A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies
ABSTRACT Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodi...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-02-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02996-22 |
| _version_ | 1811164396319145984 |
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| author | Zhi Cao Dehua Yin Lihong Zhang Shuai Ma Ke Zhang Ruimei Yang Hu Shan Zhihua Qin |
| author_facet | Zhi Cao Dehua Yin Lihong Zhang Shuai Ma Ke Zhang Ruimei Yang Hu Shan Zhihua Qin |
| author_sort | Zhi Cao |
| collection | DOAJ |
| description | ABSTRACT Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection. |
| first_indexed | 2024-04-10T15:21:56Z |
| format | Article |
| id | doaj.art-c2df4f45e1d14b138a409aced665c03a |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-10T15:21:56Z |
| publishDate | 2023-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-c2df4f45e1d14b138a409aced665c03a2023-02-14T14:15:49ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972023-02-0111110.1128/spectrum.02996-22A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus AntibodiesZhi Cao0Dehua Yin1Lihong Zhang2Shuai Ma3Ke Zhang4Ruimei Yang5Hu Shan6Zhihua Qin7College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaQingdao Animal Disease Prevention and Control Center, Qingdao, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaCollege of Veterinary Medicine, Qingdao Agricultural University, Qingdao, ChinaABSTRACT Monoclonal and polyclonal antibodies are mostly used for the development of traditional enzyme-linked immunosorbent assays (ELISAs), but the use of certain conventional antibodies may be limited by their low yield, the difficulty of their isolation, and their high cost. Heavy-chain antibodies derived from camelids with naturally missing light chains can overcome these deficiencies and are an excellent alternative to conventional antibodies. In this study, a nanobody (Nb)-AviTag fusion protein was constructed, and the feasibility of its use as a high-sensitivity probe in a blocking ELISA (bELISA) for classical swine fever virus (CSFV) was investigated. The CSFV E2 recombinant protein expressed by the CHO expression system exhibited good reactogenicity and immunogenicity and induced the production of high CSFV antibody levels in rabbits. Three different clones of Nbs were successfully isolated using a phage display system in alpaca, and an Nb1-AviTag fusion protein was successfully expressed using an Escherichia coli expression system. The purified Nb1-AviTag fusion protein was then biotinylated in vitro to obtain Nb1-biotin. A novel bELISA was developed for the detection of CSFV antibodies in clinical serum using Nb1-biotin as a probe. The cutoff value of bELISA was 32.18%, the sensitivity of bELISA was higher than that of the bELISA kit with IDEXX antibody, and the coincidence rate was 94.7%. A rapid, low-cost, highly sensitive and highly specific CSFV E2 antibody-based bELISA method was successfully established and can be used for the serological evaluation of CSFV E2 subunit vaccines and the ELISA-based diagnosis of CSFV infection. IMPORTANCE Currently, the epidemic situation of classical swine fever (CSF) is sporadic, and cases of atypical swine fever are on the rise in China. Therefore, it is necessary to accurately eliminate suspected cases by using highly sensitive and specific diagnostic techniques. In our study, a rapid, low-cost, highly sensitivity, highly reliable and reproducible, and highly specific classical swine fever virus (CSFV) E2 antibody-based blocking ELISA method was successfully established by using the phage display system and the Nb1-AviTag fusion expression platform. It provides a new technique for serological evaluation of CSFV vaccines and ELISA-based diagnosis of CSFV infection.https://journals.asm.org/doi/10.1128/spectrum.02996-22classical swine fever virusE2 recombinant proteinnanobodyblocking ELISA |
| spellingShingle | Zhi Cao Dehua Yin Lihong Zhang Shuai Ma Ke Zhang Ruimei Yang Hu Shan Zhihua Qin A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies Microbiology Spectrum classical swine fever virus E2 recombinant protein nanobody blocking ELISA |
| title | A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies |
| title_full | A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies |
| title_fullStr | A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies |
| title_full_unstemmed | A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies |
| title_short | A Novel Blocking Enzyme-Linked Immunosorbent Assay Based on a Biotinylated Nanobody for the Rapid and Sensitive Clinical Detection of Classical Swine Fever Virus Antibodies |
| title_sort | novel blocking enzyme linked immunosorbent assay based on a biotinylated nanobody for the rapid and sensitive clinical detection of classical swine fever virus antibodies |
| topic | classical swine fever virus E2 recombinant protein nanobody blocking ELISA |
| url | https://journals.asm.org/doi/10.1128/spectrum.02996-22 |
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