β-玉米赤霉烯醇单克隆抗体的研制及CiELISA检测方法的建立  

Preparation of β-zearalenol monoclonal antibody and establishment of CiELISA detection method

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作  者:朱根生 王凤丽[1,2] 夏苏干 原俊朝 邹辉 顾建红[1] 袁燕[1] 刘学忠[1] 刘宗平[1,2] 卞建春[1,2] ZHU Gen-sheng;WANG Feng-li;XIA Su-gan;YUAN Jun-zhao;ZOU Hui;GU Jian-hong;YUAN Yan;LIU Xue-zhong;LIU Zong-ping;BIAN Jian-chun(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区:[1]扬州大学兽医学院,江苏扬州225009 [2]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出  处:《中国兽医科学》2020年第11期1469-1474,共6页Chinese Veterinary Science

基  金:国家重点研发计划支持项目(2016YFD0501208);江苏高校优势学科建设工程资助项目(PAPD)。

摘  要:为建立一种针对β-玉米赤霉烯醇(β-ZOL)快速准确的检测方法,笔者用活性酯法将β-ZOL与牛血清白蛋白(BSA)和卵清白蛋白(OVA)偶联合成人工完全抗原,纯化鉴定后免疫BALB/c小鼠。经筛选最终获得1株稳定分泌抗β-ZOL抗体的细胞株1F5。利用该抗体建立检测β-ZOL的间接竞争酶联免疫吸附试验(CiELISA)方法。结果显示,β-ZOL的药物抑制标准曲线在浓度为5~500 ng/m L时线性关系良好,标准曲线方程为y=0.406 3x-0.204 2(R^2=0.990 4),IC_(50)为53.19 ng/m L,检测下限(LOD)为6 ng/m L。该单克隆抗体对β-ZOL的抑制率为100%,与α-玉米赤霉烯醇、玉米赤霉烯酮、β-雌二醇、呕吐毒素、伏马毒素等基本无交叉反应。牛奶样品β-ZOL添加回收试验显示,β-ZOL的回收率为91.24%~110.07%,变异系数为6.17%~8.73%。说明建立的检测β-ZOL的CiELISA方法稳定可靠。To establish a fast and accurate detection method forβ-zearalenol(β-ZOL),the experiment used active ester method to coupleβ-ZOL with bovine serum albumin(BSA)and ovalbumin(OVA)and synthesize artificial complete antigen.After purification and identification,BALB/c mice were immunized.Obtaining from the screening,the anti-β-ZOL antibody cell line 1F5 was used to establish an indirect competitive enzyme-linked immunosorbent assay(CiELISA)method for detectingβ-ZOL.The results showed that theβ-ZOL drug inhibition standard curve had a good linear relationship at a concentration of 5 to 500 ng/mL.The standard curve equation was y=0.4063x-0.2042(R2=0.9904),IC50 was 53.19 ng/mL,and the lower detection limit(LOD)was 6 ng/mL.The inhibition rate of this monoclonal antibody toβ-ZOL was 100%.Moreover,no cross-reaction withα-zearalenol,zearalenone,β-estradiol,vomitotoxin,fumonisin,etc.was found.Milk sampleβ-ZOL addition recovery test showed that the recovery rate ofβ-ZOL was between 91.24%—110.07%,and the coefficient of variation was between 6.17%—8.73%.It showed that the established CiELISA method for detectingβ-ZOL was stable and reliable.

关 键 词:β-玉米赤霉烯醇 单克隆抗体 间接竞争酶联免疫吸附试验 回收试验 

分 类 号:S859.87[农业科学—临床兽医学]

 

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