应用高效体积排阻色谱偶联多角度激光散射鉴定猪圆环病毒2型灭活疫苗及病毒样颗粒疫苗抗原

Characterization of the antigens in inactivated porcine circovirus type 2 vaccines and virus-like particle vaccines by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering

摘要:

本文旨在建立基于高效体积排阻色谱(high-performance size-exclusion chromatography,HPSEC)偶联多角度激光散射仪(multi-angle laser light scattering,MALLS)的猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗抗原检测方法。以纯化的PCV2灭活病毒及病毒样颗粒(virus-like particles,VLP)为参照，对4家生产企业的2种PCV2灭活病毒疫苗(a、b)及VLP疫苗(c、d)破乳后进行HPSEC-MALLS检测及分子量分析；结合PCV2抗原检测卡、Western blotting和透射电子显微镜(transmission electron microscope,TEM)，鉴定了特征色谱峰；考察了方法的重复性和检测线性。结果表明，两家企业生产的PCV2灭活病毒疫苗破乳液水相经HPSEC分离，在保留时间约13.3 min处出现抗原特征峰；MALLS计算该色谱峰分子量分别为2.61×10⁶(±4.34%) Da和2.40×10⁶(±2.51%) Da。两种VLP疫苗也在13.3 min处出现抗原特征峰，分子量分别为2.09×10⁶(±2.94%) Da和2.88×10⁶(±11.85%) Da，接近PCV2的理论分子量；同时在保留时间约11.4 min处也出现色谱峰，经检测分子量为4.37×10⁶(±0.42%) Da，TEM表征显示为VLP二聚体。取疫苗d和PCV2 VLP纯品进行重复检测，抗原色谱峰面积的RSD(n=3)均小于1.5%，重复性好；将PCV2 VLP纯品梯度稀释检测，VLP及其多聚体的色谱峰面积与浓度均呈良好的线性关系，R²分别为0.999及0.997，能够满足定量及多聚体含量分析。该方法有望成为一种准确、高效的PCV2疫苗的体外评价方法，用于质量评价与提升。

Abstract:

This paper aims to detect the antigens in porcine circovirus type 2 (PCV2) vaccines by high-performance size-exclusion chromatography (HPSEC) coupled with multi-angle laser light scattering (MALLS). With purified inactivated PCV2 and PCV2 virus-like particles (VLP) as references, two inactivated vaccines (a and b) and two VLP vaccines (c and d) for PCV2 from four manufacturers were analyzed by HPSEC-MALLS after demulsification. The antigen peaks in HPSEC-MALLS were identified by PCV2 antigen test strips, Western blotting and transmission electron microscope (TEM). The repeatability and linearity of the method were investigated. The results showed the virus antigens in the two inactivated vaccines were eluted at about 13.3 min in HPSEC. The molecular weight of these antigens was 2.61×10⁶ (±4.34%) Da and 2.40×10⁶ (±2.51%) Da, respectively, as calculated by MALLS. The antigen peaks of the two VLP vaccines also appeared at 13.3 min and the molecular weight was 2.09×10⁶ (±2.94%) Da and 2.88×10⁶(±11.85%) Da, respectively, which was close to the theoretical molecular weight of PCV2. Moreover, an antigen peak of VLP vaccine c was observed at 11.4 min and the molecular weight was 4.37×10⁶ (±0.42%) Da. The antigen was verified to be the dimer of VLP by TEM. Vaccine d and purified Cap VLP antigens were tested repeatedly, and the RSD of the peak area (n=3) was all <1.5%, indicating that the method was repeatable. The purified VLP were diluted in serial and tested for linearity. The result suggested good linear relationship between the peak area of VLP or VLP aggregates and the protein concentration of the sample with R² of 0.999 and 0.997, respectively. Thus, the method met the requirement for quantification and aggregate analysis. This method is accurate and efficient in in vitro quality evaluation and improvement of PCV2 vaccine.