Hybridoma Core Lab
Basic procedures include mouse immunization, with immunogen provided by requestor. Subsequent boost and bleeds are done to check antibody titer in the animal. Spleen cells are fused with a non-immunoglobulin secreting mouse myeloma cell line.
Hybridomas are cultured in standard selection medium. These hybridomas are screened by a variety of methods by the requesting lab, or by the Hybridoma Core by ELISA. Up to 10 are cloned by limiting dilution and screened again. Once a stable hybridoma cell line is produced and its isotype is determined, the core lab can produce ascites in vivo, or use in vitro methods of antibody production.
Cells are frozen at each stage of production and are kept in two locations. Generally, fusions are done with Balb/c mice. The Hybridoma Core Lab offers related services as time and demand permit. The Hybridoma Core also provides other tissue culture services. Endothelial cells from human umbilical vein are available upon request. Secondary passage and higher are kept in stock and can be ready with a week’s notice. Primary cultures require more advanced notice.
Ascites antibody production comes from in-house hybridoma or ATCC cell lines. If there is a sufficient supply, 3-15 ml of ascites can be provided from freezer inventory within 24 hours. If the ascites need to be produced in mice or in vitro, the wait time can be three weeks to three months, depending on demand.
New fusions are on a first-come, first-served basis. Preference is given to Versiti Blood Research Institute investigators. Generally, the requesting lab needs to provide about 1 mg of purified protein or peptide coupled to a carrier protein. The immunization period is typically 3-4 months. Screening, cloning, subcloning hybridoma and freezing of cells can take an additional 3-6 months. All hybridoma screening methods are to be worked out prior to fusion date. A technologist and the scientific director meet with each requesting lab to discuss the process and screening approach