Establishment and application of indirect ELISA for detection of bovine coronavirus antibody

Abstract:

[Background] Bovine coronavirus (BCoV) is one of the main causes of neonatal calf death, and effective detection is the prerequisite to prevent and control the disease. [Objective] At present, BCoV ELISA detection method has some defects, such as low sensitivity, instability and so on. This study aims to improve these defections to establish indirect ELISA detection method. [Methods] The method of indirect ELISA was established by using the soluble recombinant N protein of the epidemic BCoV-CD strain as an antigen. The epitope of the N protein was predicted by DNAStar soft, and was prepared by prokaryotic expression in the non-denatured condition. The seroepidemiological investigation of BCoV infection in Heilongjiang province in recent 5 years was carried out by using this method. [Results] The optimum working conditions of the ELISA method were as follows: the coating solution was 50 mmol/L pH 9.6 carbonate, and the antigen coating concentration was 2.5 μg/mL; The sample diluent was PBST, the dilution concentration was 1 μg/mL, and incubated at 37 °C for 1.5 h; The dilution concentration of HRP-labeled secondary antibody was 1:7 500, and incubated at 37 °C for 1.0 h; The blocked condition was 1% gelatin at 37 °C for 30 minutes. The negative-positive cut off value was 0.225. The method had no cross-reaction with positive serum of bovine rotavirus, bovine viral diarrhea virus, bovine respiratory syncytial body, bovine infectious rhinotracheitis, bovine parainfluenza virus type 3 and Escherichia coli. The intra-and inter-assay coefficient of variation was less than 10%, and the coincident rate with virus neutralization test was 93.5%. The results showed that the positive rate of BCoV antibody was 98.84% in 603 serum samples of cows in some areas of Heilongjiang Province. [Conclusion] The ELISA method established in this study has strong specificity, high sensitivity and good stability, which provides a technical basis for the further development of ELISA kit.

Key words:Bovine coronavirus, nucleocapsid protein, ELISA, epidemiology