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

Yılmaz, Hüseyin; Faburay, Bonto; Turan, Nuri; Cotton-Caballero, Maira; Çetinkaya, Burhan; Gürel, Aydın; Yilmaz, Aysun; Cizmecigil, Utku Y.; Aydın, Özge; Tarakci, Eda Altan; Bayraktar, Erhan; Richt, Jüergen A.

doi:10.1007/s12010-018-2815-2

Cited by 5 publications

(6 citation statements)

References 26 publications

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“…In house, ELISA methods were standardized to analyze the sera of cats for the presence of antibodies to SARS-CoV-2 by using the purified recombinant S and RBD proteins of SARS-CoV-2 as the target antigen. Indirect ELISA protocols were standardized using checkerboard titration protocol, as described previously (30). For this, two-fold dilutions of the S and RBD proteins were prepared at a range of 250-4,000 ng/ml in PBS (Sigma) on 96-well ELISA plates (MaxiSorb flat bottom).…”

Section: Standardization Of Elisa For Estimation Of S and Rbd Antibody Levels In Serum Samples Collected From Catsmentioning

confidence: 99%

Presence of Antibodies to SARS-CoV-2 in Domestic Cats in Istanbul, Turkey, Before and After COVID-19 Pandemic

et al. 2021

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Recent studies demonstrated that domestic cats can be naturally and experimentally infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This study was performed to investigate the presence of SARS-CoV-2-specific antibodies within the domestic cat population in Istanbul, Turkey, before the coronavirus disease 2019 (COVID-19) and during the COVID-19 pandemic. Overall, from 155 cat sera analyzed, 26.45% (41/155) tested positive in the spike protein-ELISA (S-ELISA), 28.38% (44/155) in the receptor-binding domain-ELISA (RBD-ELISA), and 21.9% (34/155) in both, the S- and RBD-ELISAs. Twenty-seven of those were also positive for the presence of antibodies to feline coronavirus (FCoV). Among the 34 SARS-CoV-2-positive sera, three of those were positive on serum neutralization assay. Six of the 30 cats before COVID-19 and 28 of the 125 cats during COVID-19 were found to be seropositive. About 20% of ELISA-positive cats exhibited mainly respiratory, gastrointestinal, and renal signs and skin lesions. Hematocrit, hemoglobin, white blood cells, lymphocyte, and platelet numbers were low in about 30% of ELISA-positive cats. The number of neutrophils and monocytes were above normal values in about 20% of ELISA-positive cats. The liver enzyme alanine aminotransferase levels were high in 23.5% ELISA-positive cats. In conclusion, this is the first report describing antibodies specific to SARS-CoV-2 antigens (S and RBD) in cats in Istanbul, Turkey, indicating the risk for domestic cats to contract SARS-CoV-2 from owners and/or household members with COVID-19. This study and others show that COVID-19-positive pet owners should limit their contact with companion animals and that pets with respiratory signs should be monitored for SARS-CoV-2 infections.

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Section: Standardization Of Elisa For Estimation Of S and Rbd Antibody Levels In Serum Samples Collected From Catsmentioning

confidence: 99%

Presence of Antibodies to SARS-CoV-2 in Domestic Cats in Istanbul, Turkey, Before and After COVID-19 Pandemic

et al. 2021

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“…The in-house indirect ELISA (iELISA) protocols using S, S1 and RBD proteins were validated as described previously [ 16 , 17 ]. The validation studies were performed as follows: Initially, a total of 292 sera from 222 SARS-CoV-2-vaccinated and 70 SARS-CoV-2-infected humans (clinically ill and confirmed SARS-CoV-2-RNA positive by RT-PCR) were tested using a commercial ELISA (SARS-CoV-2 IgG II Quant, Abbott Diagnostics, IL, USA) for the presence of SARS-CoV-2 IgG antibodies.…”

Section: Methodsmentioning

confidence: 99%

Development of in House ELISAs to Detect Antibodies to SARS-CoV-2 in Infected and Vaccinated Humans by Using Recombinant S, S1 and RBD Proteins

