One-Step Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein Using Screened Fv-Antibodies

IF 5.5 3区 工程技术 Q1 BIOCHEMICAL RESEARCH METHODS BioChip Journal Pub Date : 2024-04-02 DOI:10.1007/s13206-024-00151-5
Jaeyong Jung, Jeong Soo Sung, Tae-Hun Kim, Min-Jung Kang, Joachim Jose, Hyun-Jin Shin, Jae-Chul Pyun
{"title":"One-Step Immunoassay for the Detection of SARS-CoV-2 Nucleocapsid Protein Using Screened Fv-Antibodies","authors":"Jaeyong Jung, Jeong Soo Sung, Tae-Hun Kim, Min-Jung Kang, Joachim Jose, Hyun-Jin Shin, Jae-Chul Pyun","doi":"10.1007/s13206-024-00151-5","DOIUrl":null,"url":null,"abstract":"<p>Fv-antibodies against the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were screened from an Fv-antibody library, and a one-step immunoassay was performed to detect SARS-CoV-2 using real viral samples. The Fv-antibody library was prepared using site-directed mutagenesis of the CDR3 region, which was composed of 11 amino acids. To screen the target <i>Escherichia coli</i> from the Fv-antibody library, the expressed probes [N-terminal domain (NTD) labeled with GFP and C-terminal domain (CTD) labeled with GFP] were reacted separately with the Fv-antibody library. After oligonucleotide sequencing, two clones for each probe were selected as the final clones. The screened Fv-antibodies with the binding affinity to NTD (or CTD) were expressed as soluble proteins, and the affinity constant (K<sub>D</sub>) was calculated to be 25.4 nM for NTD and 26.9 nM for CTD. The expressed Fv-antibodies were used for the one-step immunoassay based on switching-peptides, which were bound to the expressed Fv-antibodies. The one-step immunoassay based on Fv-antibodies could be used for the linear detection of SARS-CoV-2 NP, and the limit of detection (LOD) was estimated to be 9.6 nM (438 ng/mL) for Anti-NTD and 14.1 nM (639 ng/mL) for Anti-CTD. For the demonstration of one-step immunoassay for SARS-CoV-2, NATtrol™ SARS-CoV-2 real sample was used, and the LOD was estimated to be 29.7 copies/mL (Ct = 39.5) using Anti-NTD and 117.8 copies/mL (Ct = 38.0) using Anti-CTD. The measured LOD for the detection of SARS-CoV-2 using a one-step immunoassay based on the switching-peptide was considered feasible for the medical diagnosis of COVID-19. Finally, the interaction between the screened Fv-antibodies and SARS-CoV-2 NP was investigated using docking simulation.</p>","PeriodicalId":8768,"journal":{"name":"BioChip Journal","volume":"14 1","pages":""},"PeriodicalIF":5.5000,"publicationDate":"2024-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"BioChip Journal","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s13206-024-00151-5","RegionNum":3,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

Fv-antibodies against the nucleocapsid protein (NP) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were screened from an Fv-antibody library, and a one-step immunoassay was performed to detect SARS-CoV-2 using real viral samples. The Fv-antibody library was prepared using site-directed mutagenesis of the CDR3 region, which was composed of 11 amino acids. To screen the target Escherichia coli from the Fv-antibody library, the expressed probes [N-terminal domain (NTD) labeled with GFP and C-terminal domain (CTD) labeled with GFP] were reacted separately with the Fv-antibody library. After oligonucleotide sequencing, two clones for each probe were selected as the final clones. The screened Fv-antibodies with the binding affinity to NTD (or CTD) were expressed as soluble proteins, and the affinity constant (KD) was calculated to be 25.4 nM for NTD and 26.9 nM for CTD. The expressed Fv-antibodies were used for the one-step immunoassay based on switching-peptides, which were bound to the expressed Fv-antibodies. The one-step immunoassay based on Fv-antibodies could be used for the linear detection of SARS-CoV-2 NP, and the limit of detection (LOD) was estimated to be 9.6 nM (438 ng/mL) for Anti-NTD and 14.1 nM (639 ng/mL) for Anti-CTD. For the demonstration of one-step immunoassay for SARS-CoV-2, NATtrol™ SARS-CoV-2 real sample was used, and the LOD was estimated to be 29.7 copies/mL (Ct = 39.5) using Anti-NTD and 117.8 copies/mL (Ct = 38.0) using Anti-CTD. The measured LOD for the detection of SARS-CoV-2 using a one-step immunoassay based on the switching-peptide was considered feasible for the medical diagnosis of COVID-19. Finally, the interaction between the screened Fv-antibodies and SARS-CoV-2 NP was investigated using docking simulation.

