Development And Application Of The Solid-phase Competition ELISA And Double Antibody Sandwich ELISA For Detection Of Bovine Rotavirus

Bovine rotavirus (BRV) is one of the major pathogens causing diarrhea among calves. BRV is one of the major pathogens causing diarrhea diseases among calves under1month of age. The clinical symptoms of calves infected the bovine rotavirus include depression, lose appetite, diarrhea, severe dehydration, acid poisoning, and even death.To establish rapid protocol for antibody anainst BRV, a new Solid-phase competition ELISA(SPCE) was developed using the G6/G10genotype monoclonal antibody(MAb) and purified polyclonal antibody against VP6protein as detector and capture antibody, respectively.The results showed that when examining test sear at1:8dilution with a cut-off point of40.32percentage inhibition(PI) of reaction, there was no overlap between the positive and negative sera in the SPCE. The specificity of SPCE was96.8%and the sensitivity of SPCE was96.8%.The coincidence rate of the SPCE,VNT, indirect ELISA and commercial ELISA Kit were higher than96%.The coefficient of variability of within-run and between-runs were less than9%. The within laboratory variation of the SPCE when tested the control samples was very small.To establish rapid protocol for bovine rotavirus (BRV) detection, a Double Antibody Sandwich ELISA for BRV was developed using the G6/G10genotype monoclonal antibody (MAb) and purified polyclonal antibody against VP6protein as detector and capture antibody, respectively. The limit detection of this assay was capable for detection of104.2TCID50BRV, whereas the detection limit of the commercial ELISA Kit and RT-PCR was10486and103.36TCID50BRV, respectively. The Double Antibody Sandwich ELISA was specific for detection of BRV, but no cross reaction with bovine enterovirus, bovine reoviruses,bovine parvovirus, bovine infectious rhinotuacheitis virus, bovine parainfluenza virus type3,bovine viral diarrhea virus and akabane virus.The coefficient variability of intra-assay and inter-assay were less than10%, indicating that the repeatability of this Double Antibody Sandwich ELISA for BRV was reproductive.The coincidence rate of the Double AntibodySandwich ELISA, RT-PCR and commercial ELISA Kit were100%.These data demonstrated that the Double Antibody Sandwich ELISA established in this study provide a useful tool for diagnosis of BRV infection in cattle.