ABSTRACT
In order to establish a rapid method for the detection of bovine parvovirus (BPV), primers were designed according to the VP2 sequence of BPV from NCBI. The VP2 gene was amplified from the viral DNA by PCR and cloned into a recombinant expression vector pProHTa. The recombinant plasmid pProHTa-BPV-VP2 was transformed into Escherichia coli Rosetta cells, which were induced to express the VP2 protein. The purified VP2 protein was immunized to mice (100 g/mouse). After cell fusion, screening, and subcloning, five positive hybridoma cell lines were obtained. They were designated as 5G9, 2B5, 6A3, 7E8, and 2B6, respectively. The subtypes of the five monoclonal antibodies (McAbs) were identified as IgG2a, IgG2b, IgM, IgM, and IgA, respectively. Indirect immunofluorescence assay showed that all McAbs reacted with BPV specifically. In the meanwhile, the purified VP2 protein was immunized to New Zealand rabbits to prepare anti-VP2 poly-antibodies (PcAbs). Subsequently, a double antibody sandwich ELISA was established for the detection of BPV using the PcAbs as capture antibody and 2B5 as detecting antibody. The specificity detection showed that only the BPV was positive, and there was no reaction with BRV, PRV, TGEV, PEDV, or BVDV. The sensitivity test showed that the minimum detection amount of this method was 3.125 x 102.8TCID50/mL, which showed high sensitivity. The results of the repetitive test showed that the intra-and inter-batch coefficients of variation were less than 10%. This method was used to detect 269 clinical diarrhea samples, in which 14 samples were positive for BPV. The coincidence rate was 100% between this method and PCR. In summary, a double antibody sandwich ELISA method was established using anti-BPV VP2 protein McAbs and anti-VP2 PcAbs. This method has good specificity, sensitivity, and reproducibility, which can provide an effective detection method for the rapid diagnosis of BPV infection and a reliable means for epidemiological investigation as well as disease prevention and control.