Production And Characterization Of Monoclonal Antibodies Against GP5 Of PRRSV BJ-4

In this study, four stable hybridoma cell lines that produced monoclonal antibodies specific for PRRSV GP5 were established with chromatography-purified recombinant GP5, the properties of these four mAbs were characterized and analysized.The Balb/C mice were immunized subcutaneously on the neck and the back with 100ug purified recombinant GP5 mixed with complete Freund's adjuvant and the other two immunizations with the same amount of antigen mixed with imcomplete Freund's adjuvant at two weeks interval. Three days before cell fusion was performed, mice were boosted with 100ug antigen intraperitoneally. Immunized splenocytes were collected and fused with myeloma cell line SP2/0, which were in the logarithmic phase of growth. An indirect ELISA was established to screen the hybridomas for production of specific antibodies in the cell culture medium, in which the partially purified recombinant protein pET32a-dORF5 was used as coating antigen. Meanwhile, the indirect ELISA based on GST-dORF2 were established to rule out the false positives. Totally 12 hybridomas with high ELISA titres were selected and subcloned by a limiting-dilution technique till the single clone producing specific antibodies were selected. In the fusion experiment, totally 4 stable hybridoma cell lines that produced monoclonal antibodies specific for GP5 were established, which designated B6A3, B6A4, F12F10, B11F11B2, respectively. Approximately 106 hybridoma cells were injected into the multiparous female mice to produce ascitic fluid, in which the monoclonal antibody titres reached 1: 320000 for B6A3 and B6A4, for F12F10 and B11F11B2, the ELISA titre was above 1: 320000. ELISA (SBA ClonotypingTM System/AP) was used to identify the subclass of these mAbs, results turned out that B6A3, B6A4, F12F10 and B11F11B2 all belong to IgG2a subclass, the light chain of these mAbs were categorized into K type. In a western-blot assay with pET32a-dORF5, mAb B6A3 and F12F10 could recognize a 31KD recombinant GP5. B6A3 and F12F10 showed strong specific perinuclear fluorescence in PRRSV(BJ-4) infected cells in IFA, but specific perinuclear fluorescence was not observed in B6A4 and B11F11B2. B6A3 was choosed to conduct virus neutralization test, but it seemed suspected neutralizing activity was observed. The epitopes that these mAbs targeted were prelimilarilly predicted by indirect ELISA, results showed that B6A3 and F12F10, B6A3 and B11F11B2, F12F10 and B11F11B2 might recognize different epitopes. Further studies are still needed to identify the virus neutralization and epitope recognition properties of these mAbs.