Development and application of nsp5-ELISA for the detection of antibody to infectious bronchitis virus

Abstract

Infectious bronchitis virus (IBV) continues to be one of the most important poultry pathogens worldwide. The current commercially available enzyme-linked immunosorbent assay (ELISA) kits for IBV specific antibody detection are mostly based on the whole virion, and few serological tests based on nonstructural proteins of IBV have been developed. Herein, an alternative indirect ELISA for detection of IBV antibody was developed with IBV nonstructural protein 5 (nsp5) produced by Escherichia coli. Using an indirect immunofluorescence assay (IFA) and a commercial ELISA kit as reference, we optimized the nsp5-ELISA and determined its cut-off as 0.12. The diagnostic sensitivity (DSN), specificity (DSP) and accuracy of the nsp5-ELISA were 93.11%, 95.38% and 93.33%, respectively, compared with IFA in 660 field serum samples, and were 98.11%, 95.00% and 97.62%, respectively, compared with the commercial IBV ELISA kit (IDEXX) in 126 field sera samples. Furthermore, a time course of IBV specific antibody level detected by nsp5-ELISA following IBV infection and vaccination is consistent with that of IBV antibody detected by the commercial ELISA kit. The results presented in this study indicate that nsp5-ELISA has the potential to serve as a rapid, reliable and cost-effective method for IBV antibody detection. This study is the first to report the development of an nsp-based ELISA to detect an antibody to IBV.

Peptides with 16R in S2 protein showed broad reactions with sera against different types of infectious bronchitis viruses

“…To detect IBV antibodies as accurately and comprehensively as possible, many ELISA antibody detection methods have been established (Chen et al, 2011;Ding et al, 2015;Lei et al, 2017). All of these methods have good efficacy, but still have some limitations.…”

Novel protein chip for the detection of antibodies against infectious bronchitis virus

“…However, the preparation of purified virions for use as an antigen is time-consuming and expensive. In the present study, recombinant nonstructural proteins expressed in Escherichia coli antigen-based protein microarray was evaluated for the first time in the serological diagnosis of IB [4]. Protein microarrays have high sensitivity and good reproducibility in quantitative and qualitative assays, and they are a valuable asset when analyzing complex biological samples [16].…”

“…To further evaluate the CIT and RDT, 186 clinical serum samples were detected by the CIT, RDT, and nsp5 ELISA antibody test kit [4]. Subsequently, the positive rate of each method was determined.…”

“…It encodes four major structural proteins, namely, glycosylated spike protein (S), membrane protein (M), phosphorylated nucleoprotein (N), and envelope protein (E) [3], and 15 nonstructural proteins (nsp2-nsp16). Generally, nonstructural proteins are present in infected cells but not in the virus, and they only play a role in the process of virus infection and replication [4]. Chickens immunized with an inactivated vaccine will produce no antibodies or low levels of antibodies against viral nonstructural proteins.…”

“…However, few IBV detection methods have been developed based on nonstructural proteins (nsps). Our laboratory has established an nsp5 ELISA to detect IBV infection [4]. The nsp5 antibodies detected are likely to be non-neutralizing and exist in lower numbers than the ones generated by other proteins.…”