Production of Monoclonal antibody by hybridoma technology
Monoclonal antibody: Monoclonal antibodies are monospecific antibodies that are made from identical immune cells which are clones of a unique parent cell. Each type of monoclonal antibody binds to the same epitope of an antigen.
Monoclonal antibodies are produce now from hybridoma technology.
Hybridoma technology: Hybridoma technology is the technology of forming immortalized cell lines by fusing B- lymphocyte cells (antibody- producing spleen cell) with myeloma tumor cells. The resulting hybrid cells produce a desirable protein, typically a monoclonal antibody. As these hybrid cells are result from fusing two cells so have the growth characteristics of the myeloma component and the antibody-secreting characteristics of the spleen cell.
Hybridoma production: the production of hybridoma that continuously Asecretes the useful monoclonal antibodies requires several steps:
1. Immunization: Mouse is immunized by giving microgram to milligram quantities of antigen injection along with an adjuvant (non antigenic in nature but stimulate the immune system) via subcutaneously or by peritoneal cavity.
After assuring that the desired antibodies are produced in specific amount, the animal is sacrificed and the spleen is dissociated into single spleenocytes by using enzymes or mechanical methods.
2. Cell fusion: At a higher concentration of polyethylene glycol , the spleenocytes are mixed with plasmacytoma cells and the mixture is allowed to take place over a period of time to form the hybridoma cells.
3. Selection and screening: After fusion the cells are transferred to HAT medium and incubated. The hybridoma cells are the viable cells .the hybridoma cells are taken to culture medium and distributed to 96 well plastic culture plates.
The medium now need to be tested for desired antibody reactivates which is done by ELISA .in this method, the antigen is allowed to adsorb to these 96 well plate and these plates are incubated for required period.
If the sample contains the desired antibody it will bind to the antigen and remain in the well. But the unbound materials are washed off.
4. Cloning: The purpose of the cloning technique is to grow a clone of cells from an isolated single cell. The method that use for cloning is limiting dilutions and soft agar method.
Limiting dilution method: This means to dilute the cells to a concentration calculated so that each well of a 96 well plate will contain one, two or no cells after plating out. Once the cells have begun growing to 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 hybridoma cells are cultured in soft agar. It is possible to simultaneously grow many cells in semisolid medium to form colonies. These colonies will be monoclonal in nature.
In actual practice, both the above techniques are combined and used for maximal production of monoclonal antibodies.
5. Characterization and Storage:
The monoclonal antibody has to be going through to biochemical and biophysical characterization for the desired specificity. Spectrometric, electrophoretic and chromatographic are used for this purpose.
The stability of the cell lines and the monoclonal antibody are important. The cells must be characterized for their ability to withstand freezing and thawing. 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 leukemia’s. Literature available till date reports over 100 unique monoclonal antibodies against human carcinomas.