动物营养学报2018，Vol.30Issue(1)：100-106,7.DOI:10.3969/j.issn.1006-267x.2018.01.014
牛乳铁蛋白多克隆抗体的制备、纯化及定量检测
Preparation, Purification and Quantitative Identification of Bovine Lactoferrin Polyclonal Antibody
摘要
Abstract
This study aimed at preparing polyclonal antibody against lactoferrin in milk with high purity and specificity to provide experimental materials for the identification and quantification of lactoferrin synthesized in milk and mammary gland. Four healthy New Zealand white rabbits were immunized with bovine lactoferrin and challenged again after 4 weeks. Thereafter,the rabbits were immunized once every 2 weeks until antibody titer was desirable. Then the blood was collected from the heart and the serum was obtained. The antibody was puri-fied by the saturated ammonium sulfate method and protein A resin. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and Western-blot were used to identify the purity and specificity of the puri-fied antibody. Antibody titer was determined and the curve of antibody inhibition was obtained. Finally, we used the obtained antibody to quantitatively determine lactoferrin in commercial liquid milk, milk powder and the homogenate of mammary gland tissue of dairy cows. The results showed that the obtained rabbit anti-bovine lactoferrin polyclonal antibody had high purity and specificity, the antibody concentration was 11.02 mg/mL, and the titer was 1:128 000;lactoferrin concentration in mammary gland tissue of dairy cows was 16.13 μg/g, that in commercial liquid milk was close to 0, and that in milk powder was 5.28 μg/g. In conclusion, lacto-ferrin polyclonal antibody with high purity and specificity was prepared by saturated ammonium sulfate method and protein A resin in the present study, which can be used to identify lactoferrin in products like milk.关键词
乳铁蛋白/多克隆抗体/抗体纯化Key words
lactoferrin/polyclonal antibody/purification of antibody分类
农业科技引用本文复制引用
王建,郝丽媛,魏婧雅,孙鹏..牛乳铁蛋白多克隆抗体的制备、纯化及定量检测[J].动物营养学报,2018,30(1):100-106,7.基金项目
国家重点研发计划(2016YFD0500507) （2016YFD0500507）
中国农业科学院科技创新工程(ASTIP-IAS07) （ASTIP-IAS07）
国家高层次人才特殊支持计划 （）
