Monoclonal antibodies are the genetically modified proteins produced from a single cell against a particular antigen. Antigens are the foreign bodies which are harmful for the human body and can be recognized by the immune system. Antibodies are produced in large numbers and are used for the medical and research purposes. They act as markers which have the ability to bind to the antigen so that it can be recognized by the immune system and then destroy it. Antibodies are attached on the antigen by a specific site called as n epitope which is a sequence of amino acid and can easily be identified by the antibodies.
There are two features of the antibody and epitope relationship which make them to be used as a molecular tool. First feature is that antibody is able to bind to the specific site of the antigen that is epitope. Secondly, it is not necessary that whole antigen is required for the epitope to attach to the antibody but only the epitope can also join with the antibody. Antibodies are actually the proteins consisting of four polypeptide chains. These four chains make a Y shape structure. In the body of an organism, one B cell produces one specific antibody. An organism contains different kinds of B cells, that is why different types of antibodies are present inside the body which act against different antigens.
To make antibodies as molecular tool, scientists need to culture a specific type of antibody. For this reason, those B cells are cultured which have the same ancestor that is they have produced from a single B cell. Only then it will be possible to get same kind of antibodies which will have the ability to bind to one type of antigen. Antibodies produced through this method will be called as monoclonal antibodies.
Synthesis of Monoclonal Antibodies:-
Commonly monoclonal antibodies are produced by the fusion of myeloma cells with the spleen cells. For this purpose, mouse is used as an experimental animal in which the specific antigen in inserted for which antibody can act. A special chemical polyethylene glycol is used to fuse B cells with the myeloma cells already present in the culture. These cells can also be fused with Electroporation, a method in which pores are produced in the cell membrane. The resulting substance after fusion is called as hybridoma.
In the next step, a medium is selected for the hybridoma. In the medium, the myeloma cells are hypoxanthine-guanine phosphoribosyl transferase and B cells are hypoxanthine-guanine phosphoribosyl transferase plus. HGPRT is an enzyme which is responsible for the synthesis of nucleotides. These nucleotides are taken from the amino acid hypoxanthine. For the culture to grow efficiently, it is grown in the HAT medium. HAT is the Hypoxanthine-aminopterin-thymine medium. Within the culture, if myeloma cells are combined with other myeloma cells, they will not survive and will die eventually. Same is the case with the b cells. Only those cells will survive in the medium which are the combination of myeloma cells and B cells.
The mixture of the cells is cloned and diluted. The clones are gotten from the single parent cells. The antibodies which will produce as a result, they will analyze to see if they have the ability to bind to the antigen. For this purpose different tests are performed like ELISA, immuno dot blot or Antigen Microarray Assay. The clone which shows best results is saved and used for the future purposes.
Along with the hybridoma, the culture also contains growth factors, hormones and enzymes. Some of the material like lipids, cell debris and dead cells are removed from the culture by filtration method. To take out the required antibodies, they are precipitated out by using the chemicals like sodium sulphate or ammonium sulphate. Usually antibodies do not need much salt concentration and they can be precipitated out at low concentrations. The level of the salt should be appropriate other wise it will be difficult to purify the antibodies. Excessive salt is then removed through dialysis. To check the purity, ELISA kits are used.
1) Monoclonal antibodies are used to treat viral infections.
2) They are also used in the treatment of cancer.
3) They can also be used in various diagnostic tests to see the effect of drugs or toxins.
4) Monoclonal antibodies can also be used for the research purposes to identify the molecules in n organism.
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