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Hybridoma Development
Mouse Monoclonal Antibody
| General Bioscience Corp offers a complete package for mouse monoclonal antibody hybridoma development project. The project is divided into three phases to meet customers's needs. If the size of the antigen is less than 20 KD, we recommend conjugation to a carrier protein such as KLH to produce the required immune response in the animal. |
Phase I: Immunization
| Procedure | Tasks |
| Step 1: injections | Immunization of 5 Balb/c mice. In general we begin with priming dose of 50 micrograms to 200 micrograms followed by several boosting doses each containing 50 to 200 micrograms. This regime will continue until sufficient antiserum titer is achieved |
| Step 2: Screening | Test bleeds are evaluated by using an indirect ELISA. Optimal ELISA conditons are developed to ensure the proper evaluation of future clones. Test bleeds can be sent to the customer for evaluation in other assays |
| Step 3: Evaluation | Data is evaluated to determine the performance of antiserum. Antiserum titers and specific assay permance must be acceptable to continue the project |
Phase II: Fusion
| Procedure | Tasks |
| Step 4: Fusions | A splenocyt fusion is performed on host animals with the best response as determined by ELISA or other assay. The lymphocytes are then fused to an Sp2/0-Ag14 cell line at a 5:1 ration using the PEG method |
| Step 5: Screening | The fusion is plated into 24 or 96 well plates. Plates are monitored for growth and fed weekly. Wells with cell growth are screened by ELISA and/or other methods at 10-20 days after fusion |
Phase III: Cell Line Cloning
| Procedure | Tasks |
| Step 6: Subcloning | Cells from up to 5 strongly positive parental clones from fusion wells are expanded, isotyped and subcloned by limiting dilution. More subclones can be processed at your request for an additonal charge |
| Step 7: Screening | Subclones are screened by ELISA, positives expanded, and screened again. Up to two positive subclones from each parental subclone will be expanded, frozen and stored for up to six months |
