Generation of Mouse MAbs Using Standard Hybridoma Technology
The technical staff performs mouse immunizations and all cell culture procedures required to construct antibody secreting hybridoma cell lines. The investigator assays 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.
Expression of MAbs from Existing Hybridomas (In Vitro)
Small scale production of Mabs in volumes that range from 0.5 to 3 liters are generated using traditional culture methods. The culture supernatant yields MAbs ranging from 5-50 mg/ml. Larger amounts of Mabs can be produced in CL-1000 Celline flasks with the ability to generate antibody in from 50 -300 mg or hollow fiber production if larger amounts of antibody are required.
Purification of MAbs
The facility can purify MAbs produced in vitro by chromatography purification methods including affinity, ion exchange and size exclusion. The endotoxin-free quality can be achieved upon request.