病毒学报

建立一种新型冠状病毒（Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2）S1、S2、NTD蛋白特异性B细胞及其亚型的标记技术，该技术可通过与流式细胞术联用检测抗原特异性B细胞及其亚型含量，或与10×免疫组库联用对抗原特异性B细胞进行测序。首先，测试最佳蛋白使用浓度：将生物素标记的S1、S2和NTD蛋白按照600 nmol/L、1 200 nmol/L、2 400 nmol/L、3 600 nmol/L和4 800 nmol/L分别与荧光素-亲和素偶联，通过流式检测S1+CD19+B、S2+CD19+B、NTD+CD19+B细胞占比，确定不同蛋白对应的最佳使用浓度；进一步优化生物素-蛋白与荧光素-亲和素的比例：将检测生物素-蛋白与荧光-亲和素的摩尔比分别按照4∶1,5∶1和6∶1比例测试，对小鼠脾脏S1+CD19+B、S2+CD19+B、NTD+CD19+B细胞及S1+CD19+CD27+B、S2+CD19+CD27+B、NTD+CD19+CD27+B细胞的检测效果，优化荧光素-亲和素的最佳使用量；通过测试BV421、Super Bright 600、PE、PE/Cy7和APC等荧光对小鼠脾细胞的非特异结合能力，选择非特异结合较弱的荧光；根据筛选的荧光，给生物素化的S1、S2和NTD均标记PE和APC荧光，建立检测这3个蛋白特异性B细胞和记忆B细胞的方法，并测试带核酸序列的荧光素-亲和素对B细胞和记忆B细胞的检测效果，测试该方法的稳定性。S1、S2和NTD蛋白浓度分别为4 800 nmol/L、3 600 nmol/L和3 600 nmol/L时，对B细胞的检测效果最佳，且对阴性小鼠的染色效果较低；生物素-S1蛋白与荧光素-亲和素的最佳摩尔比为6∶1时，检测结果最佳；生物素-S2蛋白、生物素-NTD蛋白与荧光素-亲和素均在4:1时，检测结果最佳，且在该浓度下BV421、APC、Super Bright 600、PE与细胞的非特异结合较弱；建立了一种检测SARS-CoV-2 S1、S2和NTD抗原特异性B细胞及记忆B细胞的方法，通过对一批样本的重复3次检测，发现该方法较为稳定，CV值≤20%。通过抗原特异性B细胞标记技术，实现了对SARS-CoV-2S1、S2和NTD蛋白特异性B细胞及其亚型标记技术方法，并且具有良好的适用性，可用于与流式细胞术检测抗原特异性B细胞及记忆B细胞和10×免疫组库测序联合使用。

To establish a labeling method for spike(S)1-, S2-, and N-terminal domain(NTD)-specific B cells(and their subtypes) of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2). This method could be used with flow cytometry to test, or in 10 × immune repertoire sequence to obtain the sequence of antigen-specific B cells and their subtypes. First, S1, S2, and NTD proteins were coupled with fluorescein –streptavidin(600, 1200, 2400, 3600 and 4800 nmol/L), respectively. Antigen-specific B cells were detected by flow cytometry to determine the optimal concentration. Second, the molar ratio of biotin – protein and fluorescein – streptavidin was tested at ratios of 4: 1, 5: 1, and 6: 1, respectively. According to the ratio of S1+cluster of differentiation(CD)19+B cells, S2+CD19+B cells, NTD+CD19+B cells, as well as S1+CD19+CD27+B cells, S2+CD19+CD27+B cells, and NTD+CD19+CD27+B cells, the optimal concentration was obtained. Third, the non-specific effects of different types of fluorescein(BV421, Super Bright 600, PE, PE/Cy7 and APC) were tested to evaluate their non-specific binding. Fourth, the established method was repeated thrice to test its stability in flow cytometry. We also tested it with fluorescein– avidin–biotin-containing nucleotide sequences which could be used in a 10 × immune repertoire sequence. First, the optimal concentrations(in nmol/L) of S1, S2, and NTD proteins were 4800, 3600, and 3600 nmol/L, respectively. Second, the optimal molar ratio of biotin–S1 protein to fluorescein–avidin was 6: 1, whereas the most suitable molar ratio of biotin – S2 protein and biotin – NTD protein to fluorescein – avidin was 4: 1, respectively. At this concentration, BV421, APC, Super Bright 600, and PE showed weak non-specific binding. Third, repeating this method thrice showed it to be highly stable, with a coefficient of variation of ≤20%, and it was suitable for fluorescein – avidin – biotin-containing nucleotide sequences. The established labeling technology for S1-, S2-, and NTD protein-specific B cells(and their subtypes) of SARS-CoV-2 was relatively stable, and could be used in conjunction with flow cytometry and 10 × immune repertoire sequencing.