四种重要生物恐怖毒素液相芯片多重检测方法的建立

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 toxinε,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20μg、40μg、80μg、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1 ng/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。

Establishment of a multiplex luminex-based detection method for four kinds of important biological terrorism toxins

Objective To establish a luminex-based multiplex detection method for the simultaneous detection of Ricin toxion B, Clostri botulinum toxin A, Staphylococcal enterotoxin B, Clostridium perfringens toxin ε. Methods The luminex-based multiplex detection was investigated on the double antibody sandwich theory. Through optimizing the concentration of the coupling and detection antibody as well as the incubation time of antigen and antibody, A luminex-based multiplex detection method for four important biological terrorism toxins was established eventually. Results Optimization experimental results showed that the optimum dosage for the coupling antibodies of RTB, CBTA, SEA and CPTε was 20 ug, 40 ug, 80 ug, 80 ug respectively. The optimum dilution ratio of the detection antibody of RTB, CBTA, SEA and CPTe was 1：5000, 1： 1000, 1：2000, 1：4000, respectively. The best incubation time of the antigen and antibody was 60min. Multiple test results indicated that the multiplex luminex-based detection method developed in this study could be used to detect the above toxins include any one, any two, any three of toxins combination as well as the four toxins combination at the same time. Sensitivity test results showed that the detection sensitivity of RTB., CBTA, SEA, CPT 8 was 1 ng/ml, 10 ng/ml, 5 ng/ml, 10 ng/ml, respectively. Conclusion RTB, CBTA, SEA, CPT e could be detected by this multiplex luminex-based detection method in one tube through one test. And it is a accuracy, sensitive, rapid, high throughput detection method.