol serum after repeated immunization of CL-BSA, and the average titer of antiserum was 128000, the antiserum had 0.1% cross reaction with Ractopamin and Salbutamol, this indicted that the antiserum was specific for CL. CL antisera were used to build ELISA by indirect competitive method, the standard curve regression equation of the kit was logit y=-0.8756x+1.6958, the correlation coefficient was 0.995, detectable range was 1-10μg/mL. The kit was applied to check practical samples of pig urine, the pigs were grouped by feeding with or without CL.
2. CL detection dipstick colloidal gold
Spleen cells were obtained from mice after 3 days CL-BSA immunization, then the spleen cells were fused with myeloma cells by PEG4000 fused them with myeloma cells SP2/0 to get hybridoma cell. After screening of the culture supernatant and cloning of hybridoma cell, three hybridoma cell lines, which secret clenbuterol antibody, were found, they are 1A1,1B5, and 2H9. The titer of hybridoma culture supernatant titer were 1:2000,1:1200,1:800 respectively. Finally, the hybridoma cell were induced into mice to crest ascites and get monoclonal antibodies.
Development of CL Dipstick testing:In this study, CL monoclonal was marked with 40nm colloidal gold, then it was stablely painted to NC membrane, CL-OVA and goat anti-mouse IgG in the NC membrane, CL-OVA strip as a test line, goat anti-mouse IgG as a quality control line, therefore, the dipstick colloidal gold rapid of detecting clenbuterol were prepared. Drop the sample in the sample suction area of the test paper, the sample moving along the membrane by capillary force. According the principle of Competitive immunochromatography, if the sample do not contain CL, both the quality control line and the testing line were colored, in contrast, if the sample contain CL, the line of detection wasn't colored, but the line of quality control was corlored. This method can detected CL within 10 minutes, the minimum detection limit islppb. This method is rapid, accurate, sensitive and easy to operate, suitable for non-professional checking at the slaughter house or pig farm.
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