直接竞争ELISA检测拟柱孢藻毒素的方法建立和评价

杨丹; 刘金平; 赖淑燕; 肖利娟; 雷腊梅

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中国环境科学 ›› 2023, Vol. 43 ›› Issue (10) : 5543-5549.

环境微生物

直接竞争ELISA检测拟柱孢藻毒素的方法建立和评价

杨丹, 刘金平, 赖淑燕, 肖利娟, 雷腊梅

作者信息 +

Establishment and assessment of a competitive direct enzyme-linked immunosorbent assay (cdELISA) for the determination of cylindrospermopsin

YANG Dan, LIU Jin-ping, LAI Shu-yan, XIAO Li-juan, LEI La-mei

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文章历史 +

摘要

拟柱孢藻毒素（CYN）对人和动物具有高毒性而在世界上广受关注，快速检测方法的建立可为蓝藻毒素的风险评估提供有效的技术手段.本研究基于前期制备的CYN的单克隆抗体N8，旨在建立一种CYN的快速免疫检测方法-直接竞争ELISA.在对HRP-CYN酶标记物和N8单抗的稀释度进行优化后，最适反应条件下建立的直接竞争ELISA在0~200ng/mL CYN浓度下呈现典型的S型反应曲线，该方法的灵敏度为0.069ng/mL，经logit-log拟合的线性范围为0.1~10ng/mL，检出限远低于WHO建议的0.7ng/mL CYN的安全限值.本研究建立的ELISA方法对水库水样和小球藻稀释液中加标回收率分别为80.44%~125.4%和52.97%~155.19%，在测定CYN时与商业Beacon试剂盒的检测结果高度一致.对大沙河水库中CYN的监测表明该毒素在水体中常年存在，但未超过WHO建议的安全限值.本研究发展的ELISA可实现水样和藻样中CYN的快速检测，为供水安全保障提供一种新的技术手段.

Abstract

Cylindrospermopsins(CYN) has been widely concerned in the world for its high toxicity to humans and animals. The establishment of a rapid detection method can provide an effective tool for the risk assessment of CYN. Based on the monoclonal antibody N8 against CYN prepared, a direct competitive ELISA has been developed. After optimization of the dilution of HRP-CYN conjugate and N8 monoclonal antibody, the newly developed ELISA exhibited a typical sigmoidal response for CYN at concentrations of 0~200ng/mL, with a wide quantitative range between 0.1 and 10ng/mL by using logit-log method for fitting the curve. The detection limit of the method was calculated to be 0.069ng/mL, which is well below guideline value of 0.7ng/mL CYN recommended by WHO. Recoveries ranging from 80.44% to 125.4% and 52.97% to 155.19% were observed upon analyzing CYN-spiked water samples and Chlorellae diluent, respectively. Moreover, CYN concentrations obtained from our cdELISA method through testing different samples was highly consistent with the Beacon ELISA kit. It was found that CYN can be detected year around in Dashahe reservoir but never beyond the guideline value of 0.7ng/mL. Therefore, the developed ELISA will achieve the rapid detection of CYN in water samples and algae samples and may provide a reliable technical tool for drinking water safety.

导出引用

杨丹, 刘金平, 赖淑燕, 肖利娟, 雷腊梅. 直接竞争ELISA检测拟柱孢藻毒素的方法建立和评价[J]. 中国环境科学. 2023, 43(10): 5543-5549

YANG Dan, LIU Jin-ping, LAI Shu-yan, XIAO Li-juan, LEI La-mei. Establishment and assessment of a competitive direct enzyme-linked immunosorbent assay (cdELISA) for the determination of cylindrospermopsin[J]. China Environmental Science. 2023, 43(10): 5543-5549

中图分类号： X171.5 X835

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