高滴度特异性卵黄抗体在天花病毒检测中的研究(英文)

Application of high-titred IgY antibodies in orthopox virus diagnostics

天花是人类第一个消灭的疾病,但生物界中仍广泛存在天花类病毒。目前人类还缺乏对其有效、快捷的诊断、检测和治疗措施。在全球面临生物恐怖袭击的时代,这方面的研究工作尤显迫切。以3种灭活的天花病毒(vaccinia virus,calpox virus和cowpox virus)为免疫原,分别免疫注射3组实验蛋鸡,以改良的聚乙二醇法将相应特异性抗体从卵黄中提取。利用免疫荧光实验、抗体中和实验及免疫电镜等实验持续检测抗体滴度变化以及抗体交叉反应。最后,以特异性的卵黄抗体与磁珠(Dynabead^(TM))相结合,用于天花病毒的纯化,并在PCR实验中验证其检测效果。抗vaccinia virus抗体和抗calpox virus抗体在高度稀释的情况下(分别以1:10~6,1:10~5稀释),仍在免疫荧光实验中呈阳性反应,且抗体的这种高表达水平在持续5次免疫注射下,能维持达10个月。抗体的中和能力和抗体与抗原反应的超微结构也在相应实验中观察到。抗体与病毒蛋白结合的特殊条带也在免疫印记实验中观察到。实验显示,不同天花病毒之间存在强而明显的交叉反应。最后,特异性的卵黄抗体能够与磁珠结合,并在模拟样本中用于天花病毒的PCR检测。达到纯化病毒、增强PCR实验敏感度的效果。该研究显示,抗天花病毒卵黄抗体能够应用于天花病毒的诊断,有望以此为基础,开发出快速、有效的天花病毒检测手段。

Layer chickens were immunized with three species of inactivated orthopox virus （vaccinia virus, calpox virus and cowpox virus）. Antibodies （IgY） were purified from egg yolks by improved polyethylene glycol precipitation. The development of IgY directed against orthopox virus antigens was followed by immunofluorescence assay, plaque reduction neutraliztion test and immunoelectron microscopy. Cross-reactivity of two IgY antibodies with cells infected by the different strains of the pox viruses was also investigated by different methods （immunofluorescence assay, plaque reduction neutraliztion test and Western blot）. Even in very high dilutions in immunofluorescence assay （titres up to 1：10^6 and 1：10^5, respectively） and persisted on a plateau over 10 months after four booster injections, it was showed that anti-vaccinia virus IgY and anti-calpox virus IgY were positive. Neutralizing activity and ultra-structural detection of antigen with gold-labelled antibodies were respectively observed in plaque reduction neutralization test and immunoelectron microscopy. Western blot analysis revealed specific binding of IgY to virus proteins. Thus, there was cross-reactivity between different orthopox viruses. Finally, orthopox virns-specific IgY antibodies bounded magnetic beads （Dynabead） were used to concentration of orthopox viruses. This study suggests that anti-pox virus IgY could serve as a useful tool for orthopox viruses diagnosis.