Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan.
Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 402, Taiwan.
Poult Sci. 2019 Apr 1;98(4):1658-1663. doi: 10.3382/ps/pey537.
The infectious bursal disease (IBD) causes immunosuppression in chicken of all ages and high mortality in young chicken, posing serious threat to poultry industry worldwide. One promising strategy for preventing this highly contagious disease is using recombinant subunit vaccine, employing VP2 subviral particles (SVP) as epitomic antigen. Analytical techniques of viral-like particles such as SDS-PAGE, western blot, or high-performance size-exclusion chromatography have been widely applied, but mostly unsatisfactory. In the present study, a simple, fast and cost-effective capillary zone electrophoresis (CZE) method with UV-detection was developed to analyze purified IBDV-SVP (expressed by Escherichia coli system) using commercial monoclonal antibody (mAb) against VP2. To find satisfying CZE conditions, injection mode, separation voltage, and separation buffer were explored. Through the modified CZE, mAb and SVP could be well separated and shown distinct peaks in the electropherogram. Furthermore, to determine the stoichiometry, the area of the mAb peak versus SVP/mAb binding ratio was plotted and indicated that 2 or 3 receptor molecules were bound per SVP. The purity and integrity of SVP and the interactions between SVP and mAb could be analyzed by the developed simple CZE-UV method in less than half hour. This CZE-UV method proved to be a valuable and useful tool in detection, characterization, and quantification of IBDV-SVP and the mAb, offering potential applications of in-process quality control of vaccine production, surveillance of serum antibody produced against IBDV infection, or vaccine immunization.
传染性腔上囊炎 (IBD) 可导致各年龄段鸡的免疫抑制，并且在雏鸡中死亡率很高，对全球家禽业构成严重威胁。预防这种高度传染性疾病的一种有前途的策略是使用重组亚单位疫苗，采用 VP2 亚病毒颗粒 (SVP) 作为表位抗原。病毒样颗粒的分析技术，如 SDS-PAGE、western blot 或高效尺寸排阻色谱法已被广泛应用，但大多不尽人意。在本研究中，开发了一种简单、快速且具有成本效益的毛细管区带电泳 (CZE) 方法，使用针对 VP2 的商业单克隆抗体 (mAb) 分析纯化的 IBDV-SVP（由大肠杆菌系统表达）。为了找到令人满意的 CZE 条件，探索了进样模式、分离电压和分离缓冲液。通过改良的 CZE，mAb 和 SVP 可以很好地分离，并在电泳图谱中显示出明显的峰。此外，为了确定化学计量比，绘制了 mAb 峰的面积与 SVP/mAb 结合比的关系图，表明每个 SVP 结合 2 或 3 个受体分子。通过开发的简单 CZE-UV 方法，可以在不到半小时的时间内分析 SVP 和 mAb 的纯度和完整性以及它们之间的相互作用。该 CZE-UV 方法已被证明是一种有价值且有用的工具，可用于检测、表征和定量 IBDV-SVP 和 mAb，为疫苗生产过程中的质量控制、针对 IBDV 感染产生的血清抗体的监测或疫苗免疫提供了潜在的应用。
