微柱离心结合双波长考马斯亮蓝法/酶标仪法测定牛血清白蛋白脂质体的包封率

Determination of Encapsulation Efficiency of BSA Liposome by Minicolumn Centrifuge Combined with Two-colorimetric Coomassie Brilliant Blue Protein Assay/ELIASA

目的:测定牛血清白蛋白(BSA)脂质体的包封率。方法:采用微柱离心结合双波长考马斯亮蓝法/酶标仪法测定,即先用葡聚糖凝胶DEAE-Sephadex A50微柱离心法将游离药物与脂质体进行分离,再筛选样品与染料体积比及反应时间,确定了双波长(595、450nm)考马斯亮蓝法/酶标仪法的测定条件并进行了方法学考察;考察了微柱对空白脂质体的回收率和微柱的分离效率;对BSA脂质体的包封率进行了测定。结果:确定的测定条件为样品与染料体积比为1∶3、反应时间10min,该法平均回收率为99.56%,RSD小于2.73%;微柱对空白脂质体的平均回收率为97.78%,分离效率大于90%;BSA脂质体的平均包封率为32.91%(n=3)。结论:本法实现了游离蛋白质与脂质体快速有效的分离,方法简单易行,与普通凝胶层析柱分离法相比,本法对样品的稀释倍率大大降低,适用于测定痕量大分子蛋白质脂质体的包封率。

OBJECTIVE：To determine the encapsulation efficiency（EE） of BSA liposome.METHODS：Liposome and free BSA were separated through DEAE-Sephadex A50 minicolumn centrifuge.The volume ratio of sample to dyestuff and reaction time were screened.EE was determined by minicolumn centrifuge combined with two-colorimetric coomassie brilliant blue protein assay/ELIASA（595 nm,450 nm） .RESULTS：The volume ratio of sample to dyestuff was 1 ∶ 3,and reaction time was 10 min.The average recovery rate was 99.56%（RSD2.73%） .The average recovery rate of blank liposome in minicolumn was 97.78% and separation efficiency was more than 90%.The EE of BSA liposome was 32.91%（n=3） .CONCLUSIONS：The method is rapid,effective,simple and feasible for the separation of free protein and liposome.Compared with common gel filtration column centrifuge,established method increases diluent fold of sample significantly and it is suitable for the EE determination of trace larger proteins molecule liposome.