et al. 2022

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(1) Background: The aim of this study was to produce in-house ELISAs which can be used to determine SARS-CoV-2-specific antibody levels directed against the spike protein (S), the S1 subunit of S and the receptor binding domain (RBD) of S in SARS-CoV-2 vaccinated and infected humans. (2) Methods: Three in-house ELISAs were developed by using recombinant proteins of SARS-CoV-2, namely the S, S1 and RBD proteins. Specificity and sensitivity evaluations of these tests were performed using sera from SARS-CoV-2-infected (n = 70) and SARS-CoV-2-vaccinated (n = 222; CoronaVac vaccine) humans in Istanbul, Turkey. The analyses for the presence of SARS-CoV-2-specific antibodies were performed using the in-house ELISAs, a commercial ELISA (Abbott) and a commercial surrogate virus neutralization test (sVNT). We also analyzed archival human sera (n = 50) collected before the emergence of COVID-19 cases in Turkey. (3) Results: The sensitivity of the in-house S, S1 and RBD ELISAs was found to be 88.44, 90.17 and 95.38%, while the specificity was 72.27, 89.08 and 89.92%, respectively, when compared to the commercial SARS-CoV-2 antibody test kit. The area under curve (AUC) values were 0.777 for the in-house S ELISA, 0.926 for the S1 ELISA, and 0.959 for the RBD ELISA. The kappa values were 0.62, 0.79 and 0.86 for the S, S1 and RBD ELISAs, respectively. (4) Conclusions: The in-house S1 and RBD ELISAs developed in this study have acceptable performance characteristics in terms of sensitivity, specificity, AUC and kappa values. In particular, the RBD ELISA seems viable to determine SARS-CoV-2-specific antibody levels, both in infected and vaccinated people, and help mitigate SARS-CoV-2 outbreaks and spread.

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“…Infectious bronchitis (IB) is caused by the avian coronavirus-infectious bronchitis virus (AvCoV-IBV), which is recognized as a severe threat to the poultry industry globally owing to that it affects chickens of all ages and causes respiratory and nephrotic syndromes in broilers, as well as impairs egg production in layers and breeders [ 1 , 2 , 3 ]. AvCoV-IBV that belongs to the genus Gamma-coronavirus within the family of Coronaviridae in the order Nidovirales, is an enveloped, positive single-stranded RNA virus [ 4 ]. The 27 to 28 kb genome of AvCoV-IBV encodes several structural proteins, including spikes (S), membranes (M), envelopes (E), and nucleocapsids (N) protein [ 5 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

“…Nevertheless, whole IBV particles is tedious for producing procedures including virus proliferation and purification to cause the high price of the commercial ELISA kits, which limits its application [ 9 ]. Many studies indicated that recombinant S and N proteins from prokaryotic, yeast, or baculovirus systems could provide a rapid and large-scale detection method for IBV antibody infection [ 4 ]. The S protein of IBV has highly mutation in different IBV strains, which restricted its application in for detecting the IBV antibody.…”

Section: Introductionmentioning

confidence: 99%

“…The S protein of IBV has highly mutation in different IBV strains, which restricted its application in for detecting the IBV antibody. However, the N protein is the optimal candidate antigen for early diagnosis, both serological and molecular diagnosis, since it is highly conservative among the most IBV strains and can induce strong immune response for producing amount of antibody against N protein in early infection or immunization [ 4 , 8 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

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A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum

Song

et al. 2022

IJMS

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Avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is the causative agent of infectious bronchitis (IB) that has brought great threat and economic losses to the global poultry industry. Rapid and accurate diagnostic methods are very necessary for effective disease monitoring. At the present study, we screened a novel nanobody against IBV-N protein for development of a rapid, simple, sensitive, and specific competitive ELISA for IBV antibody detection in order to enable the assessment of inoculation effect and early warning of disease infection. Using the phage display technology and bio-panning, we obtained 7 specific nanobodies fused with horseradish peroxidase (HRP) which were expressed in culture supernatant of HEK293T cells. Out of which, the nanobody of IBV-N-Nb66-vHRP has highly binding with IBV-N protein and was easily blocked by the IBV positive serums, which was finally employed as an immunoprobe for development of the competitive ELISA (cELISA). In the newly developed cELISA, we reduce the use of enzyme-conjugated secondary antibody, and the time of whole operation process is approximately 1 h. Moreover, the IBV positive serums diluted at 1:1000 can still be detected by the developed cELISA, and it has no cross reactivity with others chicken disease serums including Newcastle disease virus, Fowl adenovirus, Avian Influenza Virus, Infectious bursal disease virus and Hepatitis E virus. The cut-off value of the established cELISA was 36%, and the coefficient of variation of intra- and inter-assay were 0.55–1.65% and 2.58–6.03%, respectively. Compared with the commercial ELISA (IDEXX kit), the agreement rate of two methods was defined as 98% and the kappa value was 0.96, indicating the developed cELISA has high consistency with the commercial ELISA. Taken together, the novel cELISA for IBV antibody detection is a simple, rapid, sensitive, and specific immunoassay, which has the potential to rapidly test IBV antibody contributing to the surveillance and control of the disease.

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

Cited by 5 publications

References 26 publications

Presence of Antibodies to SARS-CoV-2 in Domestic Cats in Istanbul, Turkey, Before and After COVID-19 Pandemic

Presence of Antibodies to SARS-CoV-2 in Domestic Cats in Istanbul, Turkey, Before and After COVID-19 Pandemic

Development of in House ELISAs to Detect Antibodies to SARS-CoV-2 in Infected and Vaccinated Humans by Using Recombinant S, S1 and RBD Proteins

A Novel Nanobody-Horseradish Peroxidase Fusion Based-Competitive ELISA to Rapidly Detect Avian Corona-Virus-Infectious Bronchitis Virus Antibody in Chicken Serum

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