Development Of Hybridoma Cell Strains Secreting Monoclonal Antibodies Against Classical Swine Fever Virus And Its Application

The classical swine fever virus (CSFV) purified and mixed with the complete Freund's adjuvant and incomplete Freund's adjuvant in the same volume to prepare immune antigen and used to vaccinate the BALB/c mice. After immunized for 5 times, the mouse was inoculated intensively once, and Sp2/0 mouse myeloma cells and splenocytes isolated from a Balb/c mouse vaccinated with CSFV antigen were fused. Five hybridoma cell strains which can secrete stably monoclonal antibody (McAb) were obtained by indirect ELISA and tri-time limiting dilution.The five hybridoma cell strains are named C7,C9,G9,G10 and 4E8 respectively.Among the five McAbs,the titers of cell cultures ranged from 1:1600 to 1:3200 and the titers of ascites fluids ranged from 1:51200 to 1:102400. These hybridoma cell strains can stably secrete McAbs after thirty serial passages and freezing and anabiosis three times in six months.Cross reactivity test showed that the five McAbs reacted with CSFV,but no cross reaction was found when the five McAbs reacted with FMDV,PRV,PRRSV,PCV2,PPV. Specificity antigen interception test showed that CSFV could block hybridoma cell supernatant effectually with a max competition-inhibition ratio of 94.30% while FMDV and negative serum could not,thus it demonstrated that the hybridoma cell supernatant was specific to CSFV without crossing reaction to other viruses.Indirect enzyme-linked immunosorbent assay (indirect-ELISA) developed to detect anti-CSFV antibody from pig serum.The optimum work conditions for indirect ELISA were developed as follows: The concentration of coating antigen was 9.279μg/mL;The coating fluid was CBS (PH9.6,0.05M);The time of coating was 1 hour at 37℃and then stored at 4℃;The blocking materia was 0.5% gelatine;The dilution of the labeled goat-anti-mouse enzyme was 1:2000.Antigen capture enzyme-linked immunosorbent assay (AC ELISA) was developed to detect CSFV antigen by using the McAbs in ascite of the cell strain C7.The optimum conditions in some critical steps of the ELISA were set as follows: The optimum work concentration of anti-CSFV capture antibody which from pig serum was 1:320 and the McAbs ascite was 1:1600.The limit of quantitative detection in CSFV was 927.88ng/mL.An indirect Immunofluorescence Assay (IFA) for rapid diagnosis of CSFV was developed. The best working concentration of anti-CSFV McAbs and fluorescent antibody were 1:1000 and 1:50. The cross reaction and inhibition test showed that the method is specificity.The established method of IFA was simple,sensitive,the results will be known within a short time and easy to read,and provide an important method for rapid,sensitive detecting CSFV in clinical specimens.Development of hybridoma cell strains secreting monoclonal antibodies against classical swine fever virus and its application are important for the detection of CSFV antigen and its antibody.They provided a basis of establishing rapid diagnostic method for CSF and will benefit further studies on the biological characteristics of CSFV.