Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus

Abstract

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20 μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

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

“…reported that high levels of TGEV-specific antibodies could be induced by a combination of S-rosettes and the N protein [ 2 ]. These properties made the N protein suitable as a diagnostic antigen of PDCoV in an indirect ELISA, which was supported by other coronavirus studies [ 1 , 7 , 10 , 16 ].…”

“…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.…”

Nucleoprotein-based ELISA for detection of SARS-COV-2 IgG antibodies: Could an old assay be suitable for serodiagnosis of the new coronavirus?

“…Sera from individuals admitted to the hospital for suspected COVID-19 infection were assayed. The nucleoprotein was chosen as the target of the assay since it is an immunodominant antigen and consistently employed to detect antibodies against other coronaviruses in both humans and animals ( Guo et al, 2020a ; Abdelwahab et al, 2015 ; Leung et al, 2004 ).…”

Quantitative Fluorescence Quenching on Antibody-conjugated Graphene Oxide as a Platform for Protein Sensing

“…However, the accuracy of ELISA is affected by a few shortcomings intrinsic to the technique. For example, the uncertainty of the recognition between antibody and antigens could cause false signals123 the ELISA plates made of polystyrene lack definitive chemical surface properties to provide chemical binding of the protein4; and intricate procedures such as “plate cleaning” increase opportunities for systemic and human errors. In recent years, many research groups have tried to improve the accuracy of ELISA by synthesizing new ELISA polymer substitutes4, introducing nanomaterials56, employing more sensitive biomarkers7, and applying monoclonal antibodies8.…”