VPB 321: Animal Biotechnology (2+1)

PRODUCTION OF MONOCLONAL ANTIBODIES

Aim: To demonstrate the production of monoclonal antibodies

Materials required: Immunized mice spleen, HAT medium, Myeloma cells, PEG, Culture medium

Method

• The first step in preparing Monoclonal antibodies specific to protein x is to inject a mouse with a solution of the protein.
• The protein molecules reach the spleen, where they encounter B lymphocytes. Each B lymphocyte can produce a single type of antibody specific for a single molecular determinant.
• The B lymphocytes expressing abs specific to determinants of protein x will bind the protein. This triggers a series of events that stimulates the cell to grow and divide resulting on a colony of cells.
• Next, a population of spleen cells is removed from the mouse. This includes the B lymphocytes with protein x – specific abs and nonspecific lymphocytes.
• The spleen cells are mixed with HGPRT^- mouse myeloma cells, Fusion and the myeloma cells with the spleen cells result in hybridoma.
• The mixture of cells undergoes selection in HAT medium. Unfused lymphocytes are normal cells which cannot be cultured, so they die. Unfused myeloma cells are HGPRT^-, and thus cannot survive in HAT.
• The hybridomas are immortal and HGPRT⁺, so they are able to survive in HAT medium.
• The hybridomas are separated into single clones and cultured.
• The hybridoma clones are tested for production of antibodies specific to protein x.
• The hybridomas can now be used to produce pure monoclonal abs for use in cellular and biochemical experiments.

Questions

• Define Monoclonal antibodies.
• Write about methods employed for cell fusion.
• What is ‘Immunization’?
• Write the principle of ‘HAT’ selection.
• Define “Cloning by limiting dilution”.