Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay.
Bo Wang, Chunsheng Wang, Bo Li, Jin Yang, Pengfei Lin, Xuefeng Jin, Yaojie Niu, Wei Zhang, Xinshi Zhang, Ying Huang
{"title":"Isolation of camel single domain antibodies against Yersinia pestis V270 antigen based on a semi-synthetic single domain antibody library and development of a VHH-based lateral flow assay.","authors":"Bo Wang, Chunsheng Wang, Bo Li, Jin Yang, Pengfei Lin, Xuefeng Jin, Yaojie Niu, Wei Zhang, Xinshi Zhang, Ying Huang","doi":"10.1002/vms3.1532","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases.</p><p><strong>Objectives: </strong>To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study.</p><p><strong>Methods: </strong>The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen.</p><p><strong>Results: </strong>After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection.</p><p><strong>Conclusions: </strong>These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.</p>","PeriodicalId":23543,"journal":{"name":"Veterinary Medicine and Science","volume":"10 4","pages":"e1532"},"PeriodicalIF":1.8000,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11217587/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Veterinary Medicine and Science","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1002/vms3.1532","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"VETERINARY SCIENCES","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases.
Objectives: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi-synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study.
Methods: The semi-synthetic single domain antibody sequences consist of two parts: one is the FR1-FR3 region amplified by RT-PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3-FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy-chain antibodies (VHHs). Y. pestis low-calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi-synthetic library herein was screened using recombinant (LcrV) as a target antigen.
Results: After four cycles of panning the library, four VHH binders targeting 1-270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH-hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection.
Conclusions: These data demonstrate the great potential of the semi-synthetic library for use in isolation of antigen-specific nanobodies and the isolated specific VHHs can be used in antigen-capture immunoassays.
期刊介绍：
Veterinary Medicine and Science is the peer-reviewed journal for rapid dissemination of research in all areas of veterinary medicine and science. The journal aims to serve the research community by providing a vehicle for authors wishing to publish interesting and high quality work in both fundamental and clinical veterinary medicine and science.
Veterinary Medicine and Science publishes original research articles, systematic reviews, meta-analyses, and research methods papers, along with invited editorials and commentaries. Original research papers must report well-conducted research with conclusions supported by the data presented in the paper.
We aim to be a truly global forum for high-quality research in veterinary medicine and science, and believe that the best research should be published and made widely accessible as quickly as possible. Veterinary Medicine and Science publishes papers submitted directly to the journal and those referred from a select group of prestigious journals published by Wiley-Blackwell.
Veterinary Medicine and Science is a Wiley Open Access journal, one of a new series of peer-reviewed titles publishing quality research with speed and efficiency. For further information visit the Wiley Open Access website.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
