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

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

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
Available online: 08 September 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

Electronic Supplementary Material

10.1016j.virs.2022.09.004-ESM.docx
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.
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