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.
? Diagnostic Sector: Revolutionary changes have been brought up by use of monoclonal antibodies in various disease identification purposes. It is used as reagents of various biochemical analysis tool.
1. Monoclonal antibodies in Biochemical analysis.
• In detection Of Urinary levels of human chorionic gonadotropin hormone during pregnancy.
• In estimation of cancer specific tumor and also prostate cancer.
• In thyroid and other hormonal disorder.
• Detection of gonorrhea antigen and other infectious disease.
2. Cardiovascular Disease:
• Myocardial infraction
• Deep vein thrombosis.
3. Bacterial Infections.
? Therapeutic Application: They have various type of therapeutic application. they are used in cancer treatment, bone marrow transplantion,autoimmune disease.etc.
? Protein Purification:By immune affinity methods protein purification is done with the immobilized cells.they can be produce from any protein and be purified in this method.
? Catalytic MAb:Antibody can be made effective catalysts when they are made from reasction transiction state.Substrate hydrolysis rate can be increase thousand fold by doing incubation.
Conclusion: monoclonal antibodies now have proven to be the greatest extraordinarily tool in laboratory research and also in medicinal and diagnosis sector. These were developed around 25 years ago and their expanded scope of antibodies to ex vivo diagnosis of a great range of diseases is significantly remarkable.
The further advancement of hybridoma technology has led us to the unlimited availability of Monoclonal antibodies. A great number of monoclonal antibodies being generated by using the particular technology which now have aided us in the identification and analysis of tumor-and also tumor associated with antigens coming from several different type of human melanomas, carcinomas, lymphomas, and leukemia’s.