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.
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.
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DOI：
10.1128/spectrum.02996-22
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发表时间：
2023-02-14
影响因子：
3.7
通讯作者：
中科院分区：
文献类型：
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作者：
传统的酶联免疫吸附试验(ELISA)大多使用单抗和多克隆抗体，但某些常规抗体的产量低、分离困难、成本高，限制了其应用。从自然缺失轻链的骆驼中提取的重链抗体可以克服这些缺陷，是传统抗体的极好替代品。本研究构建了纳米抗体(Nb)-AviTag融合蛋白，并探讨了其作为猪瘟病毒(CSFV)阻断酶联免疫吸附试验(BIISA)高灵敏度探针的可行性。CHO表达系统表达的CSFV E2重组蛋白具有良好的反应性和免疫原性，可诱导兔产生高水平的CSFV抗体。利用噬菌体展示系统在羊驼体内成功分离到3个不同的NBS克隆，并利用大肠杆菌表达系统成功地表达了NB1-AviTag融合蛋白。将纯化的NB1-AviTag融合蛋白进行体外生物素化，得到NB1-生物素。以NB1-生物素为探针，建立了一种检测临床血清中猪瘟病毒抗体的新方法。BELISA的临界值为32.18%，与IDEXX抗体的敏感性高于BELISA试剂盒，符合率为94.7%。建立了一种基于CSFV E2抗体的快速、低成本、高灵敏度和高特异性的BELISA方法，可用于CSFV E2亚单位疫苗的血清学评价和基于ELISA法的CSFV感染诊断。当前，猪瘟疫情呈零星流行，中国非典型猪瘟病例呈上升趋势。因此，有必要利用高度敏感和特异的诊断技术，准确排除疑似病例。本研究利用噬菌体展示系统和NB1-AviTag融合表达平台，成功地建立了一种快速、低成本、高灵敏度、高可靠、高重复性、高特异性的猪瘟病毒E2抗体阻断ELISA方法。为CSFV疫苗的血清学评价和基于酶联免疫吸附试验的CSFV感染诊断提供了新的技术手段。
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.
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DOI：
10.3390/v9040086
发表时间：
2017-04-21
期刊：
Viruses
影响因子：
--
作者：
Blome S;Staubach C;Henke J;Carlson J;Beer M
通讯作者：
Beer M
影响因子：
4.3
作者：
Salvador, J. -Pablo;Vilaplana, Lluisa;Marco, M. -Pilar
通讯作者：
Marco, M. -Pilar
DOI：
10.13345/j.cjb.180076
发表时间：
2018-12-25
期刊：
Sheng wu gong cheng xue bao = Chinese journal of biotechnology
影响因子：
--
作者：
Kang, Xiaozhen;Cao, Jiali;Yuan, Quan
通讯作者：
Yuan, Quan
影响因子：
2.5
作者：
Chander, Vishal;Nandi, S.;Verma, Rishendra
通讯作者：
Verma, Rishendra
影响因子：
4.3
作者：
Postel, Alexander;Austermann-Busch, Sophia;Becher, Paul
通讯作者：
Becher, Paul