Preparation Of Monoclonal Antibodies Against VP2 Protein Of PPV And Preliminary Establishment Of Double-Antibody Sandwich ELISA

The purified recombinant VP2 protein of PPV(Porcine parvovirus) was used to immunize BALB/c mice, and the McAbs(monoclonal antibodies) against VP2 protein were screened by hybridoma technology and indirect ELISA. After more than three times cloning, five hybridoma cell strains were successfully constructed stably secreting anti-VP2 McAbs.the McAbs named 2D5, F11, 2B4, 3C8 and 1H3, respectively. The chromosomal average number of hybridoma cells was caculated as 88±10, the 2D5 belonging to IgG2a subtype and others to IgM subtype.Titers of cells culture supernate was reflected as 1:5000, 1:2000, 1:1000, 1:800, 1:800, respectively by indirect ELISA.Their titers of ascites were 1: 106, 1: 105, 1: 104, 1: 104, 1: 104 respectively.Western-blot assay demonstrated that all of McAbs prepared in the context could recognize VP2.The results of IFA indicated that five McAbs can react with ST cells affected with PPV showing strongly yellow and green fluorescence in the cell membranes and cytoplasm after incubating with McAbs, by contrast, the cells inoculated PPV without strongly yellow and green fluorescence after with SP2/0 cells culture supernate.The result of indirect ELISA confirmed that five McAbs had no reactive capability with TEGV, PRV and PEDV, specificity only for PPV. Five hybridoma cell lines are able to secrete stably monoclonal antibodies after serial passage in vitro for there months and resuscitation after freezeing.All these results showed that the McAbs against PPV VP2 protein were highly specific against pathogen. The strong material base were made in this research, which set up the methods for PPV epidemic monitoring, rapid and accurate diagnosis of the pathogen .Inoculateing rabbit for polyclonal antibodies to set up Double-antibody sandwich ELISA. In this study, the PPV were breed with STcells and the minimum detectable amount of TCID50 is 10-4.6, 0.1mL.The PPV were purified through high-speed centrifugation and sucrose density gradient centrifugation to inoculate rabbit. Polyclonal antibodies anti-VP2 protein and monoclonal antibody were purified by caprylic acid ammonium sulphate method and affinity chromatography. The concentration of purified polyclonal and monoclonal antibody is 4.9mg/ml and 0.23mg/ml. Through the polyacrylamide gelelectrophoresis (SDS-PAGE) analyzing, there were H line and L line obviously with 100% of the antibody purity .The purified monoclonal antibody and polyclonal antibody were used as basic detection reagents to search for Double-antibody sandwich ELISA for detecting virus. The best working concentration of purified monoclonal antibody is1: 50 (4.6μg?ml) and polyclonal antibody is 1: 800 (6.1μg?ml) by the square.By the method mentioned above. The detection system for the minimum detectable amount of 155 TCID50 that is 32 times higher than the traditional hemagglutination test in sensitivity. The result of indirect ELISA confirmed that the method, with the best stability by repetitions, had no reactive capability with TEGV, PRV and PEDV. Double-antibody sandwich ELISA not only set up a simple, rapid, sensitive and widely used PPV detection method for the field application, but provided a basis for the practical production.