研究报告
李树莹,唐云飞,盛建武,刘丽,何苗.恩诺沙星完全抗原的合成及间接竞争ELISA方法的建立[J].环境科学学报,2017,37(8):2904-2910
恩诺沙星完全抗原的合成及间接竞争ELISA方法的建立
- Preparation of complete antigen and development of an indirect competitive ELISA for the detection of enrofloxacin in water samples
- 基金项目：国家高技术研究发展计划（No.2014AA06A506）；国家重大科学仪器设备开发专项（No.2012YQ030111）
- 作者
- 单位
- 李树莹
- 清华大学环境学院, 环境模拟与污染控制国家联合重点实验室, 环境与健康传感技术研究中心, 北京 100084
- 唐云飞
- 清华大学环境学院, 环境模拟与污染控制国家联合重点实验室, 环境与健康传感技术研究中心, 北京 100084
- 盛建武
- 清华大学环境学院, 环境模拟与污染控制国家联合重点实验室, 环境与健康传感技术研究中心, 北京 100084
- 刘丽
- 清华大学环境学院, 环境模拟与污染控制国家联合重点实验室, 环境与健康传感技术研究中心, 北京 100084
- 何苗
- 清华大学环境学院, 环境模拟与污染控制国家联合重点实验室, 环境与健康传感技术研究中心, 北京 100084
- 摘要：为了建立环境新型抗生素类污染物恩诺沙星的免疫检测方法，采用间接竞争ELISA方法，用酶标仪测定酶标二抗与底物反应生成的有色产物量来确定恩诺沙星含量.研究了恩诺沙星完全抗原的合成方法，以及间接竞争ELISA的优化条件和实际水样的检测效果.结果表明，通过碳二亚胺法（EDC+NHS）成功合成了恩诺沙星的理想完全抗原，采用BCA法测定完全抗原浓度，紫外吸收法和MADLI-TOF MS法测得完全抗原的偶联比为14~16.建立的恩诺沙星间接竞争ELISA检测方法，检测限为1.92 μg·L-1，定量检测区间为7.33~238.24 μg·L-1.采用优化后的实验条件测定了清华大学地下水和饮用水配制的恩诺沙星加标样品，发现所有样品的变异系数和回收率分别为3%~11%和82%~122%，表明建立的间接竞争ELISA方法适用于实际水样中恩诺沙星的快速简便检测.
- Abstract：An indirect competitive enzyme-linked immunosorbent assay (ELISA) was established for the detection of enrofloxacin, an emerging environmental pollutant, in water samples. In our method, the peroxidase conjugated secondary antibody was used for signal amplification and the amount of enrofloxacin was determined by scanning the optical density of the plate. The carbodiimide method (EDC+NHS) was employed to synthesize the complete antigen of enrofloxacin. The concentration of the synthesized complete antigen was measured by the BCA method. The conjugation ratio of the complete antigen is between 14 and 16 determined by both the ultraviolet absorption and MADLI-TOF MS. The limit of detection (S/N=3) is 1.92 μg·L-1 and the quantitative detection range is 7.33~238.24 μg·L-1. The overall coefficients of variation and recoveries respectively in the ranges of 3%~11% and 82%~122% were obtained when the enrofloxacin-spiked groundwater and drinking water collected from Tsinghua University were detected under optimized conditions. The results demonstrate that the established indirect competitive ELISA is suitable for the rapid and facile detection of enrofloxacin in water samples.
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