Characterization and application of a series of monoclonal antibodies against SARS-CoV-2 nucleocapsid protein

The ongoing coronavirus disease 2019 (COVID-19) pandemic has a significant global social and economic impact, and the emergence of new and more destructive mutant strains highlights the need for accurate virus detection. Here, 90 monoclonal antibodies (MAbs) that exclusively reacted with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) were generated. These MAbs did not cross-react with NPs of common human coronaviruses (HCoVs, i.e., 229E, OC43, HKU1, and NL63) and Middle East Respiratory Syndrome Coronavirus. Subsequently, overlapped peptides in individual fragments (N1–N4) of NP were synthesized. N1-3 (25-GSNQNGERSGARSKQ-39), N3-1 (217-AALALLLLDRLNQL-230), and N4-8 (393-TLLPAADLDDFSKQL-407) were identified as major epitopes using enzyme-linked immunoassay (ELISA) and recognized by 47, 1, and 18 MAbs, respectively. The 24 remaining MAbs exhibited no reactivity with all synthetic peptides. Among MAb-epitope pairs, only MAbs targeting epitope N1-3 displayed no cross-reaction with NPs of SARS-CoV-1 and other SARS-related CoVs. All Omicron variants contained a three-amino acid deletion (31ERS33) in the N1-3 region. Thus, MAbs targeting N1-3 failed to recognize these variants. Furthermore, a double-antibody sandwich ELISA for antigen detection was established using the optimal MAbs. Overall, a series of MAbs targeting SARS-CoV-2 NP was prepared, characterized with epitope mapping, and applied for the detection of SARS-CoV-2 antigens, and some novel B-cell epitopes of the viral NP were identified.     

Generation and characterization of a human monoclonal IgG antibody specific for HLA-B12

Abstract: We describe here the generation and characterization of a human monoclonal IgG antibody (UL/F14) specific for HLA-B12. The antibody is suitable for complement-dependent lysis on lymphoblastoid cell lines and stimulated peripheral blood mononuclear cells. UL/F14 competes for antigen binding with an HLA-B12 human monoclonal IgM antibody and with a specific alloantiserum. Protein chemistry shows that the monoclonal antibody UL/F14 can precipitate solubilized HLA-B12 molecules.     

Long-term coexistence of SARS-CoV-2 with antibody response in COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing coronavirus disease 2019 (COVID-19) has spread worldwide. Whether antibodies are important for the adaptive immune responses against SARS-CoV-2 infection needs to be determined. Here, 26 cases of COVID-19 in Jinan, China, were examined and shown to be mild or with common clinical symptoms, and no case of severe symptoms was found among these patients. Strikingly, a subset of these patients had SARS-CoV-2 and virus-specific IgG coexist for an unexpectedly long time, with two cases for up to 50 days. One COVID-19 patient who did not produce any SARS-CoV-2–bound IgG successfully cleared SARS-CoV-2 after 46 days of illness, revealing that without antibody-mediated adaptive immunity, innate immunity alone may still be powerful enough to eliminate SARS-CoV-2. This report may provide a basis for further analysis of both innate and adaptive immunity in SARS-CoV-2 clearance, especially in nonsevere cases.     

Evaluation of the correlation between the access SARS-CoV-2 IgM and IgG II antibody tests with the SARS-CoV-2 surrogate virus neutralization test

Fully automated immunoassays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies that are strongly correlated with neutralization antibodies (nAbs) are clinically important because they enable the assessment of humoral immunity after infection and vaccination. Access SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) II antibody tests are semi-quantitative, fully automated immunoassays that detect anti-receptor-binding domain (RBD) antibodies and might reflect nAb levels in coronavirus disease 2019 (COVID-19). However, no studies have investigated the clinical utility of these tests in association with nAbs to date. To evaluate the clinical utility of Access SARS-CoV-2 IgM and IgG II antibody tests and their correlation with the SARS-CoV-2 surrogate virus neutralization test (sVNT) that measures nAbs in patients with COVID-19, we analyzed 54 convalescent serum samples from COVID-19 patients and 89 serum samples from non-COVID-19 patients. The presence of anti-RBD antibodies was detected using Access SARS-CoV-2 IgM and IgG II antibody tests, while nAbs were measured by sVNT. The sensitivity and specificity of sVNT were 94.4% and 98.9%, respectively. There were strong positive correlations between the inhibition values of sVNT and the results of the Access SARS-CoV-2 IgM (R = 0.95, R² = 0.90, p < 0.001) and IgG II antibody tests (R = 0.96, R² = 0.92, p < 0.001). In terms of the presence of nAbs, the sensitivity and specificity were 98.1% and 98.9% in the IgM assay and 100.0% and 100.0% in the IgG II assay, respectively. The Access SARS-CoV-2 IgM and IgG II antibody tests showed high sensitivity and specificity for the detection of nAbs in COVID-19 patients and might be alternatives for measuring nAbs.     

