Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probe to detect antibodies against Newcastle disease virus in the immunoassay

Autor: Yamin Sheng, Kun Wang, Qizhong Lu, Pinpin Ji, Baoyuan Liu, Jiahong Zhu, Qingyuan Liu, Yani Sun, Jingfei Zhang, En-Min Zhou, Qin Zhao
Jazyk: angličtina
Rok vydání: 2019
Předmět:
Zdroj: Journal of Nanobiotechnology, Vol 17, Iss 1, Pp 1-15 (2019)
Druh dokumentu: article
ISSN: 1477-3155
DOI: 10.1186/s12951-019-0468-0
Popis: Abstract Background Sensitive and specific antibodies can be used as essential probes to develop competitive enzyme-linked immunosorbent assay (cELISA). However, traditional antibodies are difficult to produce, only available in limited quantities, and ineffective as enzymatic labels. Nanobodies, which are single-domain antibodies (sdAbs), offer an alternative, more promising tool to circumvent these limitations. In the present work, a cELISA using nanobody-horseradish peroxidase (HRP) fusion protein firstly designed as a probe was developed for detecting anti-Newcastle disease virus (NDV) antibodies in chicken sera. Results In the study, a platform for the rapid and simple production of nanobody-HRP fusion protein was constructed. First, a total of 9 anti-NDV-NP protein nanobodies were screened from a immunised Bactrian camel. Then, the Nb5-HRP fusions were produced with the platform and used for the first time as sensitive reagents for developing cELISA to detect anti-NDV antibodies. The cut-off value of the cELISA was 18%, and the sensitivity and specificity were respectively 100% and 98.6%. The HI test and commercial ELISA kit (IDEXX) separately agreed 97.83% and 98.1% with cELISA when testing clinical chicken sera and both agreed 100% when testing egg yolks. However, for detecting anti-NDV antibodies in the sequential sera from the challenged chickens, cELISA demonstrated to be more sensitive than the HI test and commercial ELISA kit. Moreover, a close correlation (R2 = 0.914) was found between the percent competitive inhibition values of cELISA and HI titers. Conclusions A platform was successfully designed to easily and rapidly produce the nanobody-HRP fusion protein, which was the first time to be used as reagents for establishing cELISA. Results suggest that the platform supports the development of a cELISA with high sensitivity, simplicity, and rapid detection of anti-NDV antibodies. Overall, we believe that the platform based on nanobody-HRP fusions can be widely used for future investigations and treatment other diseases and viruses.
Databáze: Directory of Open Access Journals
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