A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

Jiakai Zhao; Jiahong Zhu; Ying Wang; Mengting Yang; Qiang Zhang; Chong Zhang; Yuchen Nan; En-Min Zhou; Yani Sun; Qin Zhao

doi:10.1016/j.virs.2022.09.004

December 2022

. doi: 10.1016/j.virs.2022.09.004

Citation: Jiakai Zhao, Jiahong Zhu, Ying Wang, Mengting Yang, Qiang Zhang, Chong Zhang, Yuchen Nan, En-Min Zhou, Yani Sun, Qin Zhao. A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus .VIROLOGICA SINICA, 2022, 37(6) : 922-933. http://dx.doi.org/10.1016/j.virs.2022.09.004

基于纳米抗体的一种简易检测非洲猪瘟抗体竞争ELISA的建立

赵加凯 ^a ,
朱家宏 ^a ,
王映 ^a ,
杨梦婷 ^a ,
张强 ^a ,
张冲 ^b ,
南雨辰 ^a ,
周恩民 ^a ,
孙亚妮 ^a,, ,
赵钦 ^a,,

西北农林科技大学动物医学院

通讯作者： 孙亚妮, sunyani@nwsuaf.edu.cn; 赵钦, qinzhao_2004@nwsuaf.edu.cn
收稿日期： 2022-05-04
录用日期： 2022-07-17

摘要

非洲猪瘟（African Swine fever，ASF）是由非洲猪瘟病毒（African Swine fever virus，ASFV）感染引起的猪的急性、高度接触性、热性传染病。猪一旦感染死亡率可达100%，是我国重点防范的一类动物疫情，同时也是世界动物卫生组织（OIE）法定报告的动物疫病。由于非洲猪瘟具有高致病性和高传染性，目前还没有针对非洲猪瘟的有效疫苗或其他治疗方法，因此需要建立一种快速、敏感、简单的疾病诊断方法。本研究选取ASFV-p30蛋白为靶蛋白，基于纳米抗体的优势，从免疫后的双峰驼中筛选抗p30蛋白的纳米抗体，然后将纳米抗体与辣根过氧化物酶（HRP）进行融合表达，利用纳米抗体与HRP融合蛋白ASFV-p30-Nb75-HRP为竞争探针，建立了一种检测猪血清中抗ASFV抗体的竞争ELISA。直接ELISA显示ASFV-p30-Nb75-HRP融合蛋白在HEK293T细胞中分泌表达。竞争ELISA的判定值值为22.7%，具有良好的敏感性、特异性和重复性。建立的cELISA与商业ELISA试剂盒的符合率为100%。更重要的是，基于开发的cELISA的商业试剂盒显示出低成本和易于生产的优势。综上所述，我们开发了一种简单的基于纳米体的检测ASFV抗体的竞争ELISA，为监测猪群的ASFV感染提供了一种新的方法。
- 非洲猪瘟
- , ASFV
- , 纳米抗体
- , 竞争ELISA
- , 抗体检测

A simple nanobody-based competitive ELISA to detect antibodies against African swine fever virus

Jiakai Zhao ^a ,
Jiahong Zhu ^a ,
Ying Wang ^a ,
Mengting Yang ^a ,
Qiang Zhang ^a ,
Chong Zhang ^b ,
Yuchen Nan ^a ,
En-Min Zhou ^a ,
Yani Sun ^a,, ,
Qin Zhao ^a,,

a Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University; Yangling Observing and Experimental Station of National Data Center of Animal Health, Ministry of Agriculture, Yangling, 712100, China;

Corresponding author: Yani Sun, sunyani@nwsuaf.edu.cn Qin Zhao, qinzhao_2004@nwsuaf.edu.cn
Received Date: 04 May 2022
Accepted Date: 17 July 2022

