Construction of New Hybridomas: The technical staff performs mouse immunizations and all cell culture procedures required to construct antibody secreting hybridoma cell lines. The investigator must assay mouse serum for specific antibody response. Specific procedures include removal of mouse spleen, PEG-mediated cell fusion with mouse myelomas, propagation of hybridomas in semi-solid methylcellulose HAT selection medium, robotic picking and transfer of individual hybridomas clones from the semi-solid methylcellulose to 96 well plates, ELISA screening of hybridoma wells, and expansion and freezing of positive clones in liquid nitrogen. Additionally, the resource performs isotyping assays of cloned cell lines. Investigators are asked to perform secondary screening assays to confirm specificity of the hybridomas selected by ELISA. Secondary screening typically involves immunoblotting, immunoprecipitation, immunocytochemistry or functional effects on the antigen. Several mouse myeloma cell lines are maintained as fusion partners including SP 2/0, P3-X63-AG8.653, NS-1, and FOX-NY.
In Vitro Production of MAbs from Existing Hybridomas: The facility mass produces monoclonal antibodies(MAbs) in vitro by three methods:
- Conventional spinner vessels in volumes that range from 0.5 to 5 liters. Investigators are provided with culture supernatant as a source of MAbs with yields ranging from 5-50 mg/ml.
- Stationary membrane bioreactor system (CELLine CL-1000, BD Biosciences). The level of production is intermediate between the hollow-fiber perfusion system and conventional spinner vessels, yielding antibody concentrations in the range of 0.25 - 1.0 mg/ml.
- Hollow-fiber bioreactors. The cells grow to a high density on the large interior surface area of the capillary fibers and are capable of secreting high concentrations of MAbs in the range of 0.5-1.5mg/ml.
Down–stream processing and purification of MAbs: The facility will purify MAbs produced in vitro by any of the above procedures by standard methods.
- Ammonium sulfate precipitation followed by affinity chromatography on Protein A/G agarose resins or DEAE ion-exchange.
- Analysis of purified MAbs by PAGE and protein Bradford assays.