Hybridoma Production and Screening

B14 Anat-Chem Building – (215) 898-2795

The Cell Center Service Facility will assist with monoclonal antibody production, including antigen preparation, immunization, fusion, screening and cloning. Our high efficiency fusion protocol typically results in about 80% hybridoma containing wells per fusion. Immunization is outsourced however injections are performed to our specifications. Isolation of spleen cells, fusion and all following cell culture work is done at the Cell Center Service Facility. We offer screening of supernatants by ELISA for subcloning of positive clones and other purposes.

These are the steps involved

1. Immunization (outsourced)
   • 2 mice – up to 5 injections per mouse – 1 spleen removal – isolation of spleen cells
2. Fusion (in-house)
   • Lymphocytes from 1 spleen into 10 x 96-well plates
   • Lymphocytes from 1 spleen into 20 x 96-well plates
3. Fusion maintenance and harvesting of supernatants for testing (in-house)
   • 10 plates or 20 plates
4. Screening of supernatants by ELISA
   • 10 plate or 20 plate fusion
5. Expansion of positive clones
   • Into 2 wells of a 24-well plate and freezing of 2 vials per clone
6. Subcloning by limiting dilution
   • 2 subclones per original clone – each subclone expanded into 2 wells of a 24-well plate
   • 2 vials frozen for each subclone
7. Antibody Fragmentation
   • The facility can generate Fab fragments from purified monoclonal antibodies. The antibodies are digested by Papain overnight at 37oC. Fab and Fc portions of the digested antibodies are purified over a Protein A column, dialyzed into PBS and run on SDS-PAGE for verification.

Please contact Sabine Baxter (215-898-2795) for details.