四种重要生物恐怖毒素液相芯片多重检测方法的建立

   徐华1，顾大勇2，胡春凌3，李永进4，史蕾2，刘春晓2，赵纯中2，张妮奇2，徐云庆2，姜声扬1

    1.南通大学公共卫生学院职业卫生与环境毒理学教研室，江苏 南通226000；2.深圳出入境检验检疫局；

    3.深圳博睿祥晖生物技术有限公司；4.深圳博尔美生物科技有限公司

    摘要：目的 建立包括蓖麻毒素B（Ricin toxion B,RTB）、肉毒梭菌毒素A（Clostri botulinum toxin A,CBTA）、金黄色葡萄球菌肠毒素B（Staphylococcal enterotoxin B,SEB）及产气荚膜毒素ε（Clostridium perfringens toxin ε,CPTε）在内的4种重要生物恐怖因子的多重液相芯片检测方法。方法采用双抗夹心法原理和液相芯片技术平台，通过优化偶联抗体浓度和检测抗体浓度以及抗原抗体最佳孵育时间，建立4种生物恐怖因子液相芯片多重检测方法。结果反应条件优化实验结果表明，RTB、CBTA、SEA和CPTε偶联抗体的最佳用量分别为20 μg、40 μg、80 μg、80μg；RTB、CBTA、SEA和CPTε检测抗体的最佳稀释比分别为1∶5000、1∶1000、1∶2000、1∶4000；4种毒素抗原抗体最佳孵育时间为60min。多重检测结果表明，本研究建立的毒素液相芯片多重检测方法可有效检测上述任意1种毒素、任意2种毒素组合、任意3种毒素组合并能同时检测4种毒素。灵敏度检测结果表明，RTB、CBTA、SEA、CPTε检测灵敏度分别为1ng/ml、10 ng/ml、5 ng/ml、10 ng/ml。结论本研究建立的4种常见生物恐怖毒素液相芯片多重检测方法通过1次检测能同时检测RTB、CBTA、SEA、CPTε，是一种准确、灵敏、快速和高通量的检测方法。

   关键词：蓖麻毒素B；肉毒梭菌毒素A；金黄色葡萄球菌肠毒素B；产气荚膜毒素ε；液相芯片；生物恐怖；毒素；检测

    中图分类号：R446　文献标识码：B

    Establishment of a multiplex luminex-baseddetection method for four

    kinds of important biological terrorismtoxins

    XU Hua*，GU Da-yong，HU Chun-ling，LIYong-jin，SHI Lei，LIU Chun-xiao，

    ZHAO Chun-zhong，ZHANG Ni-qi，XU Yun-qing，JIANGSheng-yang

    *School of Public Health，NantongUniversity，Nantong, Jiangsu 226000， China.

    Abstract:   Objective  To establish a luminex-based multiplex detection method for thesimultaneous detection of Ricin toxion B、Clostri botulinum toxinA、Staphylococcal enterotoxin B、Clostridium perfringens toxinε.   Methods   The luminex-based multiplexdetection was investigated on the double antibody sandwich theory.Through optimizing the concentration of the coupling and detectionantibody as well as the incubation time of antigen and antibody, Aluminex-based multiplex detection method for four importantbiological terrorism toxins was established eventually. Results   Optimization experimental results showed thatthe optimum dosage for the coupling antibodies of RTB, CBTA, SEAand CPTε was 20 μg, 40 μg, 80 μg, 80 μg respectively. The optimumdilution ratio of the detection antibody of RTB, CBTA, SEA and CPTεwas 1∶5000, 1∶1000, 1∶2000, 1∶4000, respectively. The bestincubation time of the antigen and antibody was 60min. Multipletest results indicated that the multiplex luminex-based detectionmethod developed in this study could be used to detect the abovetoxins include any one, any two, any three of toxins combination aswell as the four toxins combination at the same time. Sensitivitytest results showed that the detection sensitivity of RTB., CBTA,SEA, CPT ε was 1 ng/ml, 10 ng/ml, 5 ng/ml, 10 ng/ml, respectively.Conclusion   RTB, CBTA, SEA, CPT ε could be detected bythis multiplex luminex-based detection method in one tube throughone test. And it is a accuracy, sensitive, rapid, high throughputdetection method.

    Key words:   Ricin Toxion B；ClostriBotulinum toxin A；Staphylococcal Enterotoxin B；Clostridiumperfringens toxin ε；Luminex Assay; Biological terrorism; Toxin;Detection

    《中国国境卫生检疫杂志》2012年6月刊