一株新型鼠抗人PD-L2单克隆抗体的研制及其生物学特性的鉴定

为了研制特异性识别人PD-L2(B7-DC,CD273)的鼠单克隆抗体,并对其生物学特性和PD-L2分子的表达特性进行初步分析。以高表达人PD-L2分子的基因转染细胞L929/PD-L2作为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,以L929/PD-L2作为抗体筛选细胞,L929/mock为对照细胞,经间接免疫荧光标记和流式细胞术分析、反复筛选和多次克隆化培养,筛选出分泌特异性鼠抗人PD-L2单克隆抗体的杂交瘤细胞株;采用Western blot、Ig亚型快速定性试纸法、间接免疫荧光法和竞争结合抑制实验对单抗进行生物学特性的分析,继而利用该单抗进行免疫荧光标记和流式细胞术检测PD-L2在肿瘤细胞株和免疫细胞上的表达特性。结果显示,通过多次融合和反复筛选,成功获得一株特异性鼠抗人PD-L2(B7-DC)的杂交瘤,该杂交瘤分泌的单克隆抗体能特异识别人PD-L2分子。继而利用上述研制获得的单克隆抗体8F2进行免疫荧光标记和流式细胞术检测发现,PD-L2上调性表达在成熟的树突状细胞和调节性T细胞上。提示,成功地获得一株特异性鼠抗人PD-L2单克隆抗体,并对其生物学特性和表达谱进行初步分析,证明其识别抗原表位不同于商品化抗体,是一株新型鼠抗人PD-L2单抗,这为进一步研究PD-1/PD-L2信号通路在免疫应答中的生物学作用提供了有价值的物质基础。     

Persistence of the neutralizing antibody response after SARS-CoV-2 infection

ObjectiveNeutralizing antibodies are among the factors used to measure an individual's immune status for the control of infectious diseases. We aimed to confirm the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody levels in patients who had recovered from coronavirus disease 2019 (COVID-19).MethodsPlasma donors in South Korea who had completely recovered from SARS-CoV-2 infection had follow-up testing to determine the persistence of neutralizing antibodies using a plaque-reduction neutralization test and ELISA.ResultsOf the 111 participants—aged 20–29 years, 37/111 (33.3%); 30–39 years, 17/111 (15.3%); 40–49 years, 23/111 (20.7%); 50–59 years, 21/111 (18.9%); 60–65 years, 13/111 (11.7%); male, 43/111 (38.7%); female, 68/111 (61.3%)—66.1% still had neutralizing antibodies approximately 9 months (range 255–302 days) after confirmation of the diagnosis.ConclusionsIn this study we analysed the titre of neutralizing antibodies associated with predicting immune status in individuals with natural infection. Information about the persistence and change in levels of neutralizing antibodies against SARS-CoV-2 can be utilized to provide evidence for developing vaccination schedules for individuals with previous infection.     

Cartography of SARS-CoV-2 variants based on the susceptibility to therapeutic monoclonal antibodies

A comprehensive picture of a phenotypic relationship among severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has been poorly studied. Here, this study presents cartography showing how the wild-type strain of SARS-CoV-2 and 14 variants are alike or different from the perspective of the susceptibility to 12 therapeutic monoclonal antibodies. The Alpha variant is close to the wild-type strain, whereas the Beta, Gamma, and Delta variants diverge from the wild-type. The map highlights the very unique property of the Omicron variant. Interestingly, sublineages of the Omicron variants, BA.1, BA.2, and BA.4/5, differ substantially in the cartography.     

