鹅膏毒肽酶联免疫试剂盒研制及其应用  

Development and Application of Amanitin Enzyme⁃linked Immunosorbent Assay Kit

作　　者：谭建林 马雪涛 冯雷 方淑兵 张会香 牛之瑞 彭珍华 TAN Jianlin;MA Xuetao;FENG Lei;FANG Shubing;ZHANG Huixiang;NIU Zhirui;PENG Zhenhua(Yunnan Institute of Product Quality Supervision and Inspection,National Tropical Agricultural Byproducts Quality Inspection and Testing Center,Kunming 650223,Yunnan,China;Qingdao Pribolab Bioengineering Co.,Ltd.,Qingdao 266001,Shandong,China;Kunming Medical University,Kunming 650500,Yunnan,China)

机构地区：[1]云南省产品质量监督检验研究院/国家热带农副产品质量检验检测中心,云南昆明650223 [2]青岛普瑞邦生物工程有限公司,山东青岛266001 [3]昆明医科大学,云南昆明650500

出　　处：《食品研究与开发》2025年第7期135-140,163,共7页Food Research and Development

基　　金：国家市场监督管理总局技术保障专项项目(2022YJ25);云南省市场监督管理局科技计划项目(2023YSJK03)。

摘　　要：基于酶联免疫吸附原理,开展蘑菇中鹅膏毒肽酶联免疫试剂盒的研制。采用N-羟基琥珀酰亚胺和1-(3-二甲氨基丙基)-3-乙基卡巴朴胺作为活化剂,制备鹅膏毒肽半抗原,并进一步与钥孔帽贝血蓝蛋白和牛血清蛋白偶联制备免疫原和包被原,再利用免疫原免疫Balb/c小鼠来制备单克隆抗体。通过对抗原最佳包被量以及酶标抗体最佳稀释倍数等进行优化,研制鹅膏毒肽酶联免疫试剂盒,并对灵敏度、正确度、精密度进行评价。该试剂盒的线性范围为0.1~3.6 ng/mL,半抑制浓度(IC_(50))为0.5 ng/mL。试剂盒对于α-鹅膏毒肽、γ-鹅膏毒肽的交叉反应率分别为32%和38%,而结构类似物鬼笔毒肽的交叉反应率<0.50%,试剂盒具有良好的特异性。对于β-鹅膏毒肽的添加回收率为101.0%~106.0%,批内变异系数≤5.22%,批间变异系数≤9.24%。该试剂盒适用于蘑菇中鹅膏毒肽的现场筛查和快速检测。Based on the principle of enzyme-linked immunosorbent assay(ELISA),an ELISA kit for detecting amanitin in mushrooms was developed.Using N-hydroxysuccinimide(NHS)and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(EDC)as activators,a semi-antigen of amanitin was prepared and then coupled with keyhole limpet hemocyanin(KLH)and bovine serum albumin(BSA)to prepare immunogens and coating antigens.The immunogens were used to immunize Balb/c mice,which were then used to produce monoclonal antibodies.Through optimization of the optimal antigen coating concentration and the optimal dilution ratio of enzyme-linked antibodies,an ELISA kit for amanitin was successfully developed.The sensitivity,accuracy,and precision of the kit were evaluated.The linear range of the reagent kit was 0.1-3.6 ng/mL,with a half-maximal inhibitory concentration(IC_(50))of 0.5 ng/mL.The cross-reactivity of the kit forα-amanitin andγ-amanitin was 32%and 38%,respectively,while the cross-reactivity with the structurally similar phalloidin was less than 0.50%,demonstrating good specificity of the kit.The recovery rate for addedβ-amanitin was between 101.0%and 106.0%,with an intra-run coefficient of variation(CV)of≤5.22%and an inter-run CV of≤9.24%.This is reagent kit for on-site screening and rapid detection of amanita toxin peptides in mushrooms.

关 键 词：蘑菇 鹅膏毒肽 酶联免疫吸附 试剂盒 快速检测 

分 类 号：TS201.3[轻工技术与工程—食品科学]