时间分辨免疫荧光法测定食物中鸡蛋过敏原蛋白成分

Determination of egg allergen protein trace in food products by time-resolved fluoroimmunoassay

目的:建立双抗体夹心时间分辨免疫荧光法(TRFIA)用于测定食物中鸡蛋过敏原蛋白成分,为进出口食品鸡蛋过敏原成分检测提供技术基础。方法:提取鸡蛋过敏原总蛋白,免疫小鼠获得多克隆抗体,并以此抗体包被酶标板,采用生物素标记抗鸡蛋过敏原总蛋白抗体、Eu3+标记的链酶亲和素和以β-二酮体为主的增强液等试剂,建立双抗夹心TRFIA方法,用于检测10种食品中是否含有鸡蛋蛋白成分。结果:鸡蛋总蛋白SDS-PAGE显示带大致有10条,其中主要条带有4条,分子量分别为71、55、38、14kDa;成功地研制出双抗体夹心TRFIA法检测食品中鸡蛋过敏原蛋白成分,具有特异性,其最低检出限为0·625ng/mL,标准曲线在12·5～640ng/mL范围内线性良好;8种食品检测结果与食物过敏原标签标注内容相符,而2种标示不详的食品检测结果均呈现阳性。结论:初步建立了检测食品中鸡蛋过敏原蛋白成分的双抗体夹心TRFIA,具有较好的特异性和灵敏度;该方法对于未标注鸡蛋过敏原成分的食品检测具有一定的实际应用价值。

Objective ：To develop a new method with sandwich-antibody time-resolved fluoroimmunoassay （TRFIA） is to detect egg allergen protein trace in food products.Method：The total egg protein was extracted and further analyzed by SDS-PAGE.The Balb/c mice were immune with the extracts of egg proteins and the highly-titrated polyclonal IgG antibodies against them were isolated and coupled with biotin for TRFIA kit.Ten food products were tested using this method.Result： About 10 protein bands were observed on the SDS-PAGE gel of egg total proteins.The molecular weights of the major bands were 71, 55,38 and 14kDa.The results indicated that the standard curve was linear with egg allergen protein concentration from 12.5-640ng/mL and the detection limit was 0.625ng/mL of egg allergen protein.We found that 8 foods were detected with the egg allergen protein,which were consistent with the egg allergen labels from their manufactures.However,two foods without clear food allergen label were detected to contain egg allergen protein.Conclusion： Thus we concluded that sandwich-antibody TRFIA could be used to detect egg allergen protein trace in food products.