A cytotoxic monoclonal human hybridoma antibody (TrJ3) against HLA-B44(12) and -B45(12)

We have generated a human monoclonal cytotoxic IgM lambda antibody (TrJ3) that reacted specifically with all lymphoblastoid B-cell lines expressing HLA-B44(12) and B45(12). TrJ3 hybridoma supernatant was suitable for HLA-B12 typing of freshly isolated blood mononuclear cells. Analysis of available amino acid sequences of HLA-B molecules indicated that the alpha 1 domain does not contain the TrJ3 serological epitope. Since HLA-B44 is associated with a unique serine residue at position 167 that points towards the peptide binding groove, we propose that S167 of the alpha 2 domain helix is a critical part of the TrJ3 epitope.     

一株鼠抗人TLT-2单克隆抗体的研制及其生物学特性的鉴定

为了制备鼠抗人TLT-2单克隆抗体(mAb)并对其生物学特性进行初步的研究。以TLT-2基因转染细胞株免疫BALB/c小鼠,采用杂交瘤技术制备鼠抗人TLT-2 mAb,收集腹水,并用ProteinG亲和层析法纯化。以流式细胞术、IP和Western blot等方法鉴定该单抗的特异性。并通过流式细胞术、CCK8和ELISA等方法对单抗的生物学特性进行了初步分析。结果:获得一株持续稳定分泌鼠抗人TLT-2 mAb的杂交瘤细胞株,命名为8C10。杂交瘤细胞分泌的mAb的Ig亚类(型)为IgG1(κ)。IP和Western blot分析证明,8C10能特异性识别人TLT-2分子。经8C10荧光染色后流式细胞术的结果表明:TLT-2在T细胞表面呈弱阳性表达,在单核细胞和B细胞表面呈强阳性表达。细胞增殖实验结果显示,该mAb对T细胞的增殖有抑制作用。提示,成功获得了一株特异性鼠抗人TLT-2 mAb,该mAb对T细胞体外增殖及其细胞因子IFN-γ、IL-2、和IL-10的分泌均有明显的抑制作用。     

Longitudinal neutralizing antibody responses after SARS-CoV-2 infection: A convalescent cohort study in Taiwan

BackgroundUnderstanding the neutralizing antibody (NAb) titer against COVID-19 over time is important to provide information for vaccine implementation. The longitudinal NAb titer over one year after SARS-CoV-2 infection is still unclear. The purposes of this study are to evaluate the duration of the neutralizing NAb titers in COVID-19 convalescents and factors associated with the titer positive duration.MethodsA cohort study followed COVID-19 individuals diagnosed between 2020 and 2021 May 15th from the COVID-19 database from the Taiwan Centers for Disease Control. We analyzed NAb titers from convalescent SARS-CoV-2 individuals. We used generalized estimating equations (GEE) and a Cox regression model to summarize the factors associated with NAb titers against COVID-19 decaying in the vaccine-free population.ResultsA total of 203 convalescent subjects with 297 analytic samples were followed for a period of up to 588 days. Our study suggests that convalescent COVID-19 in individuals after more than a year and four months pertains to only 25% of positive titers. The GEE model indicates that longer follow-up duration was associated with a significantly lower NAb titer. The Cox regression model indicated the disease severity with advanced condition was associated with maintaining NAb titers (adjusted hazard ratio: 2.01, 95% CI: 1.11–3.63) and that smoking was also associated with higher risk of negative NAb titers (adjusted hazard ratio: 0.55, 95% CI: 0.33–0.92).ConclusionsNeutralizing antibody titers diminished after more than a year. The antibody titer response against SARS-CoV-2 in naturally convalescent individuals provides a reference for vaccinations.     

