蘑菇中鹅膏毒肽间接竞争ELISA检测方法的建立

Establishment of Indirect Competitive ELISA Method for Detecting Amanitin in Mushroom

目的:旨在建立一种检测蘑菇中鹅膏毒肽(Amanitin,AMA)的间接竞争酶联免疫吸附方法(Indirect competitive enzyme-linked immunosorbent assay,ic-ELISA)。方法:通过EDC/NHS在α-鹅膏毒肽的羧基位置引入6-氨基己酸得到半抗原,并进一步与不同的载体蛋白偶联制备免疫原和包被原。之后,利用免疫原免疫Balb/c小鼠制备单克隆抗体。基于所获得的抗体,并通过对包被原和抗体工作浓度、包被条件、封闭条件、酶标二抗工作浓度和孵育时间等条件的优化,建立了蘑菇中鹅膏毒肽间接竞争ELISA检测方法,最后对所建立方法的灵敏度、添加回收率、批内及批间变异等参数进行了评价。结果:合成的半抗原分子量为1033.12,经MALDI-TOF鉴定免疫原的偶联比约为10.03。基于杂交瘤技术制备、筛选出鼠单克隆抗体13H4的IC50为1.91μg/L。基于所获得单克隆抗体,所建立蘑菇中鹅膏毒肽间接竞争ELISA方法的检出限为0.88μg/kg,添加回收率为85.66%~113.05%,批内变异系数为5.35%~9.54%,批间变异系数小于15%。结论:本研究所建立的ic-ELISA方法准确度、精密度、灵敏度较高、性能稳定,为突发毒蘑菇中毒事件的毒原分析提供了一种简便、可靠的快速检测方法。

Objective:This study aims to establish an indirect competitive enzyme-linked immunosorbent assay for the detection of amanitin(AMA) in mushroom.Methods:In this study,the hapten was obtained by introducing 6-aminocaproic acid into the carboxyl position of the α-amanotropin molecule through EDC/NHS,and further coupled with different carrier proteins to prepare immunogens and coating antigen.Afterwards,Balb/c mice were immunized with the immunogen to prepare monoclonal antibodies.Based on the obtained antibody,an indirect competitive ELISA method for the detection of amanitin in mushroom was established by optimizing the working concentrations of coating antigen and antibody,coating conditions,blocking conditions,working concentrations of enzyme labeled secondary antibody and incubation time.Finally,the sensitivity,recovery rate,intra batch and inter batch variation of the established method were evaluated.Results:The molecular weight of the synthesized hapten in this study was 1033.12,and the coupling ratio of the immunogen identified by MALDI-TOF was about 10.03.Based on hybridoma technology,the IC50 of the mouse monoclonal antibody 13 H4 was 1.91 μg/L.Based on the obtained monoclonal antibody,the detection limit of the established ic-ELISA method for amanitin in mushroom was 0.88 μg/kg,the recovery rate was 85.66%~113.05%,the intraassay coefficient of variation was 5.35%~9.54%,and the inter-assay coefficient of variation was less than 15%.Conclusion:The ic-ELISA method established in this study had high accuracy,precision,sensitivity,and stable performance.It provided a simple,reliable and rapid detection method for the analysis of the toxicogen of sudden mushroom poisoning.