Biochemical Techniques and Instrumentation (1+2)

6.4.2. Technique

The production of hybridomas involves eight steps.

1. Immunization of a mouse: Laboratory animals such as mice or rabbits are first immunized through repeated injection of a specific antigen for the production of specific antibody over the course of several weeks.
2. Isolation of B cells from the spleen: Splenocytes are then isolated from the animal’s spleen as they produce specific antibodies. Spleen cells are rich in lymphocyte B cells and T cells.
3. Cultivation of myeloma cells: Tumours are produced in the bone marrow of a mouse or a rabbit. These myeloma cells do not secrete antibody themselves. They also lack the hypoxanthine-guanine phophoribosyl transferase (HGPRT) gene.
4. Fusion of myeloma and B cells: The lymphocyte B cells are then fused with immortalized myeloma cells to produce the hybrid cells. The fusion is accomplished using polyethylene glycol (PEG) or the Sendai virus. The hybrid cells are then grown in selective hypoxanthine aminopterin thymidine (HAT) medium for 10 to 14 days. The HAT medium contains a drug aminopterin, which blocks one pathway for nucleotide synthesis, making the cells dependent on another pathway that needs HGPRT gene, which is absent in myeloma cells. Therefore, myeloma cells that do not fuse with B cells will die as they cannot produce nucleotides. Removal of the un-fused myeloma cells is necessary because they have the potential to outgrow other cells. The B cells that do not fuse will also die because they have a short life span and lack tumorigenic property for immortal growth. Therefore, HAT medium allows selection of hybridoma cells, which inherit HGPRT gene from B cells and tumorigenic property from myeloma cells.
5. Separation of cell lines: The incubated HAT medium is then diluted into multi-well plates so that each well contains only one cell. As the antibodies in a well are produced by the same B cell directed towards the same epitope, and are thus monoclonal antibodies. Only one in several hundred cell hybrids will produce antibodies of the desired specificity.
6. Screening of suitable cell lines: The hybridoma culture supernatant, secondary enzymes labelled conjugate, and chromogenic substrate, are then incubated, and the formation of a coloured product indicates a positive hybridoma.
7. in vitro (a) or in vivo (b) multiplication: Multi-well plates are used to grow the selected hybridomas. B cell that produces the desired antibodies can be cloned to produce many identical daughter clones. Supplemental media containing interleukin-6 such as briclone are essential for cloning. Once a hybridoma colony is established, it will continually grow in culture medium RPMI-1640 that contain antibiotics and foetal bovine serum and produce antibodies.
8. Harvesting: The culture supernatant can yield 1 to 60 µg/ml of monoclonal antibody. Hybridoma cells can be cryopreserved or frozen for future use or can be injected in the body of an animal so that antibodies will be produced in the body and can be recovered later from the body fluid.