一株抗双链DNA单克隆抗体的制备及其特性研究

制备抗双链DNA(dsDNA)单克隆抗体并对其生物学特性进行初步研究。以活化淋巴细胞的DNA为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术制备单克隆抗体;用体内诱生法制备腹水,以间接酶链反应吸附试验(ELISA)法分析抗体的亚类、特异性和亲和力;用免疫荧光法检测抗体的荧光核型;用考马斯亮蓝法检测注入杂交瘤细胞的小鼠的尿蛋白含量。结果显示,成功获得了1株持续稳定分泌抗dsDNA单克隆抗体的杂交瘤细胞株,命名为6C2;该细胞株所分泌的抗体亚类为小鼠IgG2a,能特异性结合dsDNA,亲和力常数为2.67×10~8mol/L,免疫荧光核型为均质型;将6C2细胞注入正常BALB/c小鼠腹腔后,小鼠的尿蛋白含量较对照组明显增高。结果表明,抗dsDNA单克隆抗体6C2是致病性抗体,对于进一步研究抗dsDNA抗体的致病机制具有重要的价值。     

Peptide microarray-based analysis of antibody responses to SARS-CoV-2 identifies unique epitopes with potential for diagnostic test development

Humoral immunity to the Severe Adult Respiratory Syndrome (SARS) Coronavirus (CoV)-2 is not fully understood yet but is a crucial factor of immune protection. The possibility of antibody cross-reactivity between SARS-CoV-2 and other human coronaviruses (HCoVs) would have important implications for immune protection but also for the development of specific diagnostic ELISA tests. Using peptide microarrays, n = 24 patient samples and n = 12 control samples were screened for antibodies against the entire SARS-CoV-2 proteome as well as the Spike (S), Nucleocapsid (N), VME1 (V), R1ab, and Protein 3a (AP3A) of the HCoV strains SARS, MERS, OC43, and 229E. While widespread cross-reactivity was revealed across several immunodominant regions of S and N, IgG binding to several SARS-CoV-2-derived peptides provided statistically significant discrimination between COVID-19 patients and controls. Selected target peptides may serve as capture antigens for future, highly COVID-19-specific diagnostic antibody tests.     

The third inactivated vaccine booster dramatically enhanced SARS-CoV-2 antibody responses and did not influence the profile of prothrombotic antibody

The purpose of this study is to investigate the production of both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-specific antibodies and autoantibodies in serum following the third booster vaccination of the inactivated COVID-19 vaccine, and to study the effect of B cell subsets with CD27 and CD38 phenotypes in peripheral blood on antibody production. Routine blood indexes, SARS-CoV-2 antibodies, platelet factor 4 and seven antiphospholipid antibodies were detected both before and 2 months after vaccination in the medical staff of the Zhongnan Hospital of Wuhan University. Peripheral blood B cell subtypes were detected before vaccination. Following immunization, the positive rate of anti-N-S1 immunoglobulin (IgG) had increased from 24.8% to 91.3% and the average antibody concentration had increased by 11 times. The positive rate of neutralizing antibody had increased from 24.8% to 91.3%, the average antibody concentration had increased by 12 times, and the primary increased anti-S1 IgG subtype was that of IgG1. Peripheral blood CD27 + CD38+ B cells were positively correlated with antibody levels after vaccination and were a predictor of the antibody response. In addition, although some indicators showed slight absolute changes, the blood parameters and antiphospholipid antibodies of most volunteers were normal both before and after COVID-19 inactivated vaccine inoculation, and there was no statistical difference in abnormal rates either before or after inoculation. Antibodies in vivo were increased after vaccination with the inactivated vaccine, and IgG1 was the main subtype involved in response to the vaccine. Vaccination with the inactivated COVID-19 vaccine did not appear to affect thrombus-related autoantibodies.     

A comparison study of SARS-CoV-2 IgG antibody between male and female COVID-19 patients: A possible reason underlying different outcome between sex

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China at the end of 2019 has spread throughout the world and caused many thousands of deaths. The previous study reported a higher severe status rate and mortality rate in male patients in China. However, the reason underlying this difference has not been reported. The convalescent plasma containing a high level of SARS-CoV-2 immunoglobulin G (IgG) antibody has been used in clinical therapy and achieved good effects in China. In this study, to compare the differences of the SARS-CoV-2 IgG antibody between male and female patients, a total number of 331 patients confirmed SARS-CoV-2 infection were enrolled. The serum of these patients was collected during hospitalization and detected for the SARS-CoV-2 IgG antibody. Our data showed that the concentration of IgG antibody in mild, general, and recovering patients showed no difference between male and female patients. In severe status, compared with male patients, there were more female patients having a relatively high concentration of serum SARS-CoV-2 IgG antibody. In addition, the generation of IgG antibody in female patients was stronger than male patients in disease early phase. Our study identified a discrepancy in the SARS-CoV-2 IgG antibody level in male and female patients, which may be a potential cause leading to a different outcome of Coronavirus Disease 2019 between sex.     

