Development And Primary Application Of Hybridoma Cell Strains Secreting Monoclonal Antibodies Against Porcine Circovirus Type 2

The study generated porcine circovirus 2(PCV2) on PK15 cells.After concentrating and depurating,the absorbance of the purified virus in 280nm and 260nm was measured through the ultraviolet spectrophotometer,and the concentration of antigen's protein was 25.1970 mg/ml.The complete Freund's adjuvant and incomplete Freund's adjuvant were mixed with the antigen in the same volume to prepare immune antigen and then Balb/c mice were inoculated with the antigen at 8 weeks of age.After the third immunization,the mouse was inoculated intensively once,and SP2/0 mouse myeloma cells and splenocytes isolated from a Balb/c mouse vaccinated with PCV2 ahtigen were fused.Three hybridoma cell strains secreting the monoclonal antibodies against PCV2 were developed and named as 2B₅,2D₄ and 3C₃.The optimum work conditions for indirect enzyme-linked immunosorbent assay(indirect-ELISA)were developed as follows:the concentration of coating antigen was 25μg/ml;the coating fluid is CBS(PH9.6,0.05M);the time of coating was 1 hour at 37℃and then stored at 4℃overnigh;the blocking materia was 1%gelatine;the dilution of the labeled goat-anti-mouse enzyme was 1:1000.The results indicated that PCV2 could block hybridoma cell supernatant effectually with a max competition-inhibition ratio of 80%while PRRSV,PPV and PRV could not,thus it demonstrated that the hybridoma cell supernatant was specific to PCV2 without crossing reaction to other viruses.When the purified PCV2 antigen was utilized as the coat antigen in ELISA,the serum of mouse was positive against PCV2.The maximal ELISA titers of the McAbs against PCV2 in cell supernatants and ascites were 1:512 and 1:102400,respectively.Average chromosome number of each hybridoma cell strain was between 92 and 108.These hybridoma cell strains can stably secrete McAbs after twenty-five serial passages and freezing and anabiosis three times in six months.Antigen capture enzyme-linked immunosorbent assay(AC-ELISA) was developed to detect PCV2 antigen by using the McAbs in ascite of the cell strain 3C₃ with a titer of 1:102400 diluted in 1:1600.and the sensibility for PCV 2 antigen detection was 80.63ng/ml.In addition,the device of detecting PCV2 blood serum antibody titer was established.