环丙沙星论文：环丙沙星单克隆抗体的制备及其免疫学特性的鉴定

环丙沙星论文：环丙沙星单克隆抗体的制备及其免疫学特性 的鉴定【中文摘要】环丙沙星(Crprofloxacin,CPFX)属于氟喹诺酮类广 谱高效抗菌药物，是获美国FDA批准在美国上市的第二个喹诺酮类药 物。对革兰氏阴性菌有很强的杀伤力，能够抑制细菌的DNA螺旋酶， 抗菌谱广、低毒、组织穿透力强,被用于兽医临床和畜牧生产中,用来 治疗动物疾病。但在实际应用中由于盲目及不合理应用，导致了耐药 菌株的出现，并且在动物组织内的残留经食物链进入人体后，对人体 具有潜在的致癌性，会对人体的健康造成威胁。许多国家已经对其休 药期和在畜产品中的最高残留限量做出了限制。因此，环丙沙星在动 物产品中的残留已成为一个必检项目。这就迫切需要建立一套快速检 测残留的方法。本研究的是通过制备环丙沙星单克隆抗体(Monoclonal antibody,mAb),并对其免疫学特性进行鉴定，为畜产品 中环丙沙星残留检测阻断 ELISA 试剂盒做技术支持， 使之应用于动物 性食品质量监测， 从而保障动物性食品安全。本试验采用碳二亚胺法 (EDC)将载体蛋白牛血清白蛋白(Bovine serum albumin, BSA和鸡卵 白蛋白(Ovalbumin, OVA)分别与CPFX偶联合成免疫抗原CPFX-BSA和 包被抗原CPFX-OVA,经紫外(UV)扫描和聚丙烯酰胺凝胶电泳 (SDS-PAGE)鉴定，表明人工抗原偶联成功;用免疫抗原免疫BALB/c小 鼠,免疫的5只小鼠血清抗体效价均达到了1:105左右,其中4号小鼠 血清效价最高并且半数抑制浓度(50% inhibiting concen tra tion,IC50)最低(17.21 ug/L);通过细胞融合技术并且应 用间接ELISA和阻断ELISA筛选分泌CPFX mAb的杂交瘤细胞株，细胞 融合后筛选出1株敏感特异的杂交瘤细胞株，命名为3D2;采用体内诱 生腹水法制备CPFX mAb,并对其染色体核型、效价、敏感性和特异性 等免疫学特性进行鉴定。经鉴定，其细胞培养上清效价为1:1.28X103, 腹水效价为1:1.6X105。其对CPFX的IC50为9.95 ug/L,对恩诺沙 星的交叉反应率为 128.39%, 与其它同系的喹诺酮类抗生素的交叉反 应率很低。本实验获得了高价、敏感、特异的抗CPFX mAb,为CPFX 残留快速检测ELISA试剂盒奠定了基础。【英文摘要】The ciprofloxacin (CPFX) belongs to the fluoroquinolone (FQNs) group. As the second antibacterial agent of the FQNs group that was permeated to sale. It can efficiently damage the bacteria of Gram-positiv through inhibiting the DNA helicase. It has the advantages of broad-spectrumofantibacterial, lowtoxin, broadly spread in the body.Soitwasappliedwidely especially inthe clinicof animals and production of farming, Use to treat digestive infections in farming industries as feed additives.The wide spreaduseofantibioticsinfood-producinganimalshascaused public concern due to the transfer of antibiotic-resistant bacteriatoman, It has the latent cause cancer and effect the healthofhuman.Inmanycountries,withdrawalperiodforCPFX and maximum residue limits (MRLs) in the food-producing animals have been set. So the CPFX detecting in tissues has become a necessary content. However, it demand we build a quickly detection for the ciprofloxacin residue.The objective of the paper is generate monoclonal antibodies (mAb) against CPFX and identify its immunological traits. In order to provide the technology support for detection blocking ELISA kit of CPFX residue in livestock products The CPFX was conjugated to carrier protein BSA and OVA by EDC to obtain Immunogen and coating antigen. The ultraviolet scanning and SDS-PAGE were used to identify CPFX artificial antigen. The results of scanning and SDS-PAGE showed that artificial antigen was synthesized successfully. Then immunized BALB/c mice