Introduction

Introduction:
Hybridoma technology is a process for producing large numbers of identical antibodies also known as monoclonal antibodies. A type of white blood cell which is known as B cells are able to produce millions of different antibodies. However, each cell itself individually can only produce antibodies of a certain specificity that means a lot more different B cells are required to the multitude of antibodies needed for a proper immune system. When foreign antigen attacks our body, the stimulation of B cell gets activated to identify this particular antigen. Eventually this B cell then starts to divide to form clone cells which produce antibodies that are identical in nature or “monoclonal” antibodies.
This is a natural mechanism of action by our body.But in 1975 George Kohler and Cesar Milstein, working at Laboratory of Molecular Biology in Cambridge, found a mimicking way to produce monoclonal antibodies .They did it by emerging myeloma cells – oncogenous cells resulting from the uncontrolled cells division. The result of the division of lymphocyte to form a clone of identical cells – with antibody-producing B cells. The fusion of B cell with the myeloma cell, the ability of rapid division is highly required highly to produce large numbers of identical antibody in the cell culture.
This process is called hybridoma technology. It involves in hybridization of cell to produce identical monoclonal antibodies that function against specific antigens. Kohler and Milstein worked on developing this process independently. Milstein had established cancerous forms of antibody-producing cells that grew and multiplied. But it showed unknown specificity, on the other hand Kohler was able to manage to get antibody-producing cells of specific antibodies, but the survival of these cells were not longer . By combining their discoveries, a great thing that is monoclonal antibodies come out which showed greater specificity and divided cells were effectively lived forever. For their outstanding work George Kohler and Cesar Milstein were awarded a share of the 1984 Nobel Prize. In modern medical science monoclonal antibodies are being widely used particularly in cancer treatment.1

Methods:