The word 'Antibody' must be familiar to science graduates, medical professionals and maybe even to common man due to their significant role in the defense mechanism of living body, which is otherwise termed as immunity. When a foreign molecule or protein (antigen) is detected by our body's immune system, it produces antibodies, in response to it. These are specific molecules which has the ability to recognize any foreign material in the body, by binding to it and thereby initiating a cascade of events that facilitate the removal of antigens from the system by phagocytes. It is the B lymphocytes that produce antibodies and the part of the antibody that binds to the antigen is called the epitope.
Usually, the immune response of our body is polyclonal. That is, many types of antibodies with varied diversity of structure in epitope and effector regions are produced. The two most important properties of antibodies that make them interesting candidates for human therapy are their specificity and the fact that the epitope can by itself bind to target molecules. But, the problem that we face is that even if we become successful in isolating a single antibody secreting cell, it has limited growth potential just like any other somatic cell and will eventually die after a few generations in a culture. So, in order to use antibodies as a tool in molecular biology, a technique was devised by Georges Kohler and Cesar Milstein for which they were awarded Nobel Prize in the year 1984. The method involves the production of monoclonal antibodies that has unlimited potential to multiply. A group of B cell which originates from a single ancestral B cell is cultured which enables us to obtain a single type of antibody. Such a population of B cells can be correctly defined as monoclonal, and the antibody produced is called monoclonal antibodies.
First, the specific B cell that produces the antibody of our interest is to be isolated from a living organism. For this, production of the B cell is induced by injecting a primary dose and later, a booster dose of the antigen. A sample of the B cell produced in response to the injected antigen is extracted from the spleen of the organism and these are added to a culture of myeloma cells (or cancer cells). Like any other cell, an antibody-secreting B cell can also become cancerous. Such a cell which shows the activity of unchecked proliferation is called a myeloma. What we get from such the fusion of B cells and myeloma cells is the formation of hybridomas and this technique is called somatic cell hybridization. The fusion is carried out by Polyethylene Glycol (PEG), virus or by electroporation.
Due to the reduced success rate of intended fusion, a fool proof selection procedure is the next step. To facilitate this selection, the myeloma cells selected for hybridization should possess certain special properties. Firstly, the myeloma cells used for hybridization should be incapable of synthesizing hypoxanthine-guanine-phosphoribosyl transferase (HGPRT), which is an enzyme that helps the cells to make purines using hypoxanthine from external media, as a precursor. Normally, the absence of HGPRT does not affect the cells, as they have an alternate pathway. But cells cannot use this alternate pathway in the presence of aminopterin, so this criterion helps in the selection of the rare combination of hybridomas. The culture is transferred to HAT (hypoxanthine-aminopterin-thymine) medium. Unfused myeloma cells cannot grow as they lack HGPRT and unfused normal spleen cells will die off as they cannot grow indefinitely. Thus, only the hybridoma cells can sustain in HAT medium, which can utilize hypoxanthine from the medium and has the ability to multiply indefinitely.
Each hybridoma cell needs to be separately cultured and screened for production of the desired antibody using SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) and Western Blot. The specific epitope is used as the probe which is labeled either by immunofluorescence or radioactivity. When reliable evidence is found that the required antibody is produced by one of the hybridoma, we can culture them to facilitate large scale production of monoclonal antibody.
The applications of monoclonal antibodies are not limited to human therapy alone, but are also widely used for diagnostic and research purposes. For in vivo applications, the antibody itself is enough, which can bind to its target and initiates normal effector mechanisms in the body. And sometimes the antibody is coupled to fluorescent molecule to help in imaging or a radioactive agent is attached to it, to help in killing the target molecule. This makes monoclonal antibodies an excellent weapon to fight against cancer as it can be effectively used to target cancel cells in human body.
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