Monoclonal antibodies (mAb or moAb) are antibodies that are identical because they are produced by one type of immune cell that are all clones of a single parent cell. Given (almost) any substance, it is possible to create monoclonal antibodies that specifically bind to that substance; they can then serve to detect or purify that substance. This has become an important tool in biochemistry, molecular biology and medicine.
Preparation:
Monoclonal antibodies are made by fusing the spleen cells from a mouse that has been immunized with the desired antigen with myeloma cells.
Polyethylene glycol is used to fuse of adjacent plasma membranes, but the success rate is low so a selective medium is used in which only fused cells can grow. This is because myeloma cells have lost the ability to synthesize hypoxanthine-guanine-phosphoribosyl transferase (HGPRT).
This enzyme enables cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Ordinarily, the absence of HGPRT is not a problem for the cell because cells have an alternate biochemical pathway that they can use to synthesize purines. However, when cells are exposed to aminopterin (a folic acid analogue), they are unable to use this other, rescue, pathway and are now fully dependent on HGPRT for survival. The selective culture medium is called HAT medium because it contains:• Hypoxanthine• Aminopterin• ThymidineThis medium is selective for fused, (hybridoma) cells because unfused myeloma cells cannot grow because they lack HGPRT.
Unfused normal spleen cells cannot grow indefinitely because of their limited life span. However, hybridoma cells are able to grow indefinitely because the spleen cell partner supplies HGPRT and the myeloma partner is immortal because it is a cancer cell. The fused hybrid cells are called hybridomas, and since they are derived from cancer cells, are immortal and can be grown indefinitely.
This mixture of cells is then diluted and clones are grown from single parent cells. The antibodies secreted by the different clones are then tested for their ability to bind to the antigen (for example with a test such as EIA or Antigen Microarray Assay) or immuno-dot blot, and the most productive and stable clone is then grown in culture medium to a high volume. When the hybridoma cells are injected in mice (in the peritoneal cavity, the gut), they produce tumors containing an antibody-rich fluid called ascites fluid.
The medium must be enriched during selection to further favour hybridoma growth. This can be achieved by the use of a layer of feeder fibrocyte cells or supplement medium such as briclone.
Uses:
Once monoclonal antibodies for a given substance have been produced, they can be used to detect the presence and quantity of this substance, for instance in a Western blot test (to detect a protein on a membrane) or an immunofluorescence test (to detect a substance in a cell). They are also very useful in immunohistochemistry which detect antigen in fixed tissue sections.
Monoclonal antibodies can also be used to purify a substance with techniques called immunoprecipitation and affinity chromatography.
Monoclonal antibodies for cancer treatment:
One possible treatment for cancer involves monoclonal antibodies that bind only to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Such mAb could also be modified for delivery of a toxin, radioisotope, cytokine or other active conjugate; it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cell. In fact, every intact antibody can bind to cell receeceptors or other proteins with its Fc region.