抗MRSA-PBP2a转肽酶区蛋白单克隆抗体的制备、鉴定及初步应用

目的:制备和鉴定抗MRSA-PBP2a转肽酶区蛋白单克隆抗体(mAb),初步建立检测PBP2a的乳胶凝集方法。方法:以基因工程重组PBP2a转肽酶区蛋白为抗原,免疫BALB/c小鼠,制备鼠源性mAb,间接ELISA鉴定IgG亚类、腹水效价及亲和常数,Western blot检测mAb的特异性,用所得mAb致敏聚苯乙烯乳胶,建立检测PBP2a的乳胶凝集方法。结果:筛选出2株能稳定分泌抗PBP2a mAb的杂交瘤细胞株F1和F2,免疫球蛋白亚类均为IgG1类;腹水效价为0.5×106~1×106,亲和力常数分别为1.57×108M-1和5.43×109M-1。Western blot显示F1、F2抗体均能有效识别重组PBP2a转肽酶区蛋白及MRSA临床分离株中的天然PBP2a,用mAb F2建立了乳胶凝集法,敏感性达1mg/L。结论:获得2株特异性好、亲和力高、能稳定分泌抗PBP2a mAb的杂交瘤细胞株,为研制MRSA快速鉴定试剂盒奠定了良好的基础。     

抗人白细胞单克隆抗体的特异性及其特性鉴定

目的制备抗人白细胞单克隆抗体（ｍＡｂ），并对其识别抗原及组织的特异性进行鉴定。方法采用分离的人全白细胞悬液免疫Ｂａｌｂ／ｃ小鼠，取脾细胞与Ｓｐ２／０细胞常规融合，用间接ＥＬＩＳＡ筛选ｍＡｂ，流式细胞仪及免疫组化染色ＳＰ法鉴定其组织特异性。　结果成功地制备１株抗人白细胞　ｍＡｂ，命名为ＳＺ－１０５。ＥＬＩＳＡ法测定其腹水效价为１×１０－５。琼脂糖双扩散鉴定为ＩｇＧ１类。流式细胞仪间接免疫荧光技术及免疫组化ＳＰ法测定表明，ｍＡｂ　ＳＺ－１０５可选择性地与外周血液的粒细胞、单核细胞和淋巴细胞呈强阳性反应，而与红细胞及血小板不出现反应。该ｍＡｂ还可与正常人骨髓的白细胞前体起反应。另外，在肝、肺、脾、胸腺及淋巴结正常组织中的巨噬细胞表面，也有ＳＺ－１０５抗原表达。ＳＺ－１０５抗原经亲和层析纯化，再经ＳＤＳ－ＰＡＧＥ　和Ｗｅｓｔｅｒｎ　ｂｌｏｔ证实，ｍＡｂ　ＳＺ－１０５识别的抗原是相对分子质量（Ｍｒ）为７５　０００左右的单链蛋白多肽。结论　获得１株具有高度免疫学活性和组织特异性的抗人白细胞ｍＡｂ　ＳＺ－１０５，其对研究白细胞的分化、免疫功能，以及隐匿性急、慢性炎症疾病的放免显像诊断都具有一定的临床意义。     

抗创伤弧菌单克隆抗体杂交瘤细胞系的建立及特性鉴定

目的:制备高效、特异的抗创伤弧菌的单克隆抗体(mAb),并进行特性鉴定。方法:用创伤弧菌菌体蛋白抗原免疫小鼠,利用杂交瘤细胞融合技术,制备抗创伤弧菌的杂交瘤细胞株。用ELISA法及Western blot等筛选、鉴定其与创伤弧菌溶血素蛋白(vvhA)及其他重要海洋细菌的交叉反应性和效价。结果:共获得5株抗创伤弧菌的mAb,鉴定结果表明,5株mAb均具有良好的特异性和免疫反应性。结论:获得5株抗创伤弧菌的特异性mAb,为建立创伤弧菌快速检测试剂盒提供了重要制剂。