Monoclonal antibodies – University of Copenhagen

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BRIC > Core Facilities > Monoclonal antibodies

Monoclonal antibodies

BRICs Antibody core facility offers help to the production of polyclonal and monoclonal antibodies.  These steps include immunization, and fusion of hybridomas.


Many variables can impede the success of an antibody development project, including the antigenicity and supply of the immunogen. BRIC is pleased to offer our knowledge and background to assist in the successful development and production of antibodies. We strongly encourage customers to directly participate in all the steps of the production, from the definition of the desired product and the antigen selection, to the screening of the hybridomas to isolate a panel of antibodies that covers as many aspects of your research as possible.

The various steps in the production of a monoclonal antibody are outlined below:

Immunization

An antibody response is generated in mice to the specific immunogen. Up to 8 animals are immunized if required with the antigen. A standard project (2 animals) requires 4-5 mgs of immunogen (typically GST-tagged protein or protein fragment). Immunogens less than 3-8 kD should be conjugated. We recommend conjugating 3 mgs to KLH for immunizations and 2 mgs to BSA for screening of test bleeds & supernatants. The customer will supply the antigen, and fill out a project plan and immunization plan. The core facility will carry out immunizations in collaboration with the EMED-Unit at the biocenter and supply serum from immunized animals.

Screening

The primary screening is a direct ELISA against your antigen to identify a suitable responder animals or hybridomas. Secondary screens may also be performed using other techniques as western blotting or immunoprecipitation, depending on the customers needs. In general, all screenings will be performed by the customer and the core facility will provide bleeds and or hybridoma supernatants.

Fusion (for mouse monoclonal antibody production)

Spleen cells from the selected animal are fused with a myeloma cell line to develop a hybridoma clone that secretes a specific antibody. Once your mice are ready for fusion as judged by the screening results, a fusion will be scheduled. The mouse will receive a final fusion boost of immunogen. The fusion is plated out and screened. The primary screening is a direct ELISA against your antigen to capture all positive fusion products. Additional screening methods may be performed (western blotting etc.) The positive fusion products are scaled up and screened again. In general, the customer will perform all screening and will choose the number of fusion wells for scale-up. The core facility will culture hybridomas and supply hybridoma supernatants for screening. Not all wells that are scaled up will survive or continue to produce specific antibody. The supernatants of the positives are shipped to the customer for testing.