收稿日期: 2015-10-08
修回日期: 2016-03-01
网络出版日期: 2016-05-15
基金资助
陕西省科技统筹创新工程计划项目(2013KTCQ02-09)资助
Competitive enzyme-linked immunosorbent assay for rapid quantitative analysis of α-lactalbumin in bovine milk
Received date: 2015-10-08
Revised date: 2016-03-01
Online published: 2016-05-15
建立一种灵敏度高和特异性好的直接竞争ELISA法来定性和定量检测牛乳中α-乳白蛋白(α-LA).通过使用牛乳α-LA多次免疫新西兰大白兔制备多克隆抗体,并采用过碘酸钠氧化法制备辣根过氧化物酶(HRP)标记的α-LA,建立检测α-LA的直接竞争ELISA法.所制备的多克隆抗体特异性较高,无明显交叉反应,效价为1:80 920.酶标抗原的标记率达66.67%.直接竞争ELISA法标准曲线相关系数为0.998 57,回收率在86.00%~117.50%之间,重复性高,变异系数<5%.与国标电泳方法进行样品检测比较,结果显示该方法可有效地用于食品中α-LA的快速定性和定量检测.
关键词： 定量分析; 蛋白质; 竞争酶联免疫吸附实验; α-乳白蛋白; 免疫试验
宋宏新 , 程妮 , 薛海燕 , 杜枘宣 . 快速定量分析牛乳中α-乳白蛋白的竞争酶联免疫吸附法[J]. 中国科学院大学学报, 2016 , 33(3) : 360 -364 . DOI: 10.7523/j.issn.2095-6134.2016.03.012
A sensitive and specific competitive enzyme-linked immunosorbent assay (cELISA) for qualitative and quantitative analysis of α-lactalbumin (α-LA) is described. New Zealand white rabbits were immunised to obtain α-LA PcAb. By using sodium periodate oxidative method,the α-LA labeled by horseradish peroxidase (HRP) was prepared. Then cELISA was developed for α-LA quantitative analyses. The prepared PcAb has high specificity without obvious cross-reactivity and its titer is 1:80 920. The labeling rate of the enzyme-labeled antigen is 66.67%. The correlation coefficient of the established cELISA was 0.998 57, and the recoveries were estimated to be in the range from 86.00% to 117.50%. The variation coefficient is less than 5%. Compared with the national standard electrophoresis method,the established cELISA was useful analytical tool for rapid qualitative and quantitative analysis of α-LA in food samples.
Key words： quantitative analysis; proteins; cELISA; α-lactalbumin; immunization
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