Preparation Of The Monoclonal Antibody Against Type A Foot-And-Mouth Disease Virus

The foot-and-mouth disease (FMD) caused by the foot-and-mouth disease virus (FMDV), with a strong infection between human and animals have a huge impact on the production of livestocks.The monoclonal antibody of FMD is useful for the detection of FMD and early prevention,it also have important influence on the diagnosis, prevention and cure of the disease. This study use inactived type A FMD vaccine immune to BALB/c mice to produce antibodies against the antigen.Cell fusion induced by PEG was conducted to prepare the monoclonal cell lines against type A FMD.The biological characteristics of the hybrid cells and monoclonal antibody identification were analysed.The results can help the later development of the diagnosis of product.1. Exploration of FMDV monoclonal antibody preparation conditionsBefore monoclonal antibody preparation, the SP2/0 cells, feeder cells and spleen cells growth characteristics in complete medium and selection medium were analysed.The PEG molecular integration and concentration were optimized. The amount of injected immunization vaccines was optimized.The results showed that:1) The PEG with molecular integration of 4000, concentration of 50% achieves the highest fusion rate.2) The amount of injected immunization vaccines was optimized, and 0.1 mL vaccine immunization can produce the best result.2. The preparation, screening and cloning of monoclonal cell linesBALB/c mice with six to eight weeks old were injected antigen ip 3 times.The serum titer was detected.Tail vein injection was conducted to enhance the immune 3 days before cell fusion.The immuned spleen cells were carried out sterilly.The titer of supernatant of culture medium was conducted after cell fusion with SP2/0,selection of HAT medium for the determination of positive clone cells.The monoclonal cell were prepared by microscopic operation after indirect ELISA selection,we obtained 33 strains of monoclonal cells.According to the OD450nm values of ELISA,we chosed 3 cell lines with higher antibody expression,which were named:9F3、9F4、10B3. Through cell fusion experiments, the following conclusions:1)After three vaccine, antibody titer in mice is 1:25600; 2) After cell fusion, through HAT medium filtering, get hybrid cells; 3) After a micromanipulation technology, preparation of 33 positive strains were monoclonal cells, including 3 strains of high expression of cell line:9F3、9F4、10B3.3. The identification of monoclonal cell lines and antibodyWe analysed the cell lines stability of 9F3,9F4,10B3. The result shows that 9F3 and 10B3 can express stably, with 9F3 cell lines higher titer. With 9F3 cell lines as appraisal target,we analysed its growth curve,chromosome numbers(94) and ascites antibody titer(1:25600).