Development And Application Of Monoclonal Antibodies Against Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) is caused by porcine reproductive and respiratory syndrome virus (PRRSV) and associated with severe reproductive failure in sows, respiratory disease in young pigs. PRRS can be transmitted in a number way, such as organic material (feces, urine, feed, bedding and body fluids) and transport vehicles. PRRS can cause susceptibility to bacterium and mycoplasma infection and is considered the most economically important viral disease.Identification of PRRSV can be accomplished by virus isolation, the detection of nucleic acids, and the detection of viral proteins. Serological diagnosis is, in general, easy to perform, with good specificity and sensitivity, especially on a herd basis. The presence of PRRSV can be confirmed by immunostaining with a specific antiserum or monoclonal antibody (MAb).First, to obtain specific and sensitive monoclonal antibodies, N-gene of PRRSV was cloned to pET-28a and expressed after induction with0.5mmol/L IPTG. The expressed protein was purified using RoboPopTMNi-NTA His-Bind Purification Kit and then intraperitoneally inoculated BALB/c mice at50μg. SP2/0myeloma cells were fused with spleen cells of the repeatedly immunized mice. After identification of hybridomas secreting antibodies against PRRSV by ELISA, six cell strains (9B12,7C2,7C6,1F11,1G1and1G3) were screened for monoclonal antibody production.Second, the titers of MAbs (9B12,7C2,7C6,1F11,1G1and1G3) in mouse ascites were respectively1:1.28×104、1:6.40×103、1:3.20×103、1:3.20×103、1:8.0×102、1:4.0×102. The six MAbs can detecte PRRSV isolates (CC-11, CH-1R and JXA1-R) and not reacted with PCV, PRV and CSFV. The range of detection using the MAbs was25TCID50/ml. The mAbs9B12,1F11and1G1belong to the IgG2b subclass, the other mAbs were of the IgG1subtype.The third, the9B12MAb was purified by affinity chromatography method and labeled with FITC for direct immunostaining. The immunofluorescent antibody was optimized as to the following conditions, i.e., fixing agents and conditions, reaction period and working concentration. If the cell culture samples were examined by the immunofluoresece,80%acetone at4℃for20min for fixation,1:400immunofluorescent antibody and incubation period of1h were administered. When detecting clinical tissue samples,80%acetone at4℃for30min for fixation,1:200immunofluorescent antibody and incubation period of2h were performed.62clinical samples collected from Foshan City were respectively detected using the immunofluorescent antibody and RT-PCR, of which the accordance was evaluated. In the immunofluorescent antibody test,37samples were positive for PRRSV and25were negative. In the PCR test,25samples were positive and37were negative. Correlation between the immunoflurescent antibody and RT-PCR was74.2%. The immunofluorescent antibody can be stored at4℃for up to6months.The results indicated that the immunoflurescent antibody method for detecting PRRSV would be used as a clinical diagnostic test.