环丙沙星论文：环丙星单克隆抗体的制备及其免疫学特性的鉴定.docVIP

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)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 concentration,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1:1.28×103,腹水效价为1:1.6×105。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-spectrum of antibacterial, low toxin, broadly spread in the body. So it was applied widely especially in the clinic of animals and production of farming, Use to treat digestive infections in farming industries as feed additives.The wide spread use of antibiotics in food-producing animals has caused public concern due to the transfer of antibiotic-resistant bacteria to man, It has the latent cause cancer and effect the health of human. In many countries, withdrawal period for CPFX 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