用于分离α1-蛋白酶抑制剂（A1PI）浓缩物中A1PI亚型的便捷高分辨率等电聚焦（IEF）方法。

Convenient high-resolution isoelectric focusing (IEF) method for the separation of alpha1-proteinase inhibitor (A1PI) isoforms in A1PI concentrates.

作者信息

Weber Alfred, Engelmaier Andrea, Owen Maurice C, Schwarz Hans-Peter, Turecek Peter L

机构信息

Baxter BioScience, Industriestrasse 67, A-1220 Vienna, Austria.

Canterbury Scientific Limited, 14 Pope Street, Christchurch 8011, New Zealand.

出版信息

J Pharm Biomed Anal. 2007 Sep 21;45(1):107-111. doi: 10.1016/j.jpba.2007.06.006. Epub 2007 Jun 13.

DOI:10.1016/j.jpba.2007.06.006

PMID:17629652

Abstract

Currently, high-resolution separation of A1PI is done in highly specialized laboratories using gels made in-house. This paper presents a high-resolution method for the separation of A1PI concentrates and human plasma using commercially available gels. Hybrid IEF was performed with carrier ampholytes and the gels were stained with Coomassie Brilliant Blue G-250. In addition, a sensitive immunoblotting procedure is described. The IEF method allowed the reproducible and convenient determination of the IEF pattern of A1PI in concentrates including resolution of glycan-dependent isoforms and isoproteins with secondary modifications such a C-terminal Lys-truncation. Furthermore, a shift in the IEF pattern of A1PI occurring upon reduction could be detected. Finally, in combination with a sample pretreatment step, the method proved able to monitor complex A1PI isoform patterns in samples with low A1PI concentrations as present for example in bronchoalveolar lavage solutions.

摘要

目前，α1抗胰蛋白酶（A1PI）的高分辨率分离是在高度专业化的实验室中使用自制凝胶完成的。本文介绍了一种使用市售凝胶分离A1PI浓缩物和人血浆的高分辨率方法。采用载体两性电解质进行混合等电聚焦（IEF），并用考马斯亮蓝G - 250对凝胶进行染色。此外，还描述了一种灵敏的免疫印迹程序。该IEF方法能够可重复且方便地测定浓缩物中A1PI的IEF图谱，包括糖基依赖性异构体和具有二级修饰（如C末端赖氨酸截短）的同型蛋白的分辨率。此外，还能检测到还原后A1PI的IEF图谱的变化。最后，结合样品预处理步骤，该方法被证明能够监测低A1PI浓度样品（如支气管肺泡灌洗溶液中存在）中的复杂A1PI异构体模式。