Abstract

African swine fever virus (ASFV) infection is a big threat to the global pig industry. Because there is no effective vaccine, rapid, low-cost, and simple diagnosis methods are necessary to detect the ASFV infection in pig herds. Nanobodies, with advantages of small molecular weight and easy genetic engineering, have been universally used as reagents for developing diagnostic kits. In this study, the recombinant ASFV-p30 was expressed and served as an antigen to immunize the Bactrian camel. Then, seven nanobodies against ASFV-p30 were screened using phage display technique. Subsequently, the seven nanobodies fused horseradish peroxidase (nanobody-HRP) were secretory expressed and one fusion protein ASFV-p30-Nb75-HRP was selected with the highest sensitivity in blocking ELISA. Using the ASFV-p30-Nb75-HRP fusion protein as a probe, a competitive ELISA (cELISA) was developed for detecting anti-ASFV antibodies in pig sera. The cut-off value of cELISA was determined to be 22.7% by testing 360 negative pig sera. The detection limit of the cELISA for positive pig sera was 1:320, and there was no cross-reaction with anti-other swine virus antibodies. The comparative assay showed that the agreement of the cELISA with a commercial ELISA kit was 100%. More importantly, the developed cELISA showed low cost and easy production as a commercial kit candidate. Collectively, a simple nanobody-based cELISA for detecting antibodies against ASFV is developed and it provides a new method for monitoring ASFV infection in the pig herds.
- African swine fever virus (ASFV)
- , Nanobody
- , ASFV-p30
- , Nanobody-HRP fusion Protein
- , Competitive ELISA

