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

[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