Vasilyeva Elena, Woodard James, Taylor Frederick R, Kretschmer Matthias, Fajardo Hans, Lyubarskaya Yelena, Kobayashi Kazumi, Dingley Amy, Mhatre Rohin
BiogenIdec, Fourteen Cambridge Center, Cambridge, MA 02142, USA.
Electrophoresis. 2004 Nov;25(21-22):3890-6. doi: 10.1002/elps.200406084.
A method based on microfluidic technology was developed to support quantitative analysis of recombinant monoclonal immunoglobulin G4 (IgG4) antibody samples. The assay was performed on an Agilent 2100 Bioanalyzer in combination with the Protein 200 Plus LabChip Kit and the Protein 200 Plus assay software. Capillary electrophoresis principles have been transferred to a chip format that integrates all separation, staining, virtual destaining, and detection steps. The method is referred to in this paper as chip-based capillary gel electrophoresis (GelChip-CE method). The GelChip-CE method under nonreducing conditions proved to be a quantitative test for half-antibody determination in IgG4 samples. Similar to the traditional nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method, the GelChip-CE method includes a denaturing step prior to separation. We showed that denaturing the sample by heating resulted in an artificial increase in the amount of half-antibody detected, which could be prevented by addition of N-ethylmaleimide to the sample buffer. The GelChip-CE method allowed for analysis of IgG4 samples with more accuracy, higher precision, and a faster turnaround time than SDS-PAGE and reversed-phase high-performance liquid chromatography (RP-HPLC).
开发了一种基于微流控技术的方法，以支持对重组单克隆免疫球蛋白G4（IgG4）抗体样品进行定量分析。该分析在安捷伦2100生物分析仪上结合Protein 200 Plus LabChip试剂盒和Protein 200 Plus分析软件进行。毛细管电泳原理已被转移到一种集成了所有分离、染色、虚拟脱色和检测步骤的芯片形式中。本文将该方法称为基于芯片的毛细管凝胶电泳（GelChip-CE方法）。在非还原条件下，GelChip-CE方法被证明是一种用于测定IgG4样品中半抗体的定量测试方法。与传统的非还原十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）方法类似，GelChip-CE方法在分离前包括一个变性步骤。我们发现，通过加热使样品变性会导致检测到的半抗体量人为增加，而在样品缓冲液中添加N-乙基马来酰亚胺可以防止这种情况。与SDS-PAGE和反相高效液相色谱（RP-HPLC）相比，GelChip-CE方法能够更准确、更精确地分析IgG4样品，且周转时间更快。
