Hybridoma Facility

Services

Immunization
When making monoclonal antibodies, the first step is to immunize the mice or rats with the desired protein. The protein is added to TiterMax Adjuvant and then injected into two animals. After two weeks, the animals are immunized for the second time.

Serum Titer
Two weeks after the second immunization, a sample of serum is obtained for testing. The serum is tested by ELISA to determine the level of IgG and IgM response. If the IgG titer is high and the IgM titer is low, then a fusion will be scheduled.

Fusion
Three days before the fusion, one of the animals is immunized by tail i.v. with the protein in PBS. This will stimulate the immune system and begin the expansion of antigen-specific B cells. At day 3, the immune response has reached its peak and B cells taken from the spleen are then fused to mouse myeloma cells using PEG. The fused cells are plated in a selective media containing azaserine to kill any myeloma cells that are not fused to a B cell.

Screening Process
After approximately 2 weeks, the supernatant from each hybridoma is tested by ELISA for antibodies that recognize the antigen. If the antigen has a tag, the purified tag protein is used as a negative control in the ELISA. Any hybridomas that show a positive response to the antigen and a negative response to the tag protein are grown up to 2 ml, then the supernatant is tested again to be sure that the hybridoma is still positive. The facility keeps 2 freezes of each positive hybridoma.

Testing by Investigator
Once the positive hybridomas have been identified and confirmed, a sample of the supernatant is given to the investigator to test. If the antibody proves to be useful to the investigator, then the next step is usually cloning.

Cloning
Cloning is performed in one of two ways. If the hybridoma is still in culture at the time that subcloning is requested, then the cells are cloned by limiting dilution. If the cells have been frozen prior to cloning, they are plated at two different concentrations (50cells/ml and 5 cells/ml). After approximately one week the supernatants are tested. Two positive clones are kept for each hybridoma. The facility keeps 2 freezes of each clone.

End Product
Once cloning is complete, there are several options for the investigator: 1) Supernatant from the hybridoma may be requested. This is usually done in volumes of about 100 ml. For this, the cells are brought up to the requested volume and then allowed to overgrow until about 50% of the cells are dead. This allows for a fairly high concentration of antibody. 2) If a higher concentration of antibody is desired, the hybridoma can be grown in a bioreactor. Some cell lines perform well in the bioreactor while others are more resistant to these growth conditions. Whether or not the bioreactor will be beneficial must be decided from project to project. 3) The cells may be expanded and freezes prepared for the investigator. Cells are frozen in FCS with 5% DMSO at a concentration of 1-3 million cells/ml. 4) Any other requests are welcome and will be evaluated on a case-by-case basis.

* As previously noted, the facility does not perform ascites production due to ethical considerations. The bioreactor is suggested as an alternative to this method of antibody production.