Preparation of monoclonal antibody against avian polyomavirus VP4 protein and preliminary establishment of an indirect immunofluorescence detection method
In order to prepare a monoclonal antibody against VP4 protein of avian polyomavirus(APV),and to initially establish an indirect immunofluorescence detection method for APV.The monoclonal antibody was prepared by prokaryotic expression of APV VP4 protein and immunising BALB/c mice after purification,and the reaction conditions were optimized with the prepared against VP4 monoclonal anti-body as the primary antibody and FITC-goat anti-mouse IgG as the secondary antibody.The results showed that 15 positive cell lines were screened and identified by subtype.The heavy chain of 2 monoclonal antibodies was IgG2b type,the heavy chain of 13 monoclonal antibodies was IgG1 type,and the light chain wasк type.Three cell strains were selected to prepare monoclonal antibodies.The concentrations of a-gainst VP4 monoclonal antibodies of the 3 strains were 2.9,2.6,and 3.2 mg/mL,respectively.The affin-ity constants were 1.06×1010,2.95×109,and 9.60×109,respectively.The titer of two strains was both 1 ∶204 800,and the titer of the other strain was 1 ∶51 200.Western-blot and indirect immunofluorescence assay(IFA)showed that all 3 monoclonal antibodies could react specifically with APV.The optimal conditions for the indirect immunofluorescence detection method established in this study were as follows:1 ∶1 000 dilu-tion of Anti-A-VP4-15,overnight incubation at 4℃,1 ∶200 dilution of secondary antibody,incubation at 37℃for 1 h.The established method was used to detect egg drop syndrome virus(EDSV),reticuloendotheliosis virus(REV),avian leukosis virus(ALV),Newcastle disease virus(NDV),avian infectious bronchitis virus(IBV)and APV in the cells.Only the results for APV were positive,and tests for other viruses were negative.The indirect immunofluorescence method established using this monoclonal antibody was able to detect at least 5 TCID50 APV infections on cells.The monoclonal antibody and the indirect immunofluores-cence method developed in this study provide a basis for epidemiological investigation and labora-tory diagnosis of APV infection.
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