自2019年以来，由严重急性呼吸系统综合征冠状病毒2（新冠病毒，Sever acute respiratory syndrome coronavirus 2, SARS-CoV-2）导致的新型冠状病毒肺炎（新冠肺炎，coronavirus disease 2019，COVID-19），在全球范围内造成严重的感染和大量的死亡病例，对全球卫生系统和人类健康造成破坏。SARS-CoV-2在广泛传播过程中产生多种谱系突变株，不同突变株其感染能力、病毒载量、致病性均有所改变。已有突变株表现出对部分自然感染和接种疫苗后诱导的中和抗体产生免疫逃逸，为疾病预防和临床治疗带来巨大挑战。因此，快速筛选和鉴定出新冠病毒全人源广谱中和抗体，能够为新冠病毒感染后的治疗提供有效策略。本研究利用新冠肺炎康复者外周血单个核细胞（peripheral blood mononuclear cell，PBMC），通过磁珠分选结合流式分选技术，获得抗原特异性记忆B细胞；继续运用单B细胞克隆技术，在体外克隆表达重组全人源抗体；通过酶联免疫检测（enzyme linked immunosorbent assay，ELISA）和假病毒微量中和实验鉴定抗体的结合及中和能力，最终鉴定出多株靶向新冠病毒刺突蛋白（Spike，S）的单克隆抗体。

首先，本研究建立了磁珠分选结合流式分选技术。通过Pan B磁珠分选提高了样品中记忆B细胞比例，从1.36%提高至7.36%，提升了后续流式分选效率。比较使用该方案处理新鲜样本和冻存样本后的差异，本方案能够有效去除死细胞，使复苏后的冻存样本中细胞活率由80%提升至95%，抗原特异性记忆B细胞比例在两种样本类型中无明显差异。其次，本研究优化了抗体基因表达载体构建流程，通过重叠延伸PCR将抗体基因直接扩增构建至表达载体中，提高了载体构建的效率。单B细胞PCR结果显示，扩增抗体基因的效率超过90%。通过比对表达载体的测序结果，本研究共获得174株重组抗体序列。

本研究利用HEK293F悬浮培养系统重组表达了174株全人源抗体，通过ELISA结合实验筛选，鉴定出33株靶向Spike蛋白的抗体，并进一步确定了其中有15株为结合NTD的抗体、19株为结合RBD的抗体、28株为结合S1的抗体和8株为结合S2的抗体。多反应性结合实验结果显示，结合S2的抗体均能交叉识别SARS-CoV-2和SARS-CoV，部分RBD抗体能够交叉结合SARS-CoV-2和SARS-CoV，后续鉴定证实其为结合Class 3区域的抗体。研究发现JYTA108和JYTA172结合RBD Class 1和Class 2区域，具有广谱中和活性，能够中和包括原型株、Alpha、Gamma、lota、Epsilon、Delta、Lambda、BA.1、BA.2、BA.2.12.1、BA.2.75及BA.4/5株在内的假病毒毒株，但其对Omicron及其亚系突变株的中和活性有所下降；JYTA034结合RBD Class 3区域，具有广谱结合活性，能够结合原型株、Beta、Delta、C.1、BA.1及XBB突变株的RBD蛋白，其中和活性在检测的突变株中无明显差异。

为进一步提高抗体检测及筛选通量，本研究利用均相时间分辨荧光技术（Homogeneous Time Resolved Fluorescence， HTRF）构建了高通量RBD特异性抗体检测体系：使用2.5 μL 40 nM RBD蛋白即可检测抗体浓度，可测得的抗体浓度范围为0.003 μg/mL  – 40.8 μg/mL。

综上，本研究优化了单B细胞PCR克隆抗体的流程，建立了HTRF高通量筛选RBD抗体技术，完善了新发突发病原体全人源抗体筛选平台。利用新冠肺炎康复者PBMC样本成功分离鉴定了多株针对S蛋白的单克隆抗体，初步阐明了作用位点和机制，为潜在的治疗方法提供数据参考和理论依据。

