Biotechnology and Bioinformatics (1+1)

3.2.2. Production of monoclonal antibodies

Monoclonal antibody production is initiated by the immunisation of BALB/c mice with immunogens. e.g., protein , carbohydrate, nucleic acid or combinations of these. They can also be produced from impure antigen by selecting single cell clone after the fusion.

• Antibodies are produced by differentiated B-cells (plasma cells) and because each parent B-cell has the capability of producing antibodies of a particular specificity, the antibodies secreted by a B-lymphocyte clone are identical and therefore, is a source of homologous antibodies.
• Plasma cells are, however, short-lived and cannot be grown in culture.
• Therefore, fusion of these cells with immortal myeloma cells produces hybridoma cells with the ability to grow in culture and to secrete antibody with a defined specificity.
• Chemical selection, screening of the antibodies produced and cloning of the hybridoma cells lead to the ultimate production MAbs.
• Myeloma cell lines used in fusions have been selected because they do not produce antibody molecules, although some of the commercially available cell lines do produce immunoglobulin heavy or light chain molecules. For this reason P3x63. Ag8-653 (653) and Sp2/0-Ag14 (Sp2/0) are the most frequently used cell lines in hybridoma technology.
• Hybridoma cells can be prepared by fusing myeloma cells and antibody – producing cells which have been isolated from different mouse species, but the success rate of fusion is greatly increased if both cell types come from the same strain of mouse (e.g., BALB/c).
• Originally, Kohler and Milstein used Sendai virus as the fusion agent, but polyethylene glycol (PEG) is now routinely being used to fuse the cells.
• Even in efficient fusions, only approximately 1% of the initial cell numbers result in fusion.
• This leaves a large number of unfused cells, both spleen and myeloma cells still present in the culture.
• The spleen cells from the mouse die within 3 days of culture and therefore, do not pose a problem.
• However, the myeloma cells quickly adapt to the culture conditions and will outgrow the hybridoma cells resulting from the fusion.
• Removal of the myeloma cells is therefore, essential and is achieved by chemical selection. Commercially available myeloma cells are defective in one of the enzymes of the salvage pathway of purine nucleotide biosynthesis. Cell lines 653 and SP2 have mutations of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene . Addition of aminopterin to the culture medium blocks the de novo nucleotide synthesis pathway and forces the cell to use the salvage pathway in which HGPRT uses exogenous hypoxanthine and thymidine. Myeloma cells defective in HGPRT are unable to use this pathway and therefore, die in culture.
• The only cells able to grow in HAT (hypoxanthine, aminopterin, thymidine) culture medium are the hybridoma cells, which are unable to synthesize DNA via de novo nucleotide synthetase pathway and rely on the salvage pathway for DNA synthesis (a characteristic provided by the spleen cell part of the hybridoma).
• Positive clones producing specific antibodies are usually identified by ELISA and are selected, expanded and cloned using a limiting dilution technique. Positive hybridomas are normally cloned three times before they are considered MAb producing cells.
• The resulting MAbs are extremely specific and are therefore, very useful diagnostic tools.
• In addition, hybridoma cell lines have the advantage of providing an unlimited supply of the antibody in the cell supernatant, which allows standardisation of the MAb reagents.