3种鸡蛋清过敏原提取方法的评价  

Evaluation of three methods for extracting hen egg white proteins

作　　者：王萍[1,2] 陈寅[1] 穆晓燕[3] 孙静[3] 杨庆红[3] 李会强[1] 

机构地区：[1]天津医科大学免疫学教研室,天津300203 [2]皖南医学院微生物学与免疫学教研室,安徽芜湖241002 [3]天津人民医院检验科,天津300121

出　　处：《临床检验杂志》2010年第4期305-307,共3页Chinese Journal of Clinical Laboratory Science

基　　金：天津医科大学科学基金(编号:2009XK11);安徽省教育厅自然科学研究计划项目(编号:KJ2009B162)

摘　　要：目的评价3种鸡蛋清过敏原提取方法,为临床检测sIgE提供最佳抗原提取方案。方法分别用丙酮沉淀法、硫酸铵盐析法、碳酸氢钠溶解法粗提鸡蛋清蛋白,用BCA法测定蛋白质浓度,并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹方法分别鉴定分离蛋清的蛋白质组分和免疫活性,并进行20例鸡蛋过敏患者血清的分析。结果每100 ml蛋清用3种方法提取的蛋白质总量分别为1.5 g、1.8 g、5.2 g。免疫印迹法检测显示,蛋清与鸡蛋过敏患者的阳性血清有2条反应条带,分子量接近72 000和17 000。碳酸氢钠溶解法提取的蛋清蛋白质与20例阳性病人血清反应均出现2条反应带,其他方法提取的蛋白质在部分病例中未出现反应带。结论 3种提取方法各有特点,碳酸氢钠溶解法操作简单、收获量高,蛋白质组分完全,能够满足临床检测蛋清sIgE的实验要求。Objective To evaluate three approaches for extracting the proteins in hen egg white and to provide an optimal antigen extraction protocol for clinical detection of allergen specific IgE.Methods Proteins were extracted from hen egg white using acetone precipitation（A）,ammonium sulfate salting-out method（B）,and sodium hydrogen carbonate solution（C）,respectively.The protein concentrations were detected by bicinchoninic acid method.The total proteins of egg white were isolated by SDS-PAGE and their allergenicity was identified with western blotting（WB）.Results The total extracted protein per 100 ml egg white by method A,B,and C was 1.5 g,1.8 g and 5.2 g,respectively.Two proteins,close to 72 000 and 17 000 respectively,reacted with the sera of persons with egg allergy in WB.Egg white proteins extracted by method C reacted to all 20 positive sera and all presented two bands.However,proteins extracted by method A or B did not react to some of the positive samples.Conclusions Each method has its own characteristics.The method using sodium hydrogen carbonate solution is simple and the protein harvest is high.The protein components purified by method C are plentiful and may meet the requirements of clinical detection of egg allergen specific IgE.

关 键 词：鸡蛋 过敏原 十二烷基硫酸钠-聚丙烯酰胺凝胶电泳 免疫印迹 

分 类 号：R392.8[医药卫生—免疫学]