鹅膏毒素胶体金酶联免疫层析试纸条的研制及其应用

Development and application of colloidal gold enzyme-linked immunochromatographic test strip for amanitin detection

中文关键词:  鹅膏毒肽  胶体金  免疫层析试纸条  蘑菇

英文关键词:amanitin  colloidal gold  immunochromatographic test strip  mushroom

中文摘要:

      目的 基于免疫层析技术, 研制检测蘑菇中鹅膏毒素的酶联免疫试纸条。方法 采用N-羟基琥珀酰亚胺(N-hydroxysuccinimide, NHS)和1-(3-二甲氨基丙基)-3-乙基卡巴朴胺[N-(3-dimethylaminopropyl)- N’-ethylcarbodiimide hydrochloride, EDC]作为活化剂, 引入6-氨基己酸得到鹅膏毒素半抗原, 并进一步与钥孔帽贝血蓝蛋白(keyhole limpet hemocyanin, KLH)和牛血清蛋白(bovine serum albumin, BSA)偶联制备免疫原和包被原, 然后利用免疫原免疫Balb/c小鼠来制备单克隆抗体。通过制备胶体金和金标抗体, 优化包被抗原、二抗浓度及提取液, 成功研制鹅膏毒素胶体金免疫层析试纸条, 并对其进行交叉反应、稳定性、重复性、正确性进行了评价。结果 试纸条对α-鹅膏毒素、β-鹅膏毒素和γ-鹅膏毒素的检出限分别为100、20和50 μg/kg。灵敏度为100%, 假阳性率和假阴性率为0%, 且与现有仪器方法检测结果一致, 同其他蘑菇毒素无交叉反应。结论 该试纸条具有操作简便, 重复性高、稳定性好的特点, 适用于蘑菇中鹅膏毒素的现场筛查和快速检测。

英文摘要:

      Objective To develop an enzyme-linked immunochromatographic test strip for the determination of amanita toxin in mushroom based on immunochromatography. Methods N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) were used as activators, 6-aminohexanoic acid was introduced to obtain amanitin peptide hapten, and further conjugated with keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare immunogen and coating antigen. Then, the immunogen was used to immunize Balb/c mice to prepare monoclonal antibodies. The colloidal gold immunochromatographic test strip for amanitin was successfully developed by preparing colloidal gold and gold labeled antibody, optimizing the concentration of coating antigen, secondary antibody and extracting solution, and then its cross reactivity, stability, repeatability, and accuracy were evaluated. Results The limits of detection of α-amatoxin, β-amatoxin, and γ-amatoxin peptide were 100, 20 and 50 μg/kg, respectively. The sensitivity was 100%, the false positive rate and false negative rate were 0%, and the results were consistent with the existing instrumental methods without cross reaction with other mushroom toxins. Conclusion The test strip has the characteristics of simple operation, high repeatability and good stability, and is suitable for on-sites creening and rapid detection of amanitin in mushrooms.