Production of Monoclonal antibody by hybridoma technology
Monoclonal antibody: Mono-specific antibodies which are made from identical immune cells that are clones from a unique parent cell. Each type of the monoclonal antibody can binds to the same epitope of an antigen.
They can now be produced from hybridoma technology. So first we need to learn about what hybridoma technology is-
The Hybridoma technology: It is the technology of creating immortalized cell lines through fusing B- lymphocyte cells (antibody- producing spleen cell) with tumor cells called myloma, which results in hybrid cells which produce a desirable protein, typically that turns out to be a monoclonal antibody. As these hybrid cells are formed by fusion of two cells, so they have the growth characteristics of the myeloma component and also the antibody-secreting characteristics seen in the spleen cell.
Production of Hybridoma : useful monoclonal antibodies requires several steps in their production which are written below:
1. Immunization: Firstly a mouse is immunized, and it is done by giving microgram to milligram quantities of antigen injection along with an adjuvant (non antigenic in nature but stimulate the immune system) via peritoneal cavity.
When it is assured that the desired antibodies production are in specific amount, then the animal is sacrificed. The spleen of the sacrificed mice or animals dissociated into single spleenocytes using enzymes/mechanical methods.
2. Fusion of the cell: In very high concentration of polyethylene glycol spleenocytes are mixed with plasmacytoma cells. Then the mixture is allowed to stay over a period of time to form the hybridoma cells.
3. Selection and screening of the cultured cells: when fusion is done then the cells are transferred to a medium called HAT medium and kept for incubation. These cells are viable cells and taken to culture medium for being distributed to 96 well plastic culture plates.
The medium need to be tested now for desired antibody reactivates and it is done by ELISA.This method allows the antigen to be adsorbed 96 well plate and then the plates are incubated for a required time period.
so if the sample contains the desired amount of antibody then it will bind to the antigen and remain in the well. But the unbound materials should be washed off.
4. Cloning: This technique is to grow cells which are said to be a clone of cells from an isolated single cell. The method that is used for cloning requires limiting dilutions and method of soft agar.
Limiting the dilution method: It is to dilute the cells in to a concentration which is calculated previously so that each well of a 96 well plate hold only one/two or no cells after plating out. Once the cells have begun growing form a clone, there is need to view each well under the microscope and discount any well with more than one clone or no clones at all.
Soft agar method: In this technique, the hybrid cells are cultured where the media is soft agar. Simultaneous growth of many cells in a medium which is semisolid in nature to form colonies is possible. These newly formed colonies will now be monoclonal in characteristics.
In actual laboratory practice, both of the above mentioned techniques are combined together and then used for maximum production of monoclonal antibodies.
5. Characterization and Storage:
The cells will bypass through various bio-chemical and bio-physical characterization to attain fo desired specificity. Spectrometric, electrophoretic and chromatographic are used for this purpose.
Cell lines and the monoclonal antibodys stability is a very important issue, as the cells must be characterized by their ability to withstand the freezing and thawing environment. The desired cell lines are frozen in liquid nitrogen at several stages of cloning and culture.
Conclusion: monoclonal antibodies have been extraordinarily tools in laboratory research for many years. Monoclonal antibodies were developed about 25 years ago and have expanded the scope of antibodies to ex vivo diagnosis of a wide range of diseases.
The advent of hybridoma technology has led to the unlimited availability of Monoclonal antibodies. Numerous monoclonal antibodies generated using this technology have aided the identification and analysis of tumor-associated antigens from several different human melanomas, carcinomas, lymphomas, and leukemias. Literature available till date reports over 100 unique monoclonal antibodies against human carcinomas.