References
1. Afonso, C.L., Alcaraz, C., Brun, A., Sussman, M.D., Onisk, D.V., Escribano, J.M., Rock, D.L., 1992. Characterization of p30, a highly antigenic membrane and secreted protein of african swine fever virus. Virology 189, 368–373.
2. Ai, Q., Lin, X., Xie, H., Li, B., Liao, M., Fan, H., 2021. Proteome analysis in pam cells reveals that african swine fever virus can regulate the level of intracellular polyamines to facilitate its own replication through arg1. Viruses 13, 1236.
3. Barderas, M.G., Wigdorovitz, A., Merelo, F., Beitia, F., Alonso, C., Borca, M.V., Escribano, J.M., 2000. Serodiagnosis of african swine fever using the recombinant protein p30 expressed in insect larvae. J. Virol. Methods 89, 129–136.
4. Barthelemy, P.A., Raab, H., Appleton, B.A., Bond, C.J., Wu, P., Wiesmann, C., Sidhu, S.S., 2008. Comprehensive analysis of the factors contributing to the stability and solubility of autonomous human vh domains. J. Biol. Chem. 283, 3639–3654.
5. Blome, S., Gabriel, C., Beer, M., 2013. Pathogenesis of african swine fever in domestic pigs and european wild boar. Virus Res. 173, 122–130.
6. Cao, Y., Han, D., Zhang, Y., Zhang, K., Du, N., Tong, W., Li, G., Zheng, H., Liu, C., Gao, F., Tong, G., 2021. Identification of one novel epitope targeting p54 protein of african swine fever virus using monoclonal antibody and development of a capable elisa. Res. Vet. Sci. 141, 19–25.
7. Conrath, K., Vincke, C., Stijlemans, B., Schymkowitz, J., Decanniere, K., Wyns, L., Muyldermans, S., Loris, R., 2005. Antigen binding and solubility effects upon the veneering of a camel VHH in framework-2 to mimic a vh. J. Mol. Biol. 350, 112–125.
8. Cubillos, C., Gómez-Sebastian, S., Moreno, N., Nuñez, M.C., Mulumba-Mfumu, L.K., Quembo, C.J., Heath, L., Etter, E.M., Jori, F., Escribano, J.M., Blanco, E., 2013. African swine fever virus serodiagnosis: a general review with a focus on the analyses of african serum samples. Virus Res. 173, 159–167.
9. de Villiers, E.P., Gallardo, C., Arias, M., da Silva, M., Upton, C., Martin, R., Bishop, R.P., 2010. Phylogenomic analysis of 11 complete african swine fever virus genome sequences. Virology 400, 128–136.
10. Dixon, L.K., Chapman, D.A., Netherton, C.L., Upton, C., 2013. African swine fever virus replication and genomics. Virus Res. 173, 3–14.
11. Dovgan, I., Koniev, O., Kolodych, S., Wagner, A., 2019. Antibody-oligonucleotide conjugates as therapeutic, imaging, and detection agents. Bioconjugate Chem. 30, 2483–2501.
12. Du, T., Zhu, G., Wu, X., Fang, J., Zhou, E.M., 2019. Biotinylated single-domain antibodybased blocking elisa for detection of antibodies against swine influenza virus. Int. J. Nanomed. 14, 9337–9349.
13. Duan, H., Ma, Z., Xu, L., Zhang, A., Li, Z., Xiao, S., 2020. A novel intracellularly expressed ns5b-specific nanobody suppresses bovine viral diarrhea virus replication. Vet. Microbiol. 240, 108449.
14. Duan, H., Chen, X., Zhao, J., Zhu, J., Zhang, G., Fan, M., Zhang, B., Wang, X., Sun, Y., Liu, B., Zhou, E.M., Zhao, Q., 2021. Development of a nanobody-based competitive enzyme-linked immunosorbent assay for efficiently and specifically detecting antibodies against genotype 2 porcine reproductive and respiratory syndrome viruses. J. Clin. Microbiol. 59, e0158021.
15. Friker, B., Schüpbach, G., 2021. Stay alert: probability of african swine fever introduction from eastern asia is almost as high as from eastern europe. Schweiz. Arch. Tierheilkd. 164, 651–659.
16. Gallardo, C., Reis, A.L., Kalema-Zikusoka, G., Malta, J., Soler, A., Blanco, E., Parkhouse, R.M., Leitão, A., 2009. Recombinant antigen targets for serodiagnosis of african swine fever. Clin. Vaccine Immunol. 16, 1012–1020.
17. Gaudreault, N.N., Richt, J.A., 2019. Subunit vaccine approaches for african swine fever virus. Vaccines (Basel) 7, 56.
18. Gómez-Puertas, P., Rodríguez, F., Oviedo, J.M., Brun, A., Alonso, C., Escribano, J.M., 1998. The african swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response. Virology 243, 461–471.
19. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E.B., Bendahman, N., Hamers, R., 1993. Naturally occurring antibodies devoid of light chains. Nature 363, 446–448.
20. Hernaez, B., Escribano, J.M., Alonso, C., 2008. African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein k (hnrnp-k) during infection. FEBS Lett. 582, 3275–3280.
21. Indrabalan, U.B., Suresh, K.P., Shivamallu, C., Patil, S.S., 2021. An extensive evaluation of codon usage pattern and bias of structural proteins p30, p54 and, p72 of the african swine fever virus (asfv). Virusdisease 32, 810–822.
