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HRP-conjugated-nanobody-based cELISA for rapid and sensitive clinical detection of ASFV antibodies

文献类型： 外文期刊

作者： Zhao, Huijun 1 ; Ren, Jiahui 1 ; Wu, Shuya 1 ; Guo, Haoran 1 ; Du, Yongkun 1 ; Wan, Bo 1 ; Ji, Pengchao 1 ; Wu, Yanan 1 ; Zhuang, Guoqing 1 ; Zhang, Angke 1 ; Zhang, Gaiping 1 ;

作者机构： 1.Henan Agr Univ, Coll Vet Med, Zhengzhou 450046, Henan, Peoples R China

2.Henan Agr Univ, Coll Vet Med, Int Joint Res Ctr Natl Anim Immunol, Zhengzhou 450046, Henan, Peoples R China

3.Henan Acad Agr Sci, Minist Agr, Key Lab Anim Immunol, Zhengzhou 450002, Henan, Peoples R China

关键词： Nanobody; cELISA; African swine fever virus; Serum antibody

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：5.56； 五年影响因子：5.365 ）

ISSN： 0175-7598

年卷期： 2022 年 106 卷 11 期

页码：

收录情况： SCI

摘要： African swine fever (ASF), which is caused by the ASF virus (ASFV), is a highly contagious hemorrhagic disease that causes high mortality to domestic porcine and wild boars and brings huge economic losses to world swine industry. Due to the lack of an effective vaccine, the control of ASF must depend on early, efficient, and cost-effective detection and strict control and elimination strategies. Traditional serological testing methods are generally associated with high testing costs, complex operations, and high technical requirements. As a promising alternative diagnostic tool to traditional antibodies, nanobodies (Nb) have the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity, although the system is dependent on the immunization of Bactrian camels to obtain the specific VHH library of the target protein. The application of Nbs in the detection of ASFV antibodies has not yet been reported yet. Using a phage display technology, one Nb against the ASFV p54 protein that exhibited high specificity and affinity, Nb8, was successfully screened. A HEK293T cell line stably expressing Nb8-horseradish peroxidase (HRP) fusion protein was established using the lentiviral expression system. Following the optimization of the reaction conditions, the Nb8-HRP fusion protein was successfully used to establish a competitive enzyme-linked immunosorbent assay (cELISA) to detect ASFV-specific antibodies in pig serum, for the first time. There was no cross-reaction with healthy pig serum, porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and classical swine fever virus (CSFV) positive sera. The optimal cut-off value for the cELISA by ROC analysis was 52.5%. A total of 209 serum samples were tested using the developed cELISA and a commercial ELISA kit. The results showed that the relative specificity of the cELISA was 98.97%, and the relative sensitivity of the cELISA was 93.3%, with the percent agreement between the two ELISA methods being 98.56%. In conclusion, a specific, sensitive, and repeatable cELISA was successfully developed based on the Nb8 as a probe, providing a promising method for the detection of anti-ASFV antibodies in clinical pig serum.

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