with immunogen. Five mice was indirect ELISA titer against CPFX were all above 1:105 and the IC50 of No. 4 mice was the lowest (17.21ug/L). The hybridoma lines that secrete monoclonal antibody against CPFX (CPFX mAb) were established through cell fusion and indirect ELISA and blocking ELISA. One hybridoma cell line of 3D2 was screened for specificity to CPFX after cell fusing. CPFX mAb was generated by inducing ascites in mice. The immunological characteristics such as Karyotype, titer, sensitivity and specificity of the mAb were characterized. The indirect ELISA titer of the mAb was 1:1.28X 103 in supernatant, 1:1.6X105 in asci tes. The mAb of 3D2 showed high sens itivity with an IC50 of 9.95 ug/L to CPFX, The rate of cross reaction to Enrofloxacin could reach to 128.39%. There were low reactions to other fluoroquinolones antibiotics.The high-titer, sensitive and specific CPFX mAb has been generated and laid a solid foundation for the detection of ELISA kit of CPFX residue.【关键词】环丙沙星 单克隆抗体 ELISA 杂交瘤【英文关键词】CiprofloxacinMabELISAHybridoma 【目录】环丙沙星单克隆抗体的制备及其免疫学特性的鉴定摘要 6-7ABSTRACT 7-8第一章 文献综述12-231.1 喹诺酮类药物简介12-151.1.1 结构和类型 12-131.1.2 理化性质 131.1.3 作用机理和临床应用 131.1.4 耐药性 13-151.2 环丙沙星药物概述15-171.2.1 环丙沙星结构和性质 151.2.2 环丙沙星的作用机理和临床应用 15-161.2.3 环丙沙星的耐药性161.2.4 环丙沙星的残留及危害 16-171.3 兽药残留的现状 17-201.3.1 兽药残留的种类 171.3.2 兽药残留的成因 17-181.3.3 兽药残留的危害 18-201.3.4 针对兽药残留应采取的对策 20 1.4 环丙沙星残留检测技术20-211.4.1生物测定法(MIA)211.4.2理化分离法211.4.3免疫学分析法(IA)211.5本课题的目的和意义 21-23 第二章 人工抗原的合成及环丙沙星多克隆抗体的 制备 23-302.1 材料 232.1.1 主要试剂232.1.2 试验仪器与设备 232.1.3 实验动物232.2 方法 23-252.2.1 人工抗原合成和鉴定23-242.2.2 多克隆抗体的制备 24-252.3 结果和分析 25-282.3.1 人工抗原合成的鉴定 25-262.3.2CPFX多克隆抗体间接ELISA效价测定 26-272.3.3 CPFX多抗血清的敏感性鉴定 27-282.4 讨论 28-292.4.1 人工抗原的合成 282.4.2 完全抗原的鉴定 28-292.4.3动物免疫方案的选择 292.5 小结 29-30 第三章 环丙沙星单克隆抗体杂交瘤细胞株的建立 30-373.1 材料303.1.1 主要试剂 303.1.2 试验仪器与设备303.1.3 实验细胞 303.2 方法 30-333.2.1细胞融合备用小鼠的选择 30-313.2.2 SP2/0 骨髓瘤细胞的复苏和培养 313.2.3 脾细胞的制备 313.2.4 细胞融合 31-323.2.5 融合细胞的培养 323.2.6 阳性杂交瘤细胞的筛选 32-333.2.7 阳性杂交瘤细胞的亚克隆333.3 结果和分析 33-353.3.1 SP2/0 细胞的培养33-343.3.2 细胞融合 34-353.3.3 阳性杂交瘤细胞的筛选与克隆 353.4 讨论 35-363.4.1 细胞融合35-36 3.4.2 阳性杂交瘤细胞的筛选及克隆 36 3.5 小结 36-37 第四章 环丙沙星单克隆抗体的制备及其免疫学特性的鉴定 37-42 4.1 材料 374.1.1 主要试剂374.1.2 主要仪器设备374.1.3 试验动物374.2 方法 37-394.2.1单克隆抗体的生产37-384.2.2 免疫学特性的鉴定38-39 4.3 结果和分析 39-41 4.3.1 杂交瘤细胞核型分析 39 4.3.2 细胞上清和腹水效价测定 39-40 4.3.3 敏感性测定 40 4.3.4特异性测定40-414.4 讨论 414.4.1 单克隆抗体的大量制备 414.4.2 抗体特异性 414.5 小结41-42结论42-43 参考文献43-47 附录47-51致谢51-52 作者简介52【住采买全文皀】1.3.99388.481_3 8 1_13.7 2.1同时提供论文写作一对一辅导和论文发表服务.保过包发.说明】本文仅为中国学术文献总库合作提供无涉版权作者如有异仪请与总库或学校联茅