Hybridoma Production
Hybridoma Development
Animal inoculation and serum collection
The MAF holds an IACUC-approved protocol using Balb/c mice or Sprague-Dawley rats for immunization. Serum collection is by retro-orbital bleed under general (inhalation) anesthesia.
Immunization Protocol
The most important factor for success in creating hybridomas between spleen cells and myeloma fusion partners is an activated, antibody producing splenocyte in a state of mitosis. The researcher increases his/her chances for success by the timing and administration of antigen. To do so, we use the RIBI adjuvant System (MPL+TDM) to enhance the immune response. Each vial of MPL+TDM lyophilized product (for mice inoculation) contains: 0.5mg Monophosphoryl lipid A (MPL), 0.5mg Trehalose dimycolate (TDM), 0.04ml Squalene (hexamethyl-tetracosahexane) and 0.004ml monooleate (Tween 80). For mice, we use 0.1ml per intraperitoneal injection, boosting on day 21 and 42, then bleed 10-14 days after the third injection to check for the production of antibody titers. If the titers are adequate as determined by the investigator's screening method, we plan the final immunization preceding the fusion.
Creation of custom hybridomas using chemical (PEG) fusion techniques and NS-1 myeloma partner.
Immunized splenocytes are washed and fused to NS-1 myeloma cells using PEG-1500 and our standard protocol. The hybridomas are exposed to HAT 24 hours later, and the non-fused NS-1 cells will die. The non-fused splenocytes also have a finite lifetime, and the hybridomas are then the only proliferating cells left in the culture. We customarily expect to see growth in 80-95% of the wells plated.
Cloning and cell isolation procedures
Supernatants are harvested for testing in the investigators' antigen-specific system. Based on the data generated, cells are selected for (statistically-based) cloning. Usually 3 rounds of cloning will isolate the monoclonal cell line adequately. We encourage investigators to select 3-5 lines at each round of cloning, and to archive other cells which may be useful in the future.
Cell archiving and supernatant collection
Once a monoclonal cell line is established a stock of archived cells is produced as soon as possible. Isotyping, antibody production and purification are the next issues to consider. Selected cell lines should be retested periodically to ensure that they are still producing the desired antibody. Hybridomas continue to "throw-out" genes as they are kept in culture (and since they don't need immunoglobulin genes to survive, those go early) and may need to be re-cloned to find the best producer after 4-6 months in continuous culture.
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