VPB 321: Animal Biotechnology (2+1)

STEPS IN PRODUCTION OF MONOCLONAL ANTIBODIES

•  Immunization
  • The animal used for immunization is BALB/c mice. The purpose of immunization is to sensitize the B lymphocytes (in the spleen) against the antigen for which monoclonal antibodies required. Immunization is done by repeated injection of antigen into the mice.
• Fusion
  • The purpose of the fusion is to produce hybrids cells between the B lymphocytes and the cancerous myeloma cells (Sp2/0). The fusion is done by mixing the immunized B lymphocytes with the myeloma cells in the presence of polyethylene glycol (PEG). The PEG helps to fuse the cells.
• Selection of hybrid cells
  • The fused cells are grown in HAT (Hypoxanthine, Aminopterin and Thymidine) medium. The selection of hybrid cells formed between only B lymphocyte and the myeloma cell is achieved by growing the fused cells in the HAT medium.
• Cloning of hybrid cells
  • The hybrid cells growing in HAT medium are then screened for their ability to produce and secrete the desired antibody. This is done be enzyme linked immunosorbent assay (ELISA). Then cloning under limiting dilution is done to ensure the monoclonality of the hybrid cells. Limiting dilution is a dilution technique by which hybrid cells are distributed at a concentration of 1 cell per well. Within 2 weeks, the ‘one’ cell in the well form a group of cells and secrete monoclonal antibodies against single epitope of the antigen used in the immunization.
• Large scale production of monoclonal antibodies
  • For large scale production, hybridomas can be grown either in tissue culture, where they secrete up to 100 ug / ml (usually 10 – 50 ug / ml) or in vivo as tumors in the peritoneal cavity of BALB/c mice (hybridoma) where they produce up to 40 mg / ml (usually 2 – 20 mg / ml)