鸡蛋致敏蛋白免疫分析方法的建立  

Establishment of Immunoassay Method for Allergy-Inducing Proteins in Eggs

作　　者：兰丽平[1] 许舒婷[1] 赵杰[1] 郑月明[1,3] 高美须[1] 潘家荣[1,2] 

机构地区：[1]中国农业科学院农产品加工研究所,农业部农产品加工与质量控制重点实验室,北京100193 [2]中国计量学院生命科学学院,浙江杭州310018 [3]中国检验检疫科学研究院,北京100025

出　　处：《食品科学》2013年第6期134-138,共5页Food Science

基　　金：“十一五”国家科技支撑计划项目(2006AA10Z449);国家公益性行业科研专项(201103007)

摘　　要：采用硫酸铵沉淀法和离子交换法分离纯化鸡蛋清得致敏蛋白后免疫兔子制备抗多克隆抗体,并建立快速、灵敏、特异性强的鸡蛋致敏蛋白酶联免疫检测方法。在优化条件下,鸡蛋致敏蛋白在1×10-4～10μg/mL质量浓度范围内与抑制率线性关系良好,其线性回归方程为y=0.74145+0.21692 lgC(r=0.9971),半抑制浓度(IC50)和最低检测限(IC10)分别为为77.076、1.104ng/mL;样品加标回收率在98.29%～101.55%之间;且抗鸡蛋致敏蛋白多克隆抗体对火鸡蛋蛋白、鸭蛋蛋白、鹅蛋蛋白均具有交叉反应,与花生、小麦蛋白无交叉反应;批内和批间变异系数分别为4.63%和5.49%,且可在4℃可保存6个月以上,能够快速灵敏检测食品中鸡蛋致敏蛋白的存在。Allergy-inducing proteins in eggs were isolated and purified by ammonium sulfate precipitation and ion exchange chromatography. Polyclonal antibody was generated by inoculated rabbits. A rapid, sensitive and specific ELISA method was developed and optimized for the detection of egg allergy proteins. Under the optimal experimental conditions, a good linear relationship between allergy-inducing proteins and inhibitory rate in the range of 1 ×10-4～10μg/mL was observed. The regression equation was y = 0.74145＋0.21692 × lgC （r = 0.9971）. IC50 and IC10 were 77.076 ng/mL and 1.104 ng/mL, respectively. The recovery rate of the ELISA method was 70.1%--117.8%. The developed method bad cross-reactivity with the proteins from turkey, duck and goose eggs, despite having no cross-reactivity with the proteins from peanut or milk. The variation coefficients of intra- assay and inter-assay were 4.63% and 5.49%, respectively. The shelf life of the prepared immunoassay kit was 6 months during the storage at 4 ℃ and met the requirements for the analysis of egg allergy proteins with fast operation and high sensitivity.

关 键 词：鸡蛋致敏蛋白 酶联免疫法 多克隆抗体 

分 类 号：R155.5[医药卫生—营养与食品卫生学]