Samples will be provided to the client for testing and decision making at the appropriate stages during hybridoma development. To minimize costs to the client, all decisions regarding the selection of cell lines for cloning, expansion, freezing, and bulk antibody production must be made by the client in a timely fashion. This will minimize misunderstandings and keep costs as low as possible. It is in the best interest of the researcher to communicate test results, problems, intended schedule or procedural changes promptly. Produce bulk antibody in a bioreactor or static culture.Once large cultures are established, cyropreserve several vials from each hybridoma cell line. Expand growing antibody-secreting cultures by transferring them to progressively larger culture vessels.Communicate results within 2 working days after receipt of the samples. Test for useful antibody in the supernatant of growing cultures. Screen monoclonal microcultures for growth.Use the single-cell deposition function of a flow cytometer/cell sorter to clone selected polyclonal hybridoma cultures and establish several monoclonal hybridoma microcultures.Once large cultures are established, cyropreserve a few vials from each culture as backups. Use naive mouse serum and myeloma culture supernatant as negative controls. Use serum from the immunized mouse as a positive control. Screen primary microcultures for growth.HAT medium selects for cells which arise from splenocyte-myeloma fusion. Establish primary microcultures in HAT medium.Cell fusion is facilitated by polyethylene glycol (PEG). Isolate mouse splenocytes and fuse them with myeloma cells.The test method must be the same as that which will be used to screen hybridomas. Test serum samples to assess the immune response. Perform primary and secondary immunizations.The form of the antigen will depend on the immunization method. The amount of antigen may be reduced if immunostimulation or in vitro stimulation techniques are used. At least 500 micrograms of antigen should be available for ordinary intraperitoneal immunization. The client must be confidant that the test system can screen 300 to 500 samples in one or two days. This may require immunizing a few mice to obtain positive mouse serum for testing and standardization of the screening assay. ELISA or western blot) that will efficiently screen for useful antibodies. This may influence how the mice will be immunized and what screening methods should be used. Determine how the end product is to be used.The facility has storage capacity for about 20,000 vials. It typically takes four weeks from the fusion of the spleen cells until the positive cultures are frozen. Positives are expanded to 24-well plates, T25 flasks, T75 flasks and then finally frozen. Supernatant fluids are commonly re-screened at this stage to confirm the positive hybridomas. (If the client can not screen the sups within a day, Hybridoma Services can do the screenings in-house.) The cells in the wells containing the positive supernatant fluid are moved up to 48-well plates and allowed to expand. The client screens the sups and provides the results to Hybridoma Services the same day. Usually, 300-800 wells produce growth and as the wells turn yellow, they are sampled and the supernatant fluids are shipped overnight for next day morning delivery. This results in 12, 96-well plates per mouse. Usually, enough spleen cells are harvested to do three fusions. When the client is satisfied with the titer, the mouse with the highest titer is killed and the spleen cells are used for a fusion. Two weeks after the last boost, they are bled and the sera is sent to the client for testing to determine the Ab titer. The mice are injected twice, two weeks apart. A cage of three to four mice is injected with 25μg of the antigen mixed with adjuvant Freund’s Complete Adjuvant is used for the first injection which is subQ, and all subsequent injections are IP using Freund’s Incomplete Adjuvant. Monoclonal antibody projects done for off-campus customers usually proceed as follows: A project is submitted through the Hybridoma Services website and an antigen is received by the Services. Bulk monoclonal antibody is produced by growing the hybridoma cells in static cultures or bioreactors. Productive hybridomas are expanded and cloned to establish monoclonal hybridoma cell lines. Successful fused cells are propagated in selective medium and screened for antibody production. Antibody-secreting hybridoma cells are produced by fusing the spleen cells from an immunized mouse e ith mouse myeloma cells (e.g.
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