查看原文
分享 分享
微信好友 朋友圈 QQ好友 复制链接
本刊更多论文
使用筛选过的 Fv 抗体检测 SARS-CoV-2 核壳蛋白的一步免疫测定法
从Fv-抗体文库中筛选出了针对严重急性呼吸系统综合征冠状病毒2(SARS-CoV-2)核壳蛋白(NP)的Fv-抗体,并使用真实病毒样本进行了一步免疫测定来检测SARS-CoV-2。Fv 抗体文库是通过定点突变 CDR3 区域制备的,该区域由 11 个氨基酸组成。为了从 Fv 抗体库中筛选目标大肠杆菌,表达的探针[标记有 GFP 的 N 端结构域(NTD)和标记有 GFP 的 C 端结构域(CTD)]分别与 Fv 抗体库反应。寡核苷酸测序后,每个探针选出两个克隆作为最终克隆。筛选出的与 NTD(或 CTD)具有结合亲和力的 Fv-抗体被表达为可溶性蛋白,计算出其亲和力常数(KD)为:NTD 25.4 nM,CTD 26.9 nM。表达的 Fv- 抗体被用于基于切换肽的一步式免疫测定,切换肽与表达的 Fv- 抗体结合。基于 Fv- 抗体的一步式免疫测定可用于线性检测 SARS-CoV-2 NP,Anti-NTD 的检测限(LOD)估计为 9.6 nM(438 ng/mL),Anti-CTD 的检测限(LOD)估计为 14.1 nM(639 ng/mL)。为了验证一步式免疫测定法检测SARS-CoV-2,使用了NATtrol™ SARS-CoV-2真实样本,使用Anti-NTD的LOD估计为29.7拷贝/毫升(Ct = 39.5),使用Anti-CTD的LOD估计为117.8拷贝/毫升(Ct = 38.0)。使用基于切换肽的一步式免疫测定法检测 SARS-CoV-2 所测得的 LOD 值被认为可用于 COVID-19 的医学诊断。最后,利用对接模拟研究了筛选出的 Fv- 抗体与 SARS-CoV-2 NP 之间的相互作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 去求助
来源期刊
BioChip Journal
BioChip Journal 生物-生化研究方法
CiteScore
7.70
自引率
16.30%
发文量
47
审稿时长
6-12 weeks
期刊介绍: BioChip Journal publishes original research and reviews in all areas of the biochip technology in the following disciplines, including protein chip, DNA chip, cell chip, lab-on-a-chip, bio-MEMS, biosensor, micro/nano mechanics, microfluidics, high-throughput screening technology, medical science, genomics, proteomics, bioinformatics, medical diagnostics, environmental monitoring and micro/nanotechnology. The Journal is committed to rapid peer review to ensure the publication of highest quality original research and timely news and review articles.
期刊最新文献
Advancing Blood–Brain Barrier-on-a-Chip Models Through Numerical Simulations Advanced Microfluidic Platform for Tumor Spheroid Formation and Cultivation Fabricated from OSTE+ Polymer Classification of DNA Mixtures by Nanoelectrokinetic Driftless Preconcentration Fabrication of Nephrotoxic Model by Kidney-on-a-Chip Implementing Renal Proximal Tubular Function In Vitro Development of Multi-HRP-Conjugated Branched PEI/Antibody-Functionalized Gold Nanoparticles for Ultra-Sensitive ELISA
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
现在去查看 取消
×
提示
确定
0
微信
客服QQ
Book学术公众号 扫码关注我们
反馈
×
意见反馈
请填写您的意见或建议
请填写您的手机或邮箱
已复制链接
已复制链接
快去分享给好友吧!
我知道了
×
扫码分享
扫码分享
Book学术官方微信
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术
文献互助 智能选刊 最新文献 互助须知 联系我们:info@booksci.cn
Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。
Copyright © 2023 Book学术 All rights reserved.
ghs 京公网安备 11010802042870号 京ICP备2023020795号-1