草甘膦人工抗原的制备及兔源多抗的ELISA鉴定

doi:10.7506/spkx1002-6630-201704011

食品科学 ›› 2017, Vol. 38 ›› Issue (4): 65-69.doi: 10.7506/spkx1002-6630-201704011

草甘膦人工抗原的制备及兔源多抗的ELISA鉴定

王晶，王耀，王方雨，邓瑞广，胡骁飞，余秋颖，郝俊芳，邢云瑞，侯玉泽

1.河南省农业科学院河南省动物免疫学重点实验室，河南郑州 450002； 2.河南科技大学食品与生物工程学院，畜禽疫病诊断与食品安全检测河南省工程实验室，河南洛阳 471023

出版日期:2017-02-25 发布日期:2017-02-28
基金资助:
“十二五”国家科技支撑计划项目（2014BAD13B05）；河南科技大学博士科研启动基金项目（13480062）；河南省基础与前沿项目（142300413222）

Preparation of Artificial Antigen and ELISA Identification of Rabbit Polyclonal Antiserum for the Detection of Glyphosate

WANG Jing, WANG Yao, WANG Fangyu, DENG Ruiguang, HU Xiaofei, YU Qiuying, HAO Junfang, XING Yunrui, HOU Yuze

1. Henan Key Laboratory of Animal Immunology, Henan Academy of Agriculture Sciences, Zhengzhou 450002, China; 2. Henan Engineering Laboratory of Livestock Disease Diagnosing and Food Safety Testing, College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471023, China

Online:2017-02-25 Published:2017-02-28

摘要/Abstract

摘要： 采用1-（3-二甲氨基丙基）-3-乙基碳二亚胺（1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride，EDC）法和戊二醛（glutaraldehyde，GA）法，合成了草甘膦（N-(phosphonomethyl) glycine，PMG）人工抗原，通过免疫制备高敏感性、特异性的兔PMG多克隆抗血清并进行鉴定。将PMG分别与牛血清白蛋白（bovine serum albumin，BSA）和鸡卵清白蛋白（ovalbumin，OVA）偶联，合成免疫抗原PMG-BSA和包被抗原PMG-OVA。经紫外扫描、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳鉴定后，免疫新西兰大白兔，制备多抗。利用间接酶联免疫吸附（enzyme linked immunosorbent assay，ELISA）方法测定多抗效价，间接竞争ELISA方法测定血清的敏感性和特异性。结果表明，免疫的3 只兔血清效价均达1∶104以上，2号兔多抗效果最好，半数抑制浓度（IC50）为30.9 μg/mL，且具备良好的特异性。本实验成功合成了PMG人工抗原，并制得了敏感性高、特异性好的兔多抗，为PMG的免疫学快速检测方法的提供参考。

关键词: 草甘膦, 人工抗原, 多抗, 酶联免疫吸附检测

Abstract: To obtain polyclonal antiserum with high sensibility and specificity, artificial antigens were synthesized by coupling glyphosate (PMG) with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) separately by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and glutaraldehyde (GA) methods in this study. As a result, the immune antigen PMG-BSA and detection antigen PMG-OVA were obtained and identified by UV spectroscopy and SDS-PAGE. New Zealand white rabbits were immunized with PMG-EDC-BSA and PMG-GA-BSA, respectively to obtain polyclonal antisera. The titer of the antisera was determined by indirect ELISA and their sensibility and specificity were determined by indirect competitive ELISA. The results showed that the titer of antiserum of all three immunized rabbits was higher than 1:104. The antiserum of rabbit 2 exhibited the highest sensibility with an IC50 of 30.9 μg/mL and good specificity. The successful synthesis of artificial antigens and the obtainment of PMG polyclonal antiserum with high sensibility and specificity may lay the foundation for rapid immunological detection of PMG.

