tetano
Editor, Senior Moderator
Int J Nanomedicine. 2019 Nov 29;14:9337-9349. doi: 10.2147/IJN.S218458. eCollection 2019. [h=1]Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.[/h]
Du T[SUP]#[/SUP][SUP]1,[/SUP][SUP]2[/SUP], Zhu G[SUP]#[/SUP][SUP]1,[/SUP][SUP]2[/SUP], Wu X[SUP]1,[/SUP][SUP]2[/SUP], Fang J[SUP]1,[/SUP][SUP]2[/SUP], Zhou EM[SUP]1,[/SUP][SUP]2[/SUP].
[h=3]Author information[/h] 1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People's Republic of China. 2 Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling 712100, Shaanxi, People's Republic of China. # Contributed equally
[h=3]Abstract[/h] [h=4]Background:[/h] 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.
[h=4]Methods:[/h] Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel V[SUB]H[/SUB]H 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.
[h=4]Results:[/h] 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.
[h=4]Conclusion:[/h] A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.
? 2019 Du et al.
[h=4]KEYWORDS:[/h] NP; SIV; sdAb; sdAb-ELISA
PMID: 31819435 PMCID: PMC6890519 DOI: 10.2147/IJN.S218458
Free PMC Article
Du T[SUP]#[/SUP][SUP]1,[/SUP][SUP]2[/SUP], Zhu G[SUP]#[/SUP][SUP]1,[/SUP][SUP]2[/SUP], Wu X[SUP]1,[/SUP][SUP]2[/SUP], Fang J[SUP]1,[/SUP][SUP]2[/SUP], Zhou EM[SUP]1,[/SUP][SUP]2[/SUP].
[h=3]Author information[/h] 1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, People's Republic of China. 2 Scientific Observing and Experimental Station of Veterinary Pharmacology and Diagnostic Technology, Ministry of Agriculture, Yangling 712100, Shaanxi, People's Republic of China. # Contributed equally
[h=3]Abstract[/h] [h=4]Background:[/h] 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.
[h=4]Methods:[/h] Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel V[SUB]H[/SUB]H 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.
[h=4]Results:[/h] 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.
[h=4]Conclusion:[/h] A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.
? 2019 Du et al.
[h=4]KEYWORDS:[/h] NP; SIV; sdAb; sdAb-ELISA
PMID: 31819435 PMCID: PMC6890519 DOI: 10.2147/IJN.S218458
Free PMC Article