抗狂犬病病毒中和抗体快速荧光灶抑制试验方法的验证  

Validation of a rapid fluorescent focus inhibition test for rabies virus neutralizing antibodies

作　　者：何颍 于鹏程[1] 朱武洋[1] He Ying;Yu Pengcheng;Zhu Wuyang(National Health Commission Key Laboratory of Biosafety,National Institute for Vital Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,国家卫生健康委员会生物安全重点实验室,北京102206

出　　处：《中国疫苗和免疫》2022年第5期559-562,共4页Chinese Journal of Vaccines and Immunization

摘　　要：目的对抗狂犬病病毒中和抗体(Rabies virus neutralizing antibody,RVNA)快速荧光灶抑制试验(Rapid fluorescent focus inhibition test,RFFIT)进行验证。方法利用已检测RVNA效价的人狂犬病疫苗免疫后血清建立血清参考品。采用参考血清和狂犬病人免疫球蛋白标准品,评价RFFIT方法的准确性、重复性、稳定性、线性和检测范围。结果共制备5批血清参考品(A、B、C、D和E),RVNA效价分别为57.39、16.54、8.76、1.38和0.08IU/mL。采用效价适中的参考血清C进行RFFIT方法的认证。参考血清样品C加标(4.5IU/mL、2.25U/mL和1.125IU/mL)后的回收率分别为91.70%、80.15%和74.96%。同一实验人员对相同样品检测6次的RVNA中和效价的变异系数(Coefficient of variation,CV)为7.61%;2名实验人员对相同样品各检测3次的RVNA中和效价的CV为3.88%。标准品效价值与RFFIT方法检测值的线性方程为y=0.9524x-0.1192(R^(2)=0.999),最低检出效价为0.04IU/mL。不同病毒-抗体中和时间、细胞-抗体-病毒孵育时间、丙酮固定时间和荧光抗体染色时间测定的参考血清C中和效价的CV均<30%。结论本研究建立的RFFIT方法对人狂犬病疫苗免疫后血清检测具有很好的准确性、重复性、线性和稳定性,推荐在相关血清检测中应用。Objective To validate a rapid fluorescent focus inhibition test(RFFIT)for rabies virus neutralizing antibody(RVNA).Methods We tested human sera and determined RVNA titers after immunization with rabies vaccine to establish a serum reference.We evaluated the accuracy,repeatability,stability,linearity,and detection range of the RFFIT method using the reference serum and a rabies human immunoglobulin standard.Results Five reference serum samples(A,B,C,D,and E)were prepared,with RVNA titers being 57.39,16.54,8.76,1.38,and 0.08 IU/mL,respectively.Reference serum C,with a moderate titer,was used for the validation of RFFIT.Recoveries of the reference serum C spiked with 4.5 IU/mL,2.25 U/mL,and 1.125 IU/mL were 91.70%,80.15%,and 74.96%,respectively.Coefficients of variation(CVs)of RVNA titers were 7.61% and 3.88% after the same sample was tested six times by the same researcher and three times by each of two researchers,respectively.A linear equation between titers of the standard and titers detected by RFFIT was y=0.9524x-0.1192(R^(2)=0.999),with the lowest detected titer being 0.04 IU/mL.CVs of titers of the reference serum C were all<30% under different times of virus-antibody neutralization,cell-antibody-virus incubation,acetone fixation,and fluorescent antibody staining.Conclusions The RFFIT method has good accuracy,repeatability,linearity,and stability in detection of serum from immunization with human rabies vaccine.It may be used for detection in related sera.

关 键 词：狂犬病病毒 抗狂犬病病毒中和抗体 快速荧光灶抑制试验 回收率 变异系数 

分 类 号：R373.9[医药卫生—病原生物学]