Key words: glyphosate, artificial antigen, polyclonal antiserum, ELISA

中图分类号:

S482.4

王晶，王耀，王方雨，邓瑞广，胡骁飞，余秋颖，郝俊芳，邢云瑞，侯玉泽. 草甘膦人工抗原的制备及兔源多抗的ELISA鉴定[J]. 食品科学, 2017, 38(4): 65-69.

WANG Jing, WANG Yao, WANG Fangyu, DENG Ruiguang, HU Xiaofei, YU Qiuying, HAO Junfang, XING Yunrui, HOU Yuze. Preparation of Artificial Antigen and ELISA Identification of Rabbit Polyclonal Antiserum for the Detection of Glyphosate[J]. FOOD SCIENCE, 2017, 38(4): 65-69.

[1]	杨梅，孙思，刘文锋，王安波，潘承丹，汪俭. 超高效液相色谱-串联质谱法测定茶叶中草甘膦和草铵膦的残留量[J]. 食品科学, 2019, 40(10): 337-343.
[2]	王静，马宁宁，宋洋. 杂环胺人工抗原的合成以及多克隆抗体的制备[J]. 食品科学, 2017, 38(4): 45-50.
[3]	王晓芬，鄢爱平，谭婷，毛雪金，刘俊俊，郭岚，万益群. 多效唑人工抗原的合成与鉴定[J]. 食品科学, 2016, 37(7): 134-139.
[4]	赖丹，郑盛武，赵肃清. 4种邻苯二甲酸酯塑化剂人工抗原的合成及免疫检测[J]. 食品科学, 2016, 37(5): 114-120.
[5]	肖石妹，鄢爱平，肖芳，万益群，郭岚. 氯吡脲人工抗原的合成与鉴定[J]. 食品科学, 2016, 37(21): 183-188.
[6]	吴春生，马良，胡媛媛，张宇昊. 真菌毒素人工抗原制备方法研究进展[J]. 食品科学, 2014, 35(5): 239-245.
[7]	刘儒彪，胡骁飞，张改平，王方雨，邓瑞广，刘璐，贾国超，刘玺. 甲硝唑人工抗原的合成及鼠源多抗的制备[J]. 食品科学, 2014, 35(19): 185-188.
[8]	王吕，邵辉峰，张斌，陈雪岚. 人工抗原苯并芘的化学合成与分析[J]. 食品科学, 2014, 35(19): 53-56.
[9]	江涛，胡媛媛，马良，张宇昊，吴春生. 细交链孢菌酮酸人工抗原的制备[J]. 食品科学, 2014, 35(19): 153-157.
[10]	奚茜李沐洁龚云飞陈宗伦方结红王唯芬张明洲. 链霉素人工抗原及多克隆抗体的制备与鉴定[J]. 食品科学, 2013, 34(5): 181-185.
[11]	邓发亮，刘晓云，唐时幸，王继华. 莱克多巴胺人工抗原的合成与鉴定[J]. 食品科学, 2013, 34(18): 146-149.
[12]	陈?臣1，罗顺菁1,*，刘成梅1，邹常春2，汪志宇1，王文飞1. 对氯甲基苯甲酸链接的莱克多巴胺人工抗原的合成与鉴定[J]. 食品科学, 2013, 34(1): 180-184.
[13]	吴昊，戴彩霞，刘佳，何计国. 盐酸西布曲明人工抗原的合成与鉴定[J]. 食品科学, 2012, 33(3): 140-145.
[14]	陈兴龙，徐?玲，刘仁荣*，裘雪梅，朱立鑫 . 基于噬菌体展示技术的赭曲霉毒素A高密度模拟表位的表达研究[J]. 食品科学, 2012, 33(19): 188-192.
[15]	王梅，徐乃丰，刘丽强，匡华，林菲，宋珊珊，王泽路，胥传来. 孔雀石绿人工抗原的合成及多克隆抗体的制备[J]. 食品科学, 2011, 32(7 ): 282-285.

草甘膦人工抗原的制备及兔源多抗的ELISA鉴定

Preparation of Artificial Antigen and ELISA Identification of Rabbit Polyclonal Antiserum for the Detection of Glyphosate

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价