| Objective: To establish hybridoma cell strains secreting Fl monoclonal antibodies (McAbs) against Y.pestis Fl antigen using hybridoma technology and let them continuously produce Fl McAbs, and provide the antibody source for establishing the rapid diagnosis methods of plague. Methods: Healthy female BALB/c mice, six to eight weeks old, were immunized with Fl antigen, the differential composition of Y.pestis. Blood was collected from the mice via caudal vein, and the sera were tested for anti-Fl antibody titres by indirect enzyme-linked immunosorbent assay (ELISA). The mouse with the highest antibody titre was enhanced the immunity again by l00μg Fl antigen injected into abdominal cavity three days before cell fusion. The spleen cells of the immunized mouse were fused with myeloma cells SP2/0 be placed in the logarithmic growth phase using polyethylene glycol (PEG) as cell fusion reagent. Hybridoma cells were selected on hypoxanthine aminopterin and thymidine (HAT) medium. The supernatants of hybridoma cells were tested using indirect ELISA for the presence of Fl antibodies. Limited dilution was used to clone the positive hybridoma cells that produced Fl McAbs. After four cloning, several hybridoma cell strains stably producing anti-Fl McAbs were obtained. Results: After thirteen cell fusion experiments, three hybridoma cell strains that could stably secret Fl McAbs were obtained, named as IVIB12 IV2C10 and IV2C6. The litres of Fl McAbs in culture supernatants of three hybridoma cell strains were respectively IVlB12 1:1280-1:10240, IV2C10 1:2560-1:10240 and IV2C6 1:5120-1:10240 by indirect ELISA. Conclusion: Three hybridoma cell strains that could stably produce Fl McAbs against Y.pestis Fl antigen were successfully established by hybridoma technology. It will lay the solid foundation for establishing the rapid diagnosis technique of plague in the future. Experimental procedures of hybridoma technique were basically set up at the same time. |
PDF Full Text Request