Preparation And Identification Of Monoclonal Antibody Against Porcine Immunoglobulin

Porcine infectious diseases have threatened swine industry enormously in China. Traditional serological detection is difficult to find out the porcine diseases earlier and distinguish the antibody's character, which cause the infection-overspreading potentially. With the large scale fostering of swine in industry, it is in great urgency to research the immune mechanism in this infection and enhance the immune surveillance and diagnosis. It is very important for the further research to develop the monoclonal antibody against porcine immunoglobulin.In this study, we obtained and identified the monoclonal antibody against porcine immunoglobulin.Fistly,IgM and IgG were isolated from porcine serum by a series procedure of precipitation with saturated ammonium sulfate and chromatography with Sephadex G-200. After SDS-PAGE electrophoreisis, purified IgM and IgG reach the electrophoretic purity, and the concentration were 0.7mg/mL and 2mg/mL respectively determined by Lowry method.Secondly,indirect ELISA was established to screen the positive hybridoma cell lines. By optimization, optimal reactive condition of I-ELISA for screening the McAb coated with porcine IgM was determined as follows, the quantity of antigen coated was 0.25μg/mL, the positive serum dilution and the negative control were both 1:2000,and the dilution of IgG-HRP was 1:20000. I-ELISA coated with poricine IgG were also established, the cover concentration of antigen was 0.1μg/mL, the positive serum dilution and the negative control were both 1:2000,and the dilution of IgG-HRP was 1:20000.Thirdly,after the 6-8 week-old female BALB/c mice were immunized three times with purified IgG, the spleen cells of the immunized mouse and SP2/0 cells were mixed in proportion of 10:1, and then were fused by 1 mL of 50% PEG3000.The supernatants secreted by the fusing cells were detected 10 to 15 days later. In this research, cell fusion was made twice successfully. The cell fusion rates were 84.4% and 75% respectively, and the positive rates were 13% and 4.5%, respectively. Two hybridoma cell lines that produced monoclonal antibodies steadily, named C11 and G3, were obtained by twice subcloning. With repetitious generation, both of the hybridoma cell lines still could secrete high titer antibodies, the titers of them were both between 1:64 and 1:128. The titer of G3 line's ascites was over 1:12800. After identification by HBT Mouse Monoclonal Antibody Isotyping Kit, the McAb secreted by C11 cell line belongs to IgM,κisotype. The titers of cell supernatant were similar mensured by indirect ELISA coating with porcine IgM and IgG, and this result was in accordance with the one detected by Weston- Blotting, which testified that McAbs secreted by two hybridoma cell lines were both against L chain of porcine Immunoglobulin.