Abstract: It was to utilize the improved intrasplenic immunization and semi-solid culture medium to reduce the immune dosage, clone the hybridoma simply, and shorten the period of cycle. The skin areas of mice spleen was anesthetized, then the spleen was injected with trace antigen, which could be immuned in a short period of time, cell fusion was prepared after intravenous injection. The three different methods（limited dilution, methylcellulose, soft agar）were used to get hybridomas which can secret monoclonal antibody. Results showed that compared with the traditional method of preparation of monoclonal antibody, the improved intrasplenic immunization was simpler operation, without the addition of adjuvant, and the high titer of antibody was getted in a short time. Positive rate was the highest when methylcellulose concentration in the 1.1% condition. It was suggested that these methods could be suitable for large scale preparation of monoclonal antibody.

Key words: Intrasplenic immunization, Soft agar, Methylcellulose