头孢曲松单克隆抗体的制备及ELISA方法的初步建立

发布时间：2019-05-15 17:42

【摘要】：头孢曲松(Ceftraxone,CTRX)是第三代头孢类抗生素,其抗菌谱广,抑菌能力强,常被用于动物感染性疾病的治疗。头孢曲松的滥用,易导致人与动物多种系统的疾病,以过敏反应和结石症最为严重。头孢曲松为人用抗生素,目前已被农业部列为禁止用于动物饲养过程的人用药品。目前检测头孢类抗生素残留可采用理化检测法、微生物检测法和免疫学检测法。理化检测法所需仪器设备价格昂贵、专业技术性强及前处理过程繁琐,以上因素决定了此法并不适用于基层大量样品的检测。微生物检测法具有细菌培养耗时较长、特异性不高、准确度低等缺点。免疫学检测法,以酶联免疫吸附法(ELISA)为主,基于抗原抗体的特异性结合,其特异性强、灵敏度高、操作方法简单,适用于大量样品的筛选。1.CTRX完全抗原的合成及鉴定。本研究采用戊二醛法合成两种完全抗原。完全抗原经紫外光谱扫描及SDS-PAGE凝胶电泳分析鉴定,可以初步判定完全抗原偶联成功。完全抗原经免疫小鼠、细胞融合及ELISA测定,最终证明两种完全抗原制备是成功的。利用BCA法测得CTRX-BSA和CTRX-OVA蛋白浓度分别为5.5 mg/mL和5.0 mg/mL。2.CTRX单抗制备。采用常规免疫方案免疫BALB/c小鼠,免疫原使用人工抗原CTRX-BSA,ELISA检测包被原使用CTRX-OVA。在小鼠五免后第7d尾静脉采血,检测血清抗体效价及其药物抑制情况,血清效价达1:16000以上。通过细胞融合技术、ELISA筛选方法和有限稀释亚克隆法获得两株可持续分泌针对CTRX单克隆抗体的杂交瘤细胞株,分别命名为3E7和7A7。通过小鼠体内诱生法制备腹水,共18 mL,BCA法测其蛋白浓度分别为20.93 mg/mL 和 21.47 mg/mL;效价分别为 32000 和 128000。采用 HiTrap Protein G HP 纯化柱纯化1mL 7A7腹水,得到纯化后的7A7腹水约5 mL,纯化后的蛋白浓度为1.1 mg/mL,蛋白回收率为5.1%。SDS-PAGE凝胶电泳结果显示,腹水纯化效果良好。交叉实验结果表明,该CTRX单抗与头孢氨苄、头孢噻呋、头孢拉定、头孢噻肟、头孢噻吩几乎没有交叉反应,且对CTRX的抑制率为100%。3.ELISA检测方法的建立。利用纯化后的7A7腹水建立检测CTRX残留的CiELISA方法。包被原最佳稀释浓度1:3000,纯化后7A7抗体的工作浓度1:16000。CTRX浓度在1.72～2000 ng/mL时,标准曲线线性关系良好。其线性方程为y=0.2541x-0.0184(R2=0.9875),IC50为108.68 ng/L,LOD 为 1.72 ng/mL,LOQ 为 13.12 ng/mL。4.猪肉中CTRX添加回收实验使用猪肉进行添加回收实验,猪肉样品经过一系列前处理后用于实验测定。实验结果显示样品处理液稀释8倍以上时,基本可以消除样品基质的干扰。在20～1000 ng/mL药物浓度范围内,线性关系良好,线性方程为y=0.4466x-0.4738,相关系数R2=0.9869,IC50 为 150.52 ng/mL,LOD 为 16.38 ng/mL,LOQ 为 44.53 ng/mL。用 50、400、800 ng/mL的CTRX标准品溶液做添加回收实验,经检测猪肉样品回收率在81%～98.7%之间。
[Abstract]:Ceftriaxone (Ceftraxone,CTRX) is the third generation of cephalosporin antibiotics. Ceftriaxone (Ceftriaxone) is often used in the treatment of animal infectious diseases because of its wide antibacterial spectrum and strong bacteriostatic ability. The abuse of ceftriaxone is easy to lead to many diseases of human and animal systems, especially allergic reaction and stone formation. Ceftriaxone, a human antibiotic, has been listed by the Ministry of Agriculture as a human drug banned from animal feeding. At present, physicochemical detection, microbial detection and immunological detection can be used to detect cephalosporin antibiotic residues. The instrument and equipment needed for physical and chemical detection are expensive, professional and technical, and the pretreatment process is tedious. The above factors determine that this method is not suitable for the detection of a large number of samples at the base level. Microbial detection has many disadvantages, such as long time consuming, low specificity and low accuracy. Immunological detection, mainly enzyme-linked immunosorbent assay (ELISA), is based on the specific binding of antigen and antibody, which has strong specificity, high sensitivity and simple operation method, and is suitable for screening a large number of samples. 1. Synthesis and identification of CTRX complete antigen. In this study, two complete antigens were synthesized by glutaraldehyde method. The complete antigen was identified by UV spectrum scanning and SDS-PAGE gel electrophoresis analysis, and the complete antigen coupling success could be preliminarily determined. The complete antigen was successfully prepared by immunizing mice, cell fusion and ELISA assay. The concentrations of CTRX-BSA and CTRX-OVA proteins were determined by BCA method to be 5.5 mg/mL and 5.0 mg/mL.2.CTRX monoclonal antibodies, respectively. BALB/c mice were immunized with routine immunization regimen, and the immunogen was detected by artificial antigen CTRX-BSA,ELISA and coated with CTRX-OVA.. On the 7th day after the fifth immunization, the blood samples were collected from the tail vein of mice. The serum antibody titer and its drug inhibition were detected. The serum titer was more than 1 鈮，

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