虾有效致敏组分的提取及在sIgE检测中的初步应用  

Extraction and Its Application of the Effective Components from Shrimp in Detection for sIgE

作　　者：任杰[1] 单立新[2] 李韶深[3] 陈凯[2] 侯丽英[1] 李会强[1] 

机构地区：[1]天津医科大学检验学院,300203 [2]天津市北辰医院检验科 [3]天津中医药研究院附属医院检验科

出　　处：《天津医药》2014年第5期462-465,共4页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(项目编号:81371882);天津市中小企业创新基金资助项目(项目编号:12ZXCXSY09500)

摘　　要：目的通过改善虾类包被抗原质量,提高血清特异性IgE(sIgE)检测的敏感性。方法制备中华对虾蛋白溶液,经酶联免疫印迹实验鉴定主要的致敏蛋白组分,葡聚糖凝胶层析法初步分离大于55 ku的蛋白组分,SDS-PAGE技术分析其蛋白。分别使用虾蛋白溶液和分离的蛋白作为包被抗原包被96微孔板,采用酶斑点印迹和间接酶联免疫吸附法(间接ELISA法)初步评价纯化后的抗原是否能够提高血清sIgE检测结果的敏感性。结果酶联免疫印迹实验显示中华对虾蛋白溶液中>55 ku的蛋白组分是主要的致敏蛋白,葡聚糖凝胶层析法初步分离了>55 ku的蛋白组分,其中主要包含10条明显蛋白条带;酶斑点印迹显示,以纯化后蛋白作为包被抗原,可提高弱阳性血清斑点光密度。同时,两种包被抗原的间接ELISA结果显示,虾有效致敏成分作为包被抗原时,92.9%的虾过敏患者血清sIgE检测值升高。结论采用中华对虾有效致敏原组分作为虾过敏检测抗原能提高血清sIgE检测结果的敏感性。Objective To improve the detecting sensitivity of serum specific IgE （sIgE） by improving the quality of coated antigen in shrimp. Methods The extracts from shrimp protein was prepared. Western blot assay was used to identify the major allergenic protein components. The protein components〉55 ku were separated by Sephadex gel chromatography. SDS-PAGE technology was used to analyze proteins. Samples of shrimp protein and proteins〉55 ku were used as the coat-ing antigen to coat 96 microplate respectively. Western blot assay and ELISA were used to evaluate preliminary sensitivity of the purified antigen for detecting sIgE. Results Immunoblot experiments showed that the protein〉55 ku was the main aller-genic protein component of shrimp. Those 〉55 ku proteins were separated successfully by Sephadex gel chromatography, showing 10 identifiable bands in SDS-PAGE. Dot-pot immunoassay showed that proteins〉55 ku used as coated antigens could improve the spots density of the weak serum. Meanwhile, the result of ELISA showed that sIgE detection value in-creased 92.9%in patients with shrimp allergy after coating effective antigens. Conclusion The detecting sensitivity of sIgE can be improved by using effective protein components of shrimp as coated antigens.

关 键 词：食物过敏 对虾科 分离和提纯 印迹法 蛋白质 

分 类 号：R446[医药卫生—诊断学]