Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Abstract

BackgroundThe avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.MethodsConstructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).ResultsThe expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.ConclusionsThe association between the ELISA and Western blot techniques developed in this study with a subunit of IBV...

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

“…Indirect ELISA based on rHc-CS was optimized and developed to complement the immunoblotting results. Previously, combined use of ELISA and immunoblotting has been reported as a powerful tool to immuno-diagnose different infections [38,41]. In this study, rHc-CS indirect-ELISA showed higher diagnostic specificity and sensitivity as compared to somatic antigen-based indirect-ELISA format reported in previous study [20].…”

“…The ELISA is a powerful tool as it provides a less time consuming, easy and safe way to perform serological detection. Therefore the association between ELISA and immunoblotting techniques can be seen as a potent way to increase sensitivity for immunodiagnosis purposes [38]. Previously, a study was conducted for prepatent detection of specific H. contortus antibody, early and late patency of H. contortus infections using ELISA based on Hc26 and immunoblotting in sheep [37].…”

“…ROC analysis was used to simulate the influence of different cut-off values on sensitivity and specificity of the test [38]. ROC curves were obtained using statistical software MedCalc (version 15; http://www.medcalc.be).…”

Efficacy of Recombinant S1 Protein Expressed in Pichia pastoris in Chicken & Using for Detection Titer Antibodies against Avian Infectious Bronchitis by ELISA

“…They indicated that S1 glycoprotein can induce immune response in chicken [16]. Finger (2018) was used N protein IBV for making ELISA and western blot. He indicated that N protein can also be used for the immunodiagnosis of avian infectious bronchitis (3).…”

“…The IBV genome is a single-stranded, positive-sense RNA that is 27.6 kb in size. It is member of Gammacoronavirus, subfamily Coronavirinae, family Coronaviridae, Order Nidovirales [2,3,4,5]. It encodes four major structural proteins, named, glycosylated spike protein (S), membrane protein (M), phosphorylated nucleoprotein (N), and envelope protein (E), and 15 nonstructural proteins (nsp2-nsp16).…”