#Western blot test and elisa test software#

It has a molecular mass of approximately 50 kDa and directly binds with the viral genome to form the virion nucleocapsid. The nucleocapsid protein, or N protein, consists of 409 amino acids. It encodes non-structural (accessory proteins) and four structural proteins: the nucleocapsid protein (N), the spike protein (S), the envelope protein (E), and the matrix protein (M). The IBV genome consists of a non-segmented positive-sense single-stranded RNA that is approximately 27.6 kb in length. The consequences of IBV are a significant threat to Brazil’s poultry industry. Brazil was once the world’s largest exporter of poultry and currently the world’s third largest producer of bird meat. Carcass condemnation due to the development of airsacculitis negatively impacts commercial sales of bird meat and eggs. Secondary bacterial infections will also contribute to economic losses. Infected birds exhibit reduced performance, consequently leading to a reduction in weight gain and deterioration in egg quality and quantity. The IBV is responsible for respiratory disease, which manifests in clinical symptoms such as sneezing and tracheal-bronchial rales that can lead to the development of more severe symptoms. The Avian coronavirus was previously classified, and is most commonly referred to, as avian infectious bronchitis virus (IBV). It is a highly contagious disease with a short incubation period. The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.Īvian infectious bronchitis (IB) is caused by a virus in the Coronaviridae family, genera Gammacoronavirus. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot. 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%.

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%. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. 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 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.

#Western blot test and elisa test software#

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). 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 were compared with those obtained from a commercial kit. Positive and negative sera for IBV were used as controls. 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. A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. 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). coli BL21(DE3) Star competent cells (Invitrogen). MethodsĬonstructed recombinant pAE/ n expression vectors were used to transform E.

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. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. 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. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The 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.