22. Ji, P., Zhu, J., Li, X., Fan, W., Liu, Q., Wang, K., Zhao, J., Sun, Y., Liu, B., Zhou, E.M., Zhao, Q., 2020. Fenobody and ranbody-based sandwich enzyme-linked immunosorbent assay to detect newcastle disease virus. J. Nanobiotechnol. 18, 44.
23. Jia, N., Ou, Y., Pejsak, Z., Zhang, Y., Zhang, J., 2017. Roles of african swine fever virus structural proteins in viral infection. J Vet Res 61, 135–143.
24. Kazakova, A.S., Imatdinov, I.R., Dubrovskaya, O.A., Imatdinov, A.R., Sidlik, M.V., Balyshev, V.M., Krasochko, P.A., Sereda, A.D., 2017. Recombinant protein p30 for serological diagnosis of african swine fever by immunoblotting assay. Transbound Emerg Dis 64, 1479–1492.
25. Ley, V., Almendral, J.M., Carbonero, P., Beloso, A., Viñuela, E., Talavera, A., 1984. Molecular cloning of african swine fever virus DNA. Virology 133, 249–257.
26. Lin, Y., Cao, C., Shi, W., Huang, C., Zeng, S., Sun, J., Wu, J., Hua, Q., 2020. Development of a triplex real-time pcr assay for detection and differentiation of gene-deleted and wild-type african swine fever virus. J. Virol. Methods 280, 113875.
27. Liu, H., Wang, Y., Duan, H., Zhang, A., Liang, C., Gao, J., Zhang, C., Huang, B., Li, Q., Li, N., Xiao, S., Zhou, E.M., 2015. An intracellularly expressed nsp9-specific nanobody in marc-145 cells inhibits porcine reproductive and respiratory syndrome virus replication. Vet. Microbiol. 181, 252–260.
28. Lu, Q., Li, X., Zhao, J., Zhu, J., Luo, Y., Duan, H., Ji, P., Wang, K., Liu, B., Wang, X., Fan, W., Sun, Y., Zhou, E.M., Zhao, Q., 2020. Nanobody-horseradish peroxidase and-egfp fusions as reagents to detect porcine parvovirus in the immunoassays. J. Nanobiotechnol. 18, 7.
29. Lv, C., Zhao, Y., Jiang, L., Zhao, L., Wu, C., Hui, X., Hu, X., Shao, Z., Xia, X., Sun, X., Zhang, Q., Jin, M., 2021. Development of a dual elisa for the detection of cd2vunexpressed lower-virulence mutational asfv. Life 11, 1214.
30. Mu, Y., Jia, C., Zheng, X., Zhu, H., Zhang, X., Xu, H., Liu, B., Zhao, Q., Zhou, E.M., 2021. A nanobody-horseradish peroxidase fusion protein-based competitive elisa for rapid detection of antibodies against porcine circovirus type 2. J. Nanobiotechnol. 19, 34.
31. Murgia, M.V., Mogler, M., Certoma, A., Green, D., Monaghan, P., Williams, D.T., Rowland, R.R.R., Gaudreault, N.N., 2019. Evaluation of an african swine fever (asf) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated asf virus. Arch. Virol. 164, 359–370.
32. Oura, C.A., Edwards, L., Batten, C.A., 2013. Virological diagnosis of african swine fever– comparative study of available tests. Virus Res. 173, 150–158.
33. Petrovan, V., Yuan, F., Li, Y., Shang, P., Murgia, M.V., Misra, S., Rowland, R.R.R., Fang, Y., 2019. Development and characterization of monoclonal antibodies against p30 protein of african swine fever virus. Virus Res. 269, 197632.
34. Posner, J., Barrington, P., Brier, T., Datta-Mannan, A., 2019. Monoclonal antibodies: past, present and future. Handb. Exp. Pharmacol. 260, 81–141.
35. Rai, A., Pruitt, S., Ramirez-Medina, E., Vuono, E.A., Silva, E., Velazquez-Salinas, L., Carrillo, C., Borca, M.V., Gladue, D.P., 2020. Identification of a continuously stable and commercially available cell line for the identification of infectious african swine fever virus in clinical samples. Viruses 12, 820.
36. Reis, A.L., Parkhouse, R.M.E., Penedos, A.R., Martins, C., Leitão, A., 2007. Systematic analysis of longitudinal serological responses of pigs infected experimentally with african swine fever virus. J. Gen. Virol. 88, 2426–2434.
37. Rowlands, R.J., Michaud, V., Heath, L., Hutchings, G., Oura, C., Vosloo, W., Dwarka, R., Onashvili, T., Albina, E., Dixon, L.K., 2008. African swine fever virus isolate, Georgia, 2007. Emerg. Infect. Dis. 14, 1870–1874.
38. Sánchez-Vizcaíno, J.M., Mur, L., Gomez-Villamandos, J.C., Carrasco, L., 2015. An update on the epidemiology and pathology of african swine fever. J. Comp. Pathol. 152, 9–21.
39. Sánchez, E.G., Quintas, A., Nogal, M., Castelló, A., Revilla, Y., 2013. African swine fever virus controls the host transcription and cellular machinery of protein synthesis. Virus Res. 173, 58–75.
40. Sheng, Y., Wang, K., Lu, Q., Ji, P., Liu, B., Zhu, J., Liu, Q., Sun, Y., Zhang, J., Zhou, E.M., Zhao, Q., 2019. Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against newcastle disease virus in the immunoassay. J. Nanobiotechnol. 17, 35.
41. Stravinskiene, D., Imbrasaite, A., Petrikaite, V., Matulis, D., Matuliene, J., Zvirbliene, A., 2019. New monoclonal antibodies for a selective detection of membrane-associated and soluble forms of carbonic anhydrase ix in human cell lines and biological samples. Biomolecules 9, 304.
