VMC 221: Veterinary Immunology and Serology (1+1)

PRODUCTION OF MONOCLONAL ANTIBODIES

• This is done by different steps like generation of antibody producing plasma cells, fusion of plasma cells to the myeloma cells and re-cloning of cloned hybrid cells for specific antibody production.

Generation of antibody producing plasma cells

•  By immunizing a mouse against the antigen of interest and repeating the process several times to ensure that it mount a good response.
• Mouse spleen is removed after 2 to 4 days of last administration of antigen and broken upto form almost single cell suspensions.

Fusion of plasma cells with myeloma cells:

• The spleen cells are suspended in cell culture medium together with the special mouse myeloma cells.
• It is usual to use myeloma cells that do not secrete immunoglobin as this may interfere the monoclonal antibody production (some examples are MOPC 21 BALB/C, MPC 11 BALB/C, SP2 /0BALB/C etc.).
• For induction of fusion, polyethylene glycol (PEG) is added to this mixture of cells (out of 2, 00,000 of spleen cells, one cell can be fused with one myeloma cells).
• The fused cell mixture is cultured for several days and the unfused spleen cells will die.
• There are three biosynthetic pathway for synthesis of nucleic acid (i.e. from Hypoxanthine, Thymidine and Uridine ).
• Myeloma cells will survive but can be eliminated as they lack two enzymes namely hypoxanthine phosphoribosyl transferase (HGPRT) and thymidine kinase (Tk) but these are present in hybridoma cells. As myeloma cells lacks these two enzymes, they must use alternative pathway to convert uridine to nucleotides.
• The fused cells are allowed to grow into a culture media containing three compounds namely hypoxanthine, aminopterin and thymidine (called HAT medium).
• Aminopterin is a drug that prevents the myeloma cells to prevent making their own nucleotides from uridine and as a result they die soon. But the hybrid cells made from a myeloma and normal cells will grow since they possess hypoxanthine phosphoribosyl transferases and thymidine kinases thus utilize hypoxanthine and thymidine from culture medium to synthesize nucleic acids and survives – this pathway is called salvage pathway.
• Hybridoma cells divides rapidly on HAT medium doubling their number in 24-48 hours.
• On an average about 300-500 different hybrids can be isolated from a mouse spleen but all of them do not make antibody of interest.
• About 2-4 weeks of cultures growing cells can be seen and supernatant fluid should be checked for the presence of antibody.
• In a fusion experiment, about 50,000 myeloma cells are seeded per well in a culture plate and one hybrid cell can be obtained from every three wells.
• Clones are sub cloned to ensure that are it is single clone producing desired antibody or monoclonal antibody and that are grown in mass culture.
• Unfortunately antibody producing hybrid cells lose their ability for culturing several times. So hybrids cells stock is aliquot into small volume and preserved frozen for future use.