About Monoclonal Antibodies:
Monoclonal Antibodies are cells derived by cell division from a single ancestral cell.Monoclonals are a class of antibodies with identical offspring of a hybridoma and are very specific for a particular location in the body derived from a single clone and can be grown indefinitely. Monoclonal Antibodies recognize and bind to antigens in order to discriminate between specific epitopes which provides protection against disease organisms.
Monoclonal antibodies target various proteins that influence cell activity such as receptors or other proteins present on the surface of normal and cancer cells.The specificity of Monoclonal Antibodies allows its binding to cancerous cells by coupling a cytotoxic agent such as a strong radioactive which then seek outs to destroy the cancer cells while not harming the healthy ones.
Tumor cells that are able to replicate endlessly are fused with mammalian cells that produce a specific antibody which result in fusion called hybridoma that continuously produce antibodies. Those antibodies are named monoclonal because they come from only 1 type of cell, which is the hybridoma cell. Antibodies that are produced by conventional methods and derived from preparations containing many kinds of cells are called polyclonal Antibodies.
Monoclonal antibodies are artificially produced against a specific antigen in order to bind to their target antigens. Laboratory production of monoclonal antibodies is produced from clones of only 1 cell which means that every monoclonal antibody produced by the cell is the same.
Fusion of cell culture myeloma cells with mammalian spleen cells antibodies result in hybrid cells/hybridomas which produces large amounts of monoclonal antibody. The cell fusion resulted in two different types of cells, one with the ability to grow continually, and the other with ability to produce bulk amounts of purified antibody. Hybrid cells produce only 1 exact antibody that is more pure than polyclonal antibodies produced by conventional techniques. Monoclonal Antibodies are far more effective than conventional drugs since drugs attack the foreign substance & the body's own cells that cause harsh side effects & the monoclonal antibody only targets the foreign antigen/target molecule, without or only minor side effects.
The presence of a large amount of a specific monoclonal antibody in the blood means that there is an abnormal protein. Typically this protein can be detected during a physical examination and is identified using a screening blood test called “protein electrophoresis. The source of abnormal production of monoclonal antibody is a small population of plasma cells in the bone marrow.
Production of Monoclonal Antibodies:
Process by which bulk quantities of targeted antibodies against a specific antigen are produced.Monoclonal antibodies are produced via multiple/identical copies of a certain cell called a hybridoma. To create Hybridoma cells the fusion of 2 cells are needed in order to combine the characteristics of the 2 cells into 1 cell. 1 of the cells is a producing cell antibody which is a B-Lymphocyte used from a laboratory mouse and the other is a tumor cell named myeloma. Tumor cells have the ability to grow indefinitely and at an exceeding rate from normal cell growth. Laboratroy produced Hybridoma cells replicate much faster than normal antibody producing cells, and the individual hybridomas produce the specific antibodies for an indefinite period of time.
Hybridoma cells manufacture the specific monoclonal antibody that was originally produced by the B-Lymphocyte cell. The original B-Lymphocyte cell will produce the Monoclonal antibody depending on the kind of antigen that was injected into the mouse just prior to the harvesting of the B-Lymphocyte cells. A small example is, if the mice were injected with a certain virus, the mouse will have B-Lymphocytes that produce those specific virual antibodies. Fusion with a tumor cell to make the hybridoma, result in the production of monoclonal antibodies against the specific virus.
The hybridoma cells are placed into media that can help them grow and produce the bulk quantities of monoclonal antibodies. There are 2 ways for growing monoclonal antibodies, 1 is to grow them in laboratory flasks meaning In Vitro, and the other is to grow them in the stomach lining of mice. Injecting the hybridomas into the mice is the familiar method of harvesting monoclonal antibodies. This method is done by mixing spleen cells from the mouse that has been immunized with the desired antigen with myeloma cells.The myeloma cells need to have lost their ability to synthesize HGPRT enzyme (hypoxanthine-guanine-phosphoribosyltransferase) which enables the cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Cells have another pathway that they synthesize purines so lack of HGPRT is not a problem for the cell but when cells are exposed to aminopterin they are unable to use this other pathway and are fully dependent on HGPRT for their survival. So to summarize, unfused myeloma cells can’t grow since they lack HGPRT and unfused normal spleen cells can’t grow since they have a limited life-span. Hybridoma cells are grow indefinitely since the spleen-cell copartner supplies HGPRT and the myeloma partner is immortal.
The first step is transferring of the cell fusion mixture to HAT culture medium which contains hypoxanthine, aminopterin & pyrimidine thymidine. The 2nd step is testing the supernatants from each culture in order to locate the producing the desired antibody. One must isolate the single cells from each antibody-positive culture and subculture them, this represents the clone which its antibodies are monoclonal. Every single cell culture secretes a specific kind of antibody that is directed against a certain determinant/selected antigen. The 3rd step is scaling up the size of the cultures of the successful clones. Hybridoma cultures can be grown indefinitely in vitro in culture vessels which yield 15-65 µg/ml and in vivo using mouse, where the antibody concentration in the serum 0.5-15 mg/ml. In the past years, animal welfare activists in worldwide are trying to limit the use of mice for the production of monoclonal antibodies. When the monoclonal antibody is produced it can be used as a probe to track down, bind to and purify the specific protein that induced its formation.