Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

Taofeng Du,1,2,* Guang Zhu,1,2,* Xiaoping Wu,1,2 Junyang Fang,1,2 En-Min Zhou1,2 1Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China; 2Scientific Observing and Experimental Sta...

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Autores principales:	Du T, Zhu G, Wu X, Fang J, Zhou EM
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2019
Materias:	siv np sdab sdab-elisa Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/2568538818ad4539996cded4504d1fe6
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spelling	oai:doaj.org-article:2568538818ad4539996cded4504d1fe62021-12-02T03:56:00ZBiotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus1178-2013https://doaj.org/article/2568538818ad4539996cded4504d1fe62019-11-01T00:00:00Zhttps://www.dovepress.com/biotinylated-single-domain-antibody-based-blocking-elisa-for-detection-peer-reviewed-article-IJNhttps://doaj.org/toc/1178-2013Taofeng Du,1,2,* Guang Zhu,1,2,* Xiaoping Wu,1,2 Junyang Fang,1,2 En-Min Zhou1,2 1Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China; 2Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling 712100, Shaanxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: En-Min ZhouDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of ChinaEmail zhouem@nwsuaf.edu.cnBackground: Enzyme-linked immunosorbent assay (ELISA) is a common method for diagnosing swine influenza. However, the production of classical antibodies is both costly and time-consuming. As a promising alternative diagnostic tool, single-domain antibodies (sdAbs) offer the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity.Methods: Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel VHH library. The sdAb5 was fused to the biotin acceptor peptide (BAP) and a His-Tag for its expression as monomeric and site-specific biotinylation in E.coli to develop an sdAb-based blocking ELISA (sdAb-ELISA). In the sdAb-ELISA, the anti-SIV antibodies from swine samples were used to block the binding between the biotinylated sdAb5 and SIV-NP protein coated on the ELISA plate. The specificity, sensitivity, and reproducibility of sdAb-ELISA were determined. In addition, consistency among sdAb-ELISA, commercial ELISA kit, and Western blot was evaluated.Results: Six SIV-NP-specific sdAbs were isolated, among which sdAb5 was identified as a dominant sdAb with higher reactivity. The cut-off value of biotinylated sdAb5-based bELISA was determined to be 29.8%. Compared with the positive reference serum against five different types of swine viruses, the developed sdAb-ELISA showed 100% specificity. The detection limit of sdAb-ELISA was 1:160 in an anti-SIV positive reference serum, which is lower than that of the commercial ELISA kit (1:20). In 78 diluted anti-SIV positive serum (1:80), 21 and 42 samples were confirmed as positive by the commercial ELISA kit and sdAb-ELISA, respectively. The coefficients of variation of intra- and inter-assay were 1.79–4.57% and 5.54–9.98%, respectively. The sdAb-ELISA and commercial ELISA kit showed a consistency of 94.17% in clinical swine serum samples. Furthermore, the coincidence rate was 96.67% between the results detected by sdAb-ELISA and Western blot.Conclusion: A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.Keywords: SIV, NP, sdAb, sdAb-ELISADu TZhu GWu XFang JZhou EMDove Medical Pressarticlesivnpsdabsdab-elisaMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol Volume 14, Pp 9337-9349 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	siv np sdab sdab-elisa Medicine (General) R5-920
spellingShingle	siv np sdab sdab-elisa Medicine (General) R5-920 Du T Zhu G Wu X Fang J Zhou EM Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus
description	Taofeng Du,1,2,* Guang Zhu,1,2,* Xiaoping Wu,1,2 Junyang Fang,1,2 En-Min Zhou1,2 1Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China; 2Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling 712100, Shaanxi, People’s Republic of China*These authors contributed equally to this workCorrespondence: En-Min ZhouDepartment of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of ChinaEmail zhouem@nwsuaf.edu.cnBackground: Enzyme-linked immunosorbent assay (ELISA) is a common method for diagnosing swine influenza. However, the production of classical antibodies is both costly and time-consuming. As a promising alternative diagnostic tool, single-domain antibodies (sdAbs) offer the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity.Methods: Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel VHH library. The sdAb5 was fused to the biotin acceptor peptide (BAP) and a His-Tag for its expression as monomeric and site-specific biotinylation in E.coli to develop an sdAb-based blocking ELISA (sdAb-ELISA). In the sdAb-ELISA, the anti-SIV antibodies from swine samples were used to block the binding between the biotinylated sdAb5 and SIV-NP protein coated on the ELISA plate. The specificity, sensitivity, and reproducibility of sdAb-ELISA were determined. In addition, consistency among sdAb-ELISA, commercial ELISA kit, and Western blot was evaluated.Results: Six SIV-NP-specific sdAbs were isolated, among which sdAb5 was identified as a dominant sdAb with higher reactivity. The cut-off value of biotinylated sdAb5-based bELISA was determined to be 29.8%. Compared with the positive reference serum against five different types of swine viruses, the developed sdAb-ELISA showed 100% specificity. The detection limit of sdAb-ELISA was 1:160 in an anti-SIV positive reference serum, which is lower than that of the commercial ELISA kit (1:20). In 78 diluted anti-SIV positive serum (1:80), 21 and 42 samples were confirmed as positive by the commercial ELISA kit and sdAb-ELISA, respectively. The coefficients of variation of intra- and inter-assay were 1.79–4.57% and 5.54–9.98%, respectively. The sdAb-ELISA and commercial ELISA kit showed a consistency of 94.17% in clinical swine serum samples. Furthermore, the coincidence rate was 96.67% between the results detected by sdAb-ELISA and Western blot.Conclusion: A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.Keywords: SIV, NP, sdAb, sdAb-ELISA
format	article
author	Du T Zhu G Wu X Fang J Zhou EM
author_facet	Du T Zhu G Wu X Fang J Zhou EM
author_sort	Du T
title	Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus
title_short	Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus
title_full	Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus
title_fullStr	Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus
title_full_unstemmed	Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus
title_sort	biotinylated single-domain antibody-based blocking elisa for detection of antibodies against swine influenza virus
publisher	Dove Medical Press
publishDate	2019
url	https://doaj.org/article/2568538818ad4539996cded4504d1fe6
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Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus

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