新冠病毒奥密克戎株感染前后血浆中的抗体变化与分布

Changes and distribution of antibodies in plasma before and after infection of SARS-CoV-2 Omicron strain

目的通过ELISA和假病毒中和实验检测奥密克戎株感染前后代表性混合血浆的SARS-CoV-2-IgG水平及针对不同病毒株的中和抗体,了解抗体变化规律及中和抗体分布,确定能用于新冠特免(静脉注射COVID-19人免疫球蛋白)制备的血浆最佳采集时间段。方法建立SARS-CoV-2-IgG的ELISA定量检测方法并进行线性范围、准确度和精密度验证;检测25名献浆者奥密克戎感染后20~40 d的单人份康复期血浆SARS-CoV-2-IgG效价,按照效价结果制备了2组混合血浆小样WP1(1000~2000 EIU/mL)和WP2(2000~4000 EIU/mL),并检测其不同病毒株(原型株、BA.1、BA.2、BA.4/5、BF.7、BQ.1.1)假病毒中和效价,确定不同病毒株中和抗体的反应性分布。检测公司下属14个浆站采集的奥密克戎株感染前后的代表性混浆的SARS-CoV-2-IgG效价,包括2022年12月—2023年5月不同浆站不同时间段采集的奥密克戎康复者血浆(OP)和2022年3—12月采集的奥密克戎感染前的正常合并血浆(VN),根据结果分析献浆者感染奥密克戎株前后SARS-CoV-2-IgG差异及随时间变化规律。结果以内控参考品B2为标准品(效价100 EIU/mL),SARS-CoV-2-IgG的线性范围在6.25~200 EIU/mL,批内准确度在81.793%~106.985%之间,批内精密度在1.100%~13.000%之间,批内总误差在2.988%~22.679%;批间准确度在90.788%~96.893%之间,批间精密度在4.870%~6.272%之间,批间总误差在9.192%~15.399%。假病毒中和抗体的结果显示,WP系列混浆中和抗体效价排序为原型株>BA.2>BA.4/5>BF.7≈BQ.1.1>BA.1,WP1和WP2两者各项检测结果的相关性高(Pearson r=0.9311,P=0.0023),说明奥密克戎感染后康复期血浆中不同病毒株中和抗体效价分布基本稳定。与奥密克戎大规模感染前康复期血浆G128相比,血浆中奥密克戎株的抗体效价明显提高,其中BA.2抗体与原型株抗体的比例由感染前的26.9%上升到感染后的82.6%~87.5%。奥密克戎感染前VN系列检测结果<100 EIU/mL,奥密克戎感染后OP系列结果显示,从2022年12月初开始采集的血浆当月即为抗体的峰值,随后急剧下降,在2023年2—3月进入短暂的平台期(效价约为峰值的40%),4月开始再次急剧下降(效价约为峰值的20%)。结论新冠奥密克戎株感染后的康复期血浆中针对奥密克戎亚型的中和抗体效价明显提高,不同地区献浆者的IgG抗体在感染当月达到峰值,随后持续下降,能用于新冠特免制备的血浆最佳采集时段为感染后1~2个月。

Objective To determine the best collection time period of plasma which can be used for human COVID-19 immunoglobulin for intravenous injection through SARS-CoV-2-IgG change and neutralizing antibody distribution against different virus strain in representative mixed plasma before and after Omicron strain infection by ELISA and pseudovirus neutralization test.Methods An ELISA method for quantitative detection of SARS-CoV-2-IgG was established and its linear range,accuracy and precision was verified.SARS-CoV-2-IgG potency was detected in 25 convalescent plasma which were collected 20-40 days after confirmed Omicron infection,two groups of mixed plasma samples WP1 and WP2 were prepared according to the SARS-CoV-2-IgG results,and pseudovirus neutralization experiments with different virus strain(prototype strain,BA.1,BA.2,BA.4/5,BF.7,BQ.1.1)were carried out to determine the distribution of neutralizing antibodies against different virus strain.SARS-CoV-2-IgG potency of representative mixed plasma collected from 14 plasma stations subordinate to the company before and after Omicron strain infection was detected,including Omicron convalescent plasma(OP)collected from different plasma stations from December 2022 to May 2023 and normal pool plasma(VN)feed in March 2023 which collected from March 2022 to December 2022.According to the results,the difference and the change rule with time of SARS-CoV-2-IgG before and after Omicron strain infection were analyzed.Results The linearity of SARS-CoV-2-IgG ranged from 6.25 to 200 EIU/mL,the accuracy in-batch ranged from 81.793%to 106.985%,the precision in-batch ranged from 1.100%to 13.000%,and the total error in-batch ranged from 2.988%to 22.679%.The accuracy between batches ranged from 90.788%to 96.893%,the precision between batches ranged from 4.870%to 6.272%,and the total error between batches ranged from 9.192%to 15.399%.The results of pseudovirus neutralizing antibody showed that the potency of different virus strain neutralizing antibodies were in the order of prototype strainBA.2BA.4/5BF.7≈BQ.1.1BA.1 and the correlation between WP1 and WP2 was high(Pearson r=0.9311,P=0.0023)which indicated that the potency distribution of neutralizing antibodies of different virus strain in Omicron convalescent plasma was basically stable.Compared with the mixed convalescent plasma sample G128 collected in June 2022,the potency of Omicron neutralizing antibodies of WP series were significantly higher,the ratio of BA.2 antibody to prototype antibody increased from 26.9%(before infection)to 82.6%-87.5%(after infection).The results of VN series before Omicron infection were 100 EIU/mL,and the results of OP series after Omicron infection showed that the plasma collected from the beginning of December 2022 was the peak of antibody in the same month,and then dropped sharply,entering a short plateau in February-March 2023(potency was about 40%of the peak value),and then dropped sharply again in April(potency was about 20%of the peak value).Conclusion The potency and proportion of neutralizing antibody against Omicron subtype in convalescent plasma after COVID-19 Omicron strain infection increased significantly.IgG antibody of plasma donors in different regions reached its peak in the month of infection,then continued to dropped sharply.The best collection period of plasma that can be used for human COVID-19 immunoglobulin for intravenous injection was 1 to 2 months after infection.