Preparation And Identification Of Monoclonal Antibody Against Classical Swine Fever Virus

Classical swine fever?CSF?,also named hog cholera?HC?,is a highly contagious and acute infectious disease caused by classical swine fever virus?CSFV?.It often causes high fever and mortality,which brings a great economic loss to the swine industry.For the purposes of studying the host's immune mechanism and developing effective diagnosis methods for CSFV,the preparation of antibodies against this virus has been made for years,yet antibodies with high sensitivity and specificity are still needed.In this paper,the growth curve of CSFV in porcine kidney 15 cells?PK 15?was established.The qualitative and quantitative detection of CSFV were also optimized based on former work.For the purification of the virus,ultrafiltration in combination with size-exclusion chromatography were introduced with tangential-flow filtration and Sepharose 4 Fast Flow column.The virus purification effect was evaluated by real-time fluorescence quantitative RT-PCR technique and immunoperoxidase monolayer cell assay?IPMA?.The real-time fluorescence quantitative RT-PCR results showed that the virus was successfully purified and the first collected peak after filtration chromatography was identified as purified CSFV;and IPMA results showed that the titter of ultrafiltration concentrate increased from 10^4.7 to 10^6.5(TCID₅₀/mL)with recover rate of 76.52%,then the virus was further purified by4FF-SEC,and the final virus recover rate of 63.2%.With purified virus as antigen,6⁸ weeks old Balb/c mice were immunized and the titer of antiserum were assayed by ELISA with E.coli-expressed CSFV E0-E2 protein.Results showed that the highest titter reached 1:25600.Spleen cells of BALB/c mouse 3 with the highest titter were interfuse with myeloma cells SP2/0 in order to prepare hybridomas.Hybridomas was further sub-cloned and identified by ELISA and Western blot.Results showed that 5 cell lines,named as 5F2A7?5F2C12?8D4G7?9A1C7?11D4B6,can positively react with CSFV E0-E2 protein expressed by E.coli.The ability to combine with CSFV was also confirmed by IPMA,and 9A1C7 showed the greatest binding capacity to virus with no cross-reactivity to Bovine viral disease virus?BVDV?,Porcine reproductive and respiratory syndrome virus?PRRSV?,Porcine circovirus type 2?PCV2?.Cell line9A1C7 can stably secrete monoclonal antibodies.The mAbs were further genotyped as immunoglobulinG2c,?-light chain isotype.In addition,ascites of 9A1C7 cell lines were prepared and purified by ammonium acid-caprylic acid precipitation.The titter of purified mAbs reached 1:5.12×10⁵.