Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry

Pradhan, Sunil Kumar; Kamble, Nitin Machindra; Pillai, Aravind S.; Gaikwad, Satish S.; Khulape, Sagar A.; Reddy, M R; Mohan, C. Madhan; Kataria, J.M.; Dey, Sohini

doi:10.1016/j.jviromet.2014.08.015

Cited by 20 publications

(17 citation statements)

References 27 publications

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“…Our results are in line with those of an indirect anti-PDCoV IgG ELISA based on the putative S1 portion of the S protein, as described by Thachil et al . [ 17 ]. In addition, the N protein exhibited a high degree of conservation, 98.8–100% identities, among different PDCoV strains.…”

Section: Discussionmentioning

confidence: 99%

“…The genome of PDCoV is approximately twenty-five kilobases in length, which is similar to those of other porcine coronaviruses, and it encodes four similar major structural proteins: the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins [ 13 , 15 ]. Reports derived from other coronaviruses indicated that the N protein is highly conserved among different strains, and it is widely used as a diagnostic antigen for the development of serologic diagnostic tools [ 1 , 8 , 11 , 17 ]. In the current study, a recombinant N protein-based indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was established to detect antibodies against porcine deltacoronavirus.…”

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confidence: 99%

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A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

Guo

et al. 2016

The Journal of Veterinary Medical Science

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Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

Guo

et al. 2016

The Journal of Veterinary Medical Science

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“…Single serum dilution ELISA has the advantage of screening 35-40 samples in one plate compared to 8-10 samples in serial dilution ELISA.Single serum dilution method was first demonstrated by Snyder et al (1983)for serological profiling of NDV using purified virus antigen by standard curve method using regression analysis.Earlier, single serum dilution method, following the same procedure has been successfully employed for developing ELISA for seromonitoring of chicken for various diseases such as ND, Infecctious bronchitis and Hydropericardium syndrome etc. (Mohan et al, 2006;Ramdass et al, 2008;Pradhan et al, 2014;Roy et al, 2014). Following the same method, using the regression equation developed and testing the serum at a single working dilution of 1:1000, a linear relationship was found between the predicted antibody titres and corresponding antibody titres.…”

Section: Diagnostic Sensitivity Specificity and Accuracy Of Con A-s-mentioning

confidence: 99%

Concanavalin A- Sandwich-ELISA for the detection of antibodies against Newcastle disease virus (NDV) in chicken

Kannaki

Priyanka

Haunshi

2019

IJAR

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Concanavalin A (Con A), a lectin interacts with carbohydrate moieties of viruses and provide stable and sensitive detection when used as a capture agent. Indirect ELISA methods need purified Newcastle disease virus (NDV) or recombinant antigens for adsorption, whereas use of Con A as capture agent will enable the use of non-purified and non-concentrated virus as antigen replacing costly and time-consuming virus purification step. Con A based sandwich ELISA with non-purified NDV whole virus antigen with single serum dilution format has been developed in this study. The optimum concentrations of the capture agent, Con A and non-purified antigen preparations were determined by checker-board titration. Briefly, microplates were coated with predetermined optimum concentration of ConA (0.5 mg/ml; 50µg per well) and incubated for 18h at 4°C. After washing, allantoic fluid with Newcastle disease virus (NDV) LaSota (HA titre, 210) at a constant predetermined dilution (1:1; 50µl) was coated and incubated for 45 min at 37°C, followed by blocking with 2 % bovine serum albumin for 45 min at 37°C. The antigen coated plates were used in the detection of antibody titre against NDV in serum samples at single serum dilution of 1: 500. Then, wells were added with goat anti-chicken IgG horseradish peroxidase conjugate and incubated for 1h at 37°C, followed by addition of TMB substrate and the plates were read spectrophotometrically at 450 nm. ELISA antibody titres were determined by standard serial dilution of positive sera and endpoints were calculated by a subtraction method. By using positive negative threshold curve (PNT), intercept and slope of the standard curve were calculated. Total of 271 random chicken serum samples were analyzed for antibodies against NDV by Haemagglutination inhibition assay (HI), indirect ELISA and compared with the Con A- S- ELISA developed in this study. The Con A-S-ELISA showed a high coefficient of correlation (r=0.85, n=271, P less than 0.01) and an agreement of ê=0.99 with the commercially available Indirect-ELISA. The relative sensitivity and specificity were 98% and 85% respectively in comparison to HI test. Hence, the Con A-S-ELISA is a simple, easy and effective for monitoring serum antibody levels against NDV.

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“…Optimization of ELISA An indirect ELISA protocol was first optimized by checkerboard titration following the methods as described previously [16,18,22]. For this, two-fold dilutions of IBV-N protein were made starting from 400 to 25 ng in a carbonate bi-carbonate coating buffer (Sigma, C-3041).…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

Production of Recombinant N Protein of Infectious Bronchitis Virus Using the Baculovirus Expression System and Its Assessment as a Diagnostic Antigen

Yılmaz

Faburay

Turan

et al. 2018

Appl Biochem Biotechnol

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The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA.

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Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry

Cited by 20 publications

References 27 publications

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus

Concanavalin A- Sandwich-ELISA for the detection of antibodies against Newcastle disease virus (NDV) in chicken

Production of Recombinant N Protein of Infectious Bronchitis Virus Using the Baculovirus Expression System and Its Assessment as a Diagnostic Antigen

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