磺胺嘧啶单克隆抗体的制备与鉴定  

Preparation and Characterization of Sulfadiazine Monoclonal Antibodies

作　　者：郭晨 尚鹤婷 霍俊奇 王培征 栗慧 魏东[1,2,3] GUO Chen;SHANG Heting;HUO Junqi;WANGPeizheng;LI Hui;WEI Dong(Hebei Key Laboratory of Quality&Safety Analysis-Testing for Agro-Products and Food,Hebei North University,Zhangjiakou,Hebei 075000,China;College of Agriculture and Forestry Technology,Hebei North University,Zhangjiakou,Hebei 075000,China;Zhang Jiakou Key Laboratory of Quality&Safety for Charactenistics Agro-Products,Hebei Narth University,Zhangjiakou,Hebei 075000,China)

机构地区：[1]河北北方学院河北省农产品食品质量安全分析检测重点实验室,河北张家口075000 [2]河北北方学院农林科技学院,河北张家口075000 [3]河北北方学院张家口市特色农产品质量安全重点实验室,河北张家口075000

出　　处：《农产品加工》2024年第16期63-70,共8页Farm Products Processing

基　　金：河北北方学院省属高校基本科研业务费项目(JYT2022010)。

摘　　要：为提高动物源性食品中SD(Sulfadiazine,SD)药物残留免疫分析方法的特异性和灵敏度,建立快速高效便捷的免疫检测方法,制备特异性强的SD单克隆抗体。采用重氮化法偶联SD和牛血清白蛋白(Bovine serum albumin,BSA)及卵清白蛋白(OVA),制备SD免疫抗原和包被抗原。经过紫外-可见分光光度仪(UV-vis)和聚丙烯酰胺凝胶电泳(SDS-PAGE)法鉴定其偶联成功;免疫原使用4周龄的BALB/c小鼠7只;通过细胞融合、阳性筛选、亚克隆、制备腹水及纯化等过程制备SD单克隆抗体,建立间接竞争酶联免疫吸附试验(Indirect competitive enzyme linked immunosorbent assay,ic-ELISA)标准曲线。结果表明,SD单克隆抗体效价为1:(8×106),IC50为78.7 ng/mL,与磺胺一甲基嘧啶、磺胺二甲基嘧啶、磺胺甲噁唑交叉反应率分别为2.78%,0.16%,0.26%,与磺胺二甲氧嘧啶、磺胺间甲氧嘧啶交叉反应率均小于0.1%。经鉴定,单克隆抗体免疫球蛋白为鼠IgG的2b亚类。获得了抗SD的单克隆抗体,特异性较高,灵敏度较高,亲和力较好,与其他结构类似物交叉反应率较低,为后续制备SD快速检测试纸条奠定基础。To improve the specificity and sensitivity of the immunoassay method for the determination of sulfadiazine(SD)in food of animal origin,to establish a rapid,efficient and convenient immunoassay method,and to prepare a monoclonal antibody against sulfadiazine with high specificity.Sulfadiazine immunoantigens and detection antigens were prepared by coupling sulfadiazine with bovine serum albumin(BSA)and ovalbumin(OVA)using diazotisation.The successful coupling was identified by ultraviolet-visible spectrophotometry(UV-vis)and polyacrylamide gel electrophoresis(SDS-PACE);five 4-week-old BALB/c mice were used as immunogens;the monoclonal antibody to sulfadiazine was prepared through the process of cell fusion,positive screening,subcloning,preparation of ascites,and purification;and the indirect competition enzyme-linked immunosorbent assay(ic-ELISA)was established.The standard curve was established.The monoclonal antibody to sulfadiazine had a potency of 1:(8 x 10),an ICso of 78.7 ng/mL,a cross-reactivity with sulfamonomethylpyrimidine,dimethylpyrimethamine,and sulfamethoxazole of 2.78%,0.16%,and 0.26%,respectively,and a cross-reactivity with sulfamonyl-dimethoxypyrimidine,and sulfamonium mesomethylpyrimethamine,which were all less than 0.1%.The monoclonal antibody immunoglobulin was identified as subclass 2b of murine IgG.The monoclonal antibody against sulfadiazine was obtained,with high specificity,high sensitivity,good affinity and low cross-reactivity with other structural analogues,which can lay the foundation for the subsequent preparation of sulfadiazine rapid test strips.

关 键 词：磺胺嘧啶 单克隆抗体 完全抗原 间接竞争 

分 类 号：S859.84[农业科学—临床兽医学]