Monoclonal antibodies

Monoclonal antibodies (mAb or moAb) are antibodies that are identical because they were produced by one type of immune cell and 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. When used as medications

The idea of a "magic bullet" was first proposed by Paul Ehrlich who at the beginning of the 20th century postulated that if a compound could be made that selectively targeted a disease-causing organism, then a toxin for that organism could be delivered along with the agent of selectivity.

In the 1970s the B-cell cancer myeloma was known, and it was understood that these cancerous B-cells all produce a single type of antibody (a paraprotein). This was used to study the structure of antibodies, but it was not yet possible to produce identical antibodies specific to a given antigen.

The process of producing monoclonal antibodies described above was invented by Georges Köhler, César Milstein, and Niels Kaj Jerne in 1975; they shared the Nobel Prize in Physiology or Medicine in 1984 for the discovery. The key idea was to use a line of myeloma cells that had lost their ability to secrete antibodies, come up with a technique to fuse these cells with healthy antibody producing B-cells, and be able to select for the successfully fused cells.

In 1988 Greg Winter and his team pioneered the techniques to humanize monoclonal antibodies,removing the reactions that many monoclonal antibodies caused in some patients.

Hybridoma

Monoclonal antibodies can be produced in cell culture or in live animals. If a foreign substance (an antigen) is injected into a vertebrate such as a mouse or a human, some of the immune system's B-cells will turn into plasma cells and start to produce antibodies that recognize that antigen. Each B-cell produces only one kind of antibody, but different B-cells will produce structurally different antibodies that bind to different parts ("epitopes") of the antigen. This natural mixture of antibodies found in serum is known as polyclonal antibodies.

To produce monoclonal antibodies, the B-cells from the spleen or lymph nodes are removed from an animal that has been challenged several times with the antigen of interest. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer or more specifically a plasmacytoma) and that have lost the ability to produce antibodies. This fusion is done by making the cell membranes more permeable by the use of polyethylene glycol (PEG), electroporation or, of historical importance, infection with some virus. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely. Large amounts of antibodies can therefore be produced. The hybridomas are sufficiently diluted to ensure clonality (all cells in the culture stem from the same single cell) and grown. The antibodies from the different clones are then tested for their ability to bind to the antigen (for example with a test such as ELISA or Antigen Microarray Assay) or immuno-dot blot, and the most sensitive one is picked out. 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.

In the above process, myeloma cell lines that have lost their ability to produce their own antibodies or antibody chain are used, so as to not contaminate the target antibody. Furthermore, only myeloma cells that have lost a specific enzyme called hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and therefore cannot grow under certain conditions (e.g. in the presence of a selection medium called HAT medium) are used; these cells are preselected by the use of either 8-azaguanine or 6-thioguanine(8-azaguanine has been shown to produce unreliable results.(van Diggelen et al 1979)) media prior to the fusion since cells that possess the HGPRT will be killed by the 8-azaguanine. During the fusion process many cells can fuse: Myeloma cell with myeloma cell, spleen cell with spleen cell, spleen cell with myeloma cell, etc. The desired fusions for making hybridomas are between a healthy B-cell, which produces antibodies against the antigen of interest, and a myeloma cell. In these relatively rare fusions, the healthy B cell will make the HGPRT enzyme that will allow the fused cell to survive in HAT medium so that only the successfully fused cells will grow in culture. The medium must be enriched during selection to favour hybridoma growth. This can be achieved by the use of a layer of feeder cells or supplement media such as briclone. Production in cell culture is usually preferred as the ascites technique may be very painful to the animal and if replacement techniques exist, may be considered unethical.

Recombinant

The production of Recombinant monoclonal antibodies involves technologies, referred to as repertoire cloning or phage display/yeast display. Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice. These techniques rely on rapid cloning of immunoglobulin gene segments to create libraries of antibodies with slightly different amino acid sequences from which antibodies with desired specificities can be selected.These techniques can be used to enhance: the specificity with which antibodies recognize antigens, their stability in various environmental conditions, their therapeutic efficacy, and their detectability in diagnostic applications. Fermentation chambers have been used to produce these antibodies on a large scale.

Applications

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.