Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus.
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.
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, United States.
Mohamed
M
Abdelwahab
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, United States. Electronic address: chienchang.loa@mylan.com.
Chien Chang
CC
Loa
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, United States.
Ching Ching
CC
Wu
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN 47907, United States.
Tsang Long
TL
Lin
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D018352:Coronavirus Infections / Q000175:diagnosis / Q000662:veterinary*; D004752:Coronavirus, Turkey / Q000276:immunology*; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019251:Nucleocapsid / Q000235:genetics / Q000276:immunology*; D011201:Poultry Diseases / Q000175:diagnosis*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D014422:Turkeys
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins
Coronavirus; Enteritis; Enzyme-linked immunosorbent assay; Nucleocapsid; Recombinant protein; Turkey