BSA-ELISA初步检测短穗鱼尾葵花粉特异性IgE  

Detection of Caryota mitis Pollen Specific IgE with BSA-ELISA

作　　者：姚敏[1] 孟光[1] 刘志刚[2] 孔小丽[2] 

机构地区：[1]海南省海口市人民医院检验科,海口570208 [2]深圳大学医学院过敏反应与免疫学研究所,深圳518060

出　　处：《热带医学杂志》2009年第10期1134-1137,共4页Journal of Tropical Medicine

基　　金：国家自然科学基金(No.30070702);海南省自然科学基金(No.80570);广东省自然科学基金(No.07301310)

摘　　要：目的对短穗鱼尾葵花粉粗浸液的主要变应原进行分析、鉴定。方法通过SDS-PAGE分析短穗鱼尾葵花粉蛋白质组份,采用Western blotting鉴定主要变应原,以短穗鱼尾葵花粉粗浸液包板,摸索出其包被浓度、血清稀释度和酶结合物浓度,采用BSA-ELISA法对短穗鱼尾葵花粉过敏患者血清进行初步检测,并与皮肤挑刺试验比较。结果短穗鱼尾葵花粉粗浸液SDS-PAGE显示有30余条蛋白条带,其中主要蛋白条带有10条,Western blotting显示5例短穗鱼尾葵花粉过敏患者的混合血清能与其中3条蛋白条带起反应,分子量分别是26000、14000和12000Mr。BSA-ELISA检测短穗鱼尾葵花粉特异性IgE,最适粗浸液稀释度为1:100,血清稀释倍数为1:5,生物素化抗体为1:1000,辣根过氧化物酶标记的链霉亲和素(strepavidin-HRP)为1:1000。在此条件下BSA-ELISA与浸液皮试比较,检出结果与皮试阳性患者血清符合率为90%,与皮试阴性患者符合率为80%,与健康人对照检测符合率为100%。结论本实验对短穗鱼尾葵花粉主要变应原进行了分离和鉴定,BSA-ELISA法测定结果与皮肤挑刺试验初步比较符合率较好。Objective To characterize allergenic components of Caryota mitis pollen.Methods Extracts from Caryota mitis were prepared and analyzed by SDS-PAGE.Proteins bands were visualized by staining the gel with Coomassie blue.Antigenic properties of the separated proteins were analyzed by immuno-blotting.The best concentration of coating Caryota mitis extracts,serum dilutions and concentration of enzyme-conjugates were pretest in BSA-ELISA,which was used to detect the Caryota mitis pollen specific IgE.And the results were compared with those of skin prick test（SPT）. Results For the pollen extract of Caryota mitis, more than 10 major protein bands were detected in SDS-PAGE. Three of the separated protein bands with a molecular weight of 26000Mr, 12000Mr and ld000Mr showed immunoreactivity with IgE in the sera from patients with allergy to pollen of Caryota mitis. It was found the best dilution of coating pollen extract was 1：100, the proper dilution of serum was 1：5, biotin labeled antibody was 1 ： 1000, and the concentration of strepavidin-HRP was 1 ： 1000. Compared with SPT under this condition the coincidence rate between the results of the SPT with positive patients serum and those with BSA-ELISA was 90%. And the coincidence rate with negative patient sera was 80%. Conclusion Pollen allergens from Caryota mitis were characterized and the coincidence rate of detection between the SPA and BSA-ELISA showed high coincidence.

关 键 词：短穗鱼尾葵花粉 SDS-PAGE WESTERN BLOTTING ELISA 

分 类 号：R392.8[医药卫生—免疫学]