Since 2019, Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has brought about serious infections and large numbers of deaths worldwide, causing damage to the global health system and human health. During the widespread transmission of SARS-CoV-2, a variety of lineage mutant strains have been produced, and their infectivity, viral load and pathogenicity have changed. Some mutant strains have shown immune escape to partial neutralizing antibodies induced by natural infection or vaccination, which poses great challenges for disease prevention and clinical treatment. Therefore, rapid screening and identification of fully human broad-spectrum neutralizing antibodies against SARS-CoV-2 could provide an effective strategy for its treatment. In this study, the peripheral blood mononuclear cells (PBMC) of recovered patients were used to obtain antigen-specific memory B cells through magnetic bead sorting combined with flow sorting technology. The recombinant fully human antibodies were cloned and expressed in vitro using single B cell cloning technology. The binding and neutralization abilities of antibodies were identified by enzyme linked immunosorbent assay (ELISA) and pseudovirus neutralization test. Finally, multiple monoclonal antibodies targeting spike (S) protein of SARS-CoV-2 were identified.

The study established the magnetic bead sorting combined with flow sorting technology. Sorted by Pan B magnetic beads, the proportion of memory B cells in the sample was increased from 1.36% to 7.36%, improving the efficiency of subsequent flow sorting. By comparing the difference between fresh blood collected from on-site and cryopreserved samples, this plan could effectively remove dead cells, and increased the viability of cell samples from 80% to 95% after cryopreservation and recovery. The proportion of antigen-specific memory B cells was not significantly different between the two sample types. The study optimized the construction process of the antibody gene expression vector, and directly amplified and extended the antibody gene into the expression vector through Overlap PCR, which improved the efficiency of vector construction. Single B cell PCR results showed that the efficiency of amplification of the antibody genes was over 90%. By sequencing and comparing the expression vectors, a total of 174 recombinant antibody sequences were obtained.

In the study, 174 strains of fully human antibodies were recombined and expressed using HEK293F suspension culture. Through ELISA binding test, 33 strains of antibodies targeting Spike protein were identified, of which 15, 19, 28 and 8 antibody strains bound to NTD, RBD, S1 and S2, respectively were further determined. The results of the polyreactive binding test showed that all S2-binding antibodies could cross-recognize SARS-CoV-2 and SARS-CoV, and some RBD antibodies could cross-recognize SARS-CoV-2 and SARS-CoV, and were subsequently identified as antibodies binding to Class 3 regions. The study found that JYTA108 and JYTA172 binding to RBD Class 1 and Class 2 regions, had broad-spectrum neutralizing activity, and could neutralize the prototype, Alpha, Gamma, lota, Epsilon, Delta, Lambda, BA.1, BA.2, BA.2.12 .1, BA.2.75 and BA.4/5 pseudoviruses, but their neutralizing activity in Omicron and its sublineage mutants was decreased. JYTA034 bound to the RBD Class 3 region and had a broad-spectrum binding activity. It could bind to all mutant RBD proteins detected by prototype, Beta, Delta, C.1, BA.1 and XBB strains. There was no significant difference in the binding activity among the tested mutant strains.

To further improve the throughput of antibody detection and screening, this study used Homogeneous Time Resolved Fluorescence (HTRF) to construct a high-throughput RBD-specific antibody detection system: Using 2.5 μL of 40 nM RBD protein, the antibody concentration ranged from 0.003 μg/mL to 40.8 μg/mL could be detected.

This study optimized the single B cell PCR cloning antibody process, established the HTRF high-throughput screening RBD antibody technology, and improved the screening platform for fully human antibodies against new pathogens. Multiple antibodies against S protein were successfully isolated and identified using PBMC samples from COVID survivors. Our study preliminarily clarified the action sites and mechanisms of those antibodies, providing data reference and theoretical basis for potential treatment.