基于磁珠分选及多肽自组装介导的SARS-CoV-2抗体荧光定量检测方法的建立及验证  

Development and verification of a fluorescence quantitative detection method for SARS⁃CoV⁃2 antibodies based on magnetic bead sorting and peptide self⁃assembly

作　　者：金育 何春艳[1] 罗忠瑞 张玉芳 马云飞 刘野 JIN Yu;HE Chunyan;LUO Zhongrui;ZHANG Yufang;MA Yunfei;LIU Ye(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《中国生物制品学杂志》2024年第10期1257-1262,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(21874033);云南省科技厅科技计划项目(202101AS070051)。

摘　　要：目的建立基于磁珠分选及多肽自组装介导的严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)抗体荧光定量检测方法,并进行验证。方法通过点击化学反应将磁珠和SARS-CoV-2核衣壳(nucleocapsid,N)蛋白特异性结合,采用Western blot和BCA法检测其结合效果。用N蛋白标记的磁珠作为包被抗原,碱性磷酸酶(alkaline phosphatase,ALP)标记的驴抗人IgG作为酶标抗体,结合可组装的多肽及硫磺素-T(thioflavine-T,ThT)发光系统,建立SARS-CoV-2抗体定量检测方法,并验证其特异性、线性范围、最低检测限(limit of detection,LOD)、精密性和耐用性。采用建立的方法和市售ELISA试剂盒分别检测8份已知背景的血清样本,并计算二者的一致性。结果大部分SARS-CoV-2 N蛋白均已稳定结合至磁珠上。建立的方法可特异性检测阳性血清样本;SARS-CoV-2抗体IgG标准品浓度在2.5~80 ng/mL范围内,与荧光值呈良好的线性关系,线性方程为y=182.11 x+5095.5,R2=0.99;LOD为2.5 ng/mL;批内变异系数(coefficient of variation,CV)在3.9%~10.6%之间,批间CV在6.0%~13.8%之间;耐用性CV在1.3%~6.2%之间。建立的方法与市售ELISA试剂盒定量检测结果具有较高的一致性(R2=0.99,P<0.0001)。结论本研究建立的基于磁珠分选及多肽自组装介导的荧光定量检测方法具有良好的特异性、精密性和耐用性,可用于SARS-CoV-2抗体水平的定量检测。Objective To develop and verify a fluorescence quantitative detection method for antibody against severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)based on magnetic bead sorting and peptide self-assembly.Methods Magnetic beads were specifically bound to nucleocapsid(N)protein of SARS-CoV-2 by click chemistry reaction,which were detected by Western blot and BCA.Using magnetic beads labeled with N protein as coating antigen and donkey anti-human IgG labeled with alkaline phosphatase(ALP)as enzyme-labeled antibody,combined with assemblable peptide and thioflavine-T(ThT)luminescence system,a quantitative detection method for SARS-CoV-2 antibody was established,and the specificity,linear range,limit of detection(LOD),precision and durability were verified.The developed method and commercial ELISA kit were used to detect 8 serum samples with known background respectively,and the consistency between them was calculated.Results Most of SARS-CoV-2 N proteins were stably bound to magnetic beads.The developed method specifically detected positive serum samples.The concentration of SARS-CoV-2 IgG antibody standard ranged from 2.5 to 80 ng/mL,showing a good linear relationship with fluorescence value,and the linear equation was y=182.11 x+5095.5,R2=0.99;The LOD was 2.5 ng/mL;The coefficient of variation(CV)within batches ranged from 3.9%to 10.6%,the CV between batches ranged from 6.0%to 13.8%,and the CV of durability was within 1.3%~6.2%.The consistency between the results of the developed method and commercial ELISA kit was high(R2=0.99,P<0.0001).Conclusion The developed fluorescence quantitative detection method based on magnetic bead sorting and peptide self-assembly showed good specificity,precisionanddurability,which might be used for quantitative detection of SARS-CoV-2 antibody.

关 键 词：严重急性呼吸综合征冠状病毒2 抗体 磁珠分选 多肽自组装 

分 类 号：R392.7[医药卫生—免疫学]