Development To Hybridoma Cell Strains Secreting Monoclonal Antibodies Against Porcine Parvovirus And Its Application

In this study, we purify porcine parvovirus vp2protein with the nickel ion metal chelating affinity chromatography.then adding an equal volume of Freund’s complete adjuvant or Freund’s complete adjuvant, made complete adjuvant, antigen and incomplete adjuvant antigen, immunization six to eight-week-old female BALB/c mice,after being immunized five times, the mice with the ELISA titer of more than1:51200were immunized intraveinally for the final immunization.Three days later.spleen cells from immunized mice were fused with SP2/0myeloma cells.The indirect ELISA method were established with sucrose embedding and differential centrifugation as detection antigens..four cell lines were developed and identified with limited dilution method after five to six cloning.named2C5.4B5.1A11and5C6.After determining.4B5and2C5only response with virus,1A11and5C6response with virus and VP2.After the class identification.mAbs from4B5were of IgG1isotype.mAbs from2C5were of IgM isotype.Chromosome analysis of four hybridoma cells indicated that average chromosome number were92～108.which is consistent with chromosome number sum of SP2/0cell and spleen cell.The result demonstrated that acquired antibody-secreting cells were hybridoma cells.The specificity assay showed that four mAbs only reacted with inactivated PPV or PPV VP2.not reacted with antigens from PRV.PRRSV.PCV2.The ELISA titers of cell cultures supernatant and ascites fluids of all4hybridoma cell lines ranged from1:800-1:8000.and1:8000-1:1024000.Stability detection of hybridoma cell was still vigorous after being cultured10,25generation or freeze-stored recovery two times.This showed that established hybridoma cell lines could secret mAbs consistently.Immunocapture-PCR for detecting Porcine Parvovirus was developed by using the mcAbs produced from hybridoma cell lines.The IC-PCR are highly specific and sensitive in detecting samples.It used as a reference test for submitted13clinical samples.the positive rate are higher than conventional PCR...