兽医科学与医学诊断杂志

Development of Recombinant Nucleocapsid Protein-based Enzyme-linked Immunosorbent Assay for Serological Detection of Winter Dysentery Disease

Kanokwan Singasa, Taweesak Songserm, Preeda Lertwatcharasarakul, Siriluk Jala, Sakuna Phattanakunanan and Pipat Arunvipas

Winter Dysentery Disease is an acute and contagious viral disease that adversely affects dairy cattle on farms in Thailand. An indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant nucleocapsid (rN) protein which has been developed for antibodies detection against Winter Dysentery Disease was produced by an Escherichia coli protein expression system. The epitope antigen N protein was designed using almost total N gene fragments (8-430 aa, N gene). It was identified to be approximately 48 kDa as the rN protein and can bind bovine coronavirus dairy cattle positive serum by western blot analysis. The conditions of the ELISA method were optimized. The rN protein was standardized with a coating antigen concentration of 5 μg/well. The rN protein was tested with sera in both infected and uninfected dairy cattle. The dilution of the primary antibodies was identified as 1:50 using a checkerboard titration. The intra- and inter-assays were repeatable. The cut-off of the corrected OD450 value from mean ± 2SD (standard deviations) was established at 0.049. The percentage of specificity, sensitivity and accuracy between the developed rNELISA and a SVANOVIR® BCV-Ab ELISA kit was 96.3, 84.8 and 86.1%, respectively. Cohen’s kappa value of the developed ELISA compared with the commercial test kit was 0.71. The correlation coefficient of absorbance values from these two tests was 0.68. The recombinant nucleocapsid protein ELISA method might be helpful for bovine coronavirus diagnosis and surveillance.