ok荞麦过敏原特异性抗体酶联免疫吸附法检测试剂制备.pdfVIP

※技术应用 食品科学 2011, Vol. 32, No. 20 32 7 荞麦过敏原特异性抗体酶联免疫 吸附法检测试剂制备 1 2 2 张 昕 ，崔晓东 ，王转花 ( 1.山西大学环境科学与工程研究中心，山西太原 030006 ； 2.山西大学生物技术研究所，山西太原 030006) 摘 要：以天然苦荞过敏蛋白包被聚苯乙烯微孔板，以鼠抗人IgE 抗体标记辣根过氧化物酶，以间接酶联免疫吸 附法(indirect enzyme-linked immunosorbent assay ，IELISA)检测病人血清中的特异性抗体，分析并建立关键操作步 骤及反应条件。结果表明：ELISA 反应的最适工作条件为包被的抗原质量浓度100 μg/mL 、阳性血清稀释度1:50 、 酶标二抗稀释度1:1000 ，37 ℃时封闭的最佳时间为4 0min ，包被抗原与阳性血清中抗体的最佳作用时间为4 0min ， 阳性血清与酶标二抗的最佳作用时间为4 0 m in 。本方法具有良好的特异性、重复性和精密度。 关键词：苦荞；过敏蛋白；试剂盒；间接酶联免疫吸附法 Preparation of an ELISA Method for Determining Buckwheat Allergen 1 2 2 ZHANG X in ，CUI X iao-dong ，WAN G Zhu an -hua (1. Research Center for Environmental Science and Engineering, Shanxi University, Taiyuan 030006, China ； 2. In stitute of Biotechnology, Shanxi University, Taiyuan 030006, China) Abstract ：An indirect enzyme-linked immunosorbent assay (IELISA) was proposed to detect specific antibodies in tartary buckwheat allergenic patients, sera using polystyrene micro-well plate coated with natural tartary buckwheat allergenic proteins and horseradish peroxidase (HRP)-labeled mouse anti-human IgE. Key operation s and reaction conditions were optimized. The results showed that the optimal coating antigen concentration , positive serum dilution, HRP-labeled mou se anti-human IgE concentration, blocking time at 37 ℃, reaction time between coating antigen and antibodies in positive sera, and reaction time between positive sera and HRP-la