42. Sun, E., Huang, L., Zhang, X., Zhang, J., Shen, D., Zhang, Z., Wang, Z., Huo, H., Wang, W., Huangfu, H., Wang, W., Li, F., Liu, R., Sun, J., Tian, Z., Xia, W., Guan, Y., He, X., Zhu, Y., Zhao, D., Bu, Z., 2021. Genotype i african swine fever viruses emerged in domestic pigs in China and caused chronic infection. Emerg. Microb. Infect. 10, 2183–2193.
43. Tabares, E., Fernandez, M., Salvador-Temprano, E., Carnero, M.E., Sanchez-Botija, C., 1981. A reliable enzyme linked immunosorbent assay for african swine fever using the major structural protein as antigenic reagent. Arch. Virol. 70, 297–300.
44. Teklue, T., Wang, T., Luo, Y., Hu, R., Sun, Y., Qiu, H.J., 2020. Generation and evaluation of an african swine fever virus mutant with deletion of the cd2v and UK genes. Vaccines (Basel) 8, 763.
45. Tesfagaber, W., Wang, L., Tsegay, G., Hagoss, Y.T., Zhang, Z., Zhang, J., Huangfu, H., Xi, F., Li, F., Sun, E., Bu, Z., Zhao, D., 2021. Characterization of anti-p54 monoclonal antibodies and their potential use for african swine fever virus diagnosis. Pathogens 10, 178.
46. Tran, H.T.T., Truong, A.D., Dang, A.K., Ly, D.V., Nguyen, C.T., Chu, N.T., Nguyen, H.T., Dang, H.V., 2021. Genetic characterization of african swine fever viruses circulating in north central region of vietnam. Transbound Emerg Dis 68, 1697–1699.
47. Vanlandschoot, P., Stortelers, C., Beirnaert, E., Ibañez, L.I., Schepens, B., Depla, E., Saelens, X., 2011. Nanobodies^®: new ammunition to battle viruses. Antivir. Res. 92, 389–407.
48. Vincke, C., Muyldermans, S., 2012. Introduction to heavy chain antibodies and derived nanobodies. Methods Mol. Biol. 911, 15–26.
49. Vincke, C., Gutiérrez, C., Wernery, U., Devoogdt, N., Hassanzadeh-Ghassabeh, G., Muyldermans, S., 2012. Generation of single domain antibody fragments derived from camelids and generation of manifold constructs. Methods Mol. Biol. 907, 145–176.
50. Wang, A., Jia, R., Liu, Y., Zhou, J., Qi, Y., Chen, Y., Liu, D., Zhao, J., Shi, H., Zhang, J., Zhang, G., 2020a. Development of a novel quantitative real-time pcr assay with lyophilized powder reagent to detect african swine fever virus in blood samples of domestic pigs in China. Transbound Emerg Dis 67, 284–297.
51. Wang, D., Yu, J., Wang, Y., Zhang, M., Li, P., Liu, M., Liu, Y., 2020b. Development of a real-time loop-mediated isothermal amplification (lamp) assay and visual lamp assay for detection of african swine fever virus (asfv). J. Virol. Methods 276, 113775.
52. Wang, J., Wang, J., Geng, Y., Yuan, W., 2017. A recombinase polymerase amplificationbased assay for rapid detection of african swine fever virus. Can. J. Vet. Res. 81, 308–312.
53. Wang, Y., Fan, Z., Shao, L., Kong, X., Hou, X., Tian, D., Sun, Y., Xiao, Y., Yu, L., 2016. Nanobody-derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications. Int. J. Nanomed. 11, 3287–3303.
54. Wang, Y., Xu, L., Noll, L., Stoy, C., Porter, E., Fu, J., Feng, Y., Peddireddi, L., Liu, X., Dodd, K.A., Jia, W., Bai, J., 2020c. Development of a real-time pcr assay for detection of african swine fever virus with an endogenous internal control. Transbound Emerg Dis 67, 2446–2454.
55. Yu, X., Zhu, X., Chen, X., Li, D., Xu, Q., Yao, L., Sun, Q., Ghonaim, A.H., Ku, X., Fan, S., Yang, H., He, Q., 2021. Establishment of a blocking elisa detection method for against african swine fever virus p30 antibody. Front. Vet. Sci. 8, 781373.
56. Yuan, F., Petrovan, V., Gimenez-Lirola, L.G., Zimmerman, J.J., Rowland, R.R.R., Fang, Y., 2021. Development of a blocking enzyme-linked immunosorbent assay for detection of antibodies against african swine fever virus. Pathogens 10, 760.
57. Zhang, X., Liu, X., Wu, X., Ren, W., Zou, Y., Xia, X., Sun, H., 2021. A colloidal gold test strip assay for the detection of african swine fever virus based on two monoclonal antibodies against p30. Arch. Virol. 166, 871–879.
58. Zhou, G., Shi, Z., Luo, J., Cao, L., Yang, B., Wan, Y., Wang, L., Song, R., Ma, Y., Tian, H., Zheng, H., 2022. Preparation and epitope mapping of monoclonal antibodies against african swine fever virus p30 protein. Appl. Microbiol. Biotechnol. 106, 1199–1210.
59. Zhou, X., Li, N., Luo, Y., Liu, Y., Miao, F., Chen, T., Zhang, S., Cao, P., Li, X., Tian, K., Qiu, H.J., Hu, R., 2018. Emergence of african swine fever in China, 2018. Transbound Emerg Dis 65, 1482–1484.
Proportional views
Electronic Supplementary Material

10.1016j.virs.2022.09.004-ESM.docx