Laboratory for the Analysis of Medicines (LAM), Dept. of Pharmaceutical Sciences, CIRM, University of Liège, Liège, Belgium.
Cellular and Molecular Immunology, GIGA-Research University of Liège, Liège, Belgium.
Talanta. 2017 Dec 1;175:325-330. doi: 10.1016/j.talanta.2017.07.046. Epub 2017 Jul 15.
Capillary electrophoresis (CE) for HPV-VLP quantitation is a very interesting alternative technique compared to those currently used in viral analysis, such as SDS-PAGE, Western blot or protein assay that are destructive and semi-quantitative or non specific. In this study, the quantitative performance of the CE method was evaluated. A main issue in virus quantitation is the absence of reference material. Therefore, the concentration of a HPV16-VLP sample produced in the laboratory was determined using ELISA with Gardasil, after adjuvant dissolution, as reference material and conformational H16.V5 antibody. HPV16-VLP concentration was found to influence particles electrophoretic mobility until a plateau was reached for concentrations ≤ 50µgml. As zeta potential is directly proportional to the electrophoretic mobility, it was measured at different HPV-VLP concentrations and the results were in complete accordance with the measured electrophoretic mobilities. The concentration dependence of the electrophoretic mobility could be explained by an overlap of the electrical double layers of adjacent particles. The HPV16-VLP peak identity was demonstrated unequivocally by the study of HPV16-VLP/H16.V5 antibody complex formation using affinity CE. Finally, the CE method was successfully validated following the ICH Q2R1 guidelines. To overcome the sample heterogeneity issue, a well-designed sample preparation was used. Considering sample complexity, validation results were satisfactory with maximum repeatability and intermediate precision RSD of 12.2% and a maximum relative bias of 1.4%.
毛细管电泳（CE）用于 HPV-VLP 定量是一种非常有趣的替代技术，与目前用于病毒分析的 SDS-PAGE、Western blot 或蛋白分析等破坏性、半定量或非特异性方法相比。在这项研究中，评估了 CE 方法的定量性能。病毒定量的一个主要问题是缺乏参考物质。因此，使用 ELISA 用 Gardasil 作为参考物质和构象 H16.V5 抗体来确定实验室中生产的 HPV16-VLP 样品的浓度。HPV16-VLP 浓度会影响颗粒的电泳迁移率，直到达到浓度≤50μg/ml 的平台。由于 ζ 电位与电泳迁移率成正比，因此在不同的 HPV-VLP 浓度下测量了 ζ 电位，结果与测量的电泳迁移率完全一致。电泳迁移率的浓度依赖性可以通过相邻颗粒的电双层重叠来解释。使用亲和 CE 研究 HPV16-VLP/H16.V5 抗体复合物的形成，明确证明了 HPV16-VLP 峰的身份。最后，根据 ICH Q2R1 指南成功验证了 CE 方法。为了克服样品异质性问题，采用了精心设计的样品制备方法。考虑到样品的复杂性，验证结果令人满意，最大重复性和中间精密度 RSD 为 12.2%，最大相对偏差为 1.4%。
