新型冠状病毒中和抗体检测方法的建立与应用  

Establishment and application of a SARS-Co V-2 neutralizing antibody detection method

作　　者：卢世栋 宋云 毋碧聪 胡晓 马红霞[1,2] 黄学勇 郭万申[1,2] LU Shidong;SONG Yun;WU Bicong;HU Xiao;MA Hongxia;HUANG Xueyong;GUO Wanshen(infectious disease prevention and control institute,henan provincial center for disease control and prevention,zhengzhou,henan 450016,china;henan provincial key laboratory of infectious diseases and pathogens)

机构地区：[1]河南省疾病预防控制中心传染病预防控制所,河南郑州450016 [2]河南省传染病病原生物重点实验室

出　　处：《河南预防医学杂志》2022年第12期881-885,890,共6页Henan Journal of Preventive Medicine

基　　金：河南省科技厅重大专项(211100311000);新型冠状病毒防控应急攻关首批项目(201100310800);河南省中青年卫生健康科技创新领军人才培养项目(YXKC2020006)。

摘　　要：目的建立基于微量细胞中和试验(MRNT)的血清抗新型冠状病毒(SARS-CoV-2)中和抗体的检测方法,并应用于检测新型冠状病毒肺炎(COVID-19)病例的血清抗SARS-CoV-2中和抗体水平。方法用Vero细胞和已知滴度的SARS-CoV-2毒株对待测血清进行基于MRNT的中和抗体检测,对中和反应作用时间、Vero细胞代次等影响试验的关键因素进行验证,并用建立的方法重复4次检测8份血清样品的中和抗体效价,计算变异系数(CV),验证方法的重复性;用建立的方法检测165份COVID-19病例血清样本的中和抗体水平并进行分析;采用磁微粒化学发光免疫分析法(CMIA)检测其中55份感染3~4周的血清样本,并与建立方法的结果比对,分析两者的分布情况及相关性。结果不同中和反应作用时间(1.0 h、1.5 h、2.0 h)及不同代次(5代、10代、15代)Vero细胞测得血清样本的中和抗体效价相同;8份血清样本重复检测4次结果的CV为0.0%~14.2%,差异无统计学意义(F=0.508,P>0.05),表明重复性良好;建立的方法测得165份血清阳性率98.18%,中和抗体效价分布范围为4~2048,中位数(M)为128,几何平均效价(GMT)为93.80;对55份感染3~4周的样本,CMIA方法与建立方法检测阳性率均为100.00%,中和抗体分布范围为36.35~2986.49 AU/mL,M为448.11 AU/mL,GMT为476.94 AU/mL。两种方法检测的中和抗体结果呈明显相关性,相关系数r=0.758。结论成功建立了血清抗SARS-CoV-2中和抗体的检测方法,该方法具有良好的重复性,可用于评价血清抗SARS-CoV-2中和抗体水平及接种疫苗后的免疫效果。Objective To establish a serum anti-SARS-CoV-2 neutralizing antibody detection method based on microneutralization tests(MRNT),and apply it to the detection of serum anti-SARS-CoV-2 in COVID-19 cases for obtaining their neutralizing antibody levels.Methods Neutralizing antibody detection based on microcell neutralization assay using Vero cells and known titers of SARS-CoV-2 strains was conducted.The key factors of the experiment(neutralization reaction time,Vero cell generation)were verified.The established method was repeated 4 times to detect the neutralizing antibody titers of 8 serum samples,and the coefficient of variation(CV)was calculated to verify the repeatability of the method.The neutralizing antibodies and analyzed antibody levels in serum samples from 165 COVID-19 cases using the established method were detected.Magnetic particle chemiluminescence immunoassay(CMIA)was used to detect 55 serum samples collected after 3 to 4 weeks of the infection,and the detection results were compared with the results using the established method.The distribution and correlation of the two were analyzed.Results The neutralizing antibody titers of serum samples measured at different neutralization reaction times(1.0 h,1.5 h,2.0 h)and different passages of Vero cells(5th,10th,and 15th passages)were the same;8 serum samples were tested repeatedly 4 times,with the CV ranging from 0.0%-14.2%,and the difference was not statistically significant(F=0.508,P>0.05),indicating good repeatability.The established method detected a seropositive rate of 98.18%in 165 samples,and the neutralizing antibody titers ranged from 4 to 2048,with the median(M)of 128 and the geometric mean titer(GMT)of 93.80.For 55 samples collected after 3-4 weeks of infection,the positive rates using CMIA method and established method were both 100.00%;the distribution range of neutralizing antibody was 36.35-2986.49 AU/mL,with the M of 448.11AU/mL,and the GMT was476.94 AU/mL.The results of neutralizing antibodies detected by the two methods were significantl

关 键 词：新型冠状病毒 VERO细胞 中和抗体 微量细胞中和 

分 类 号：R373.9[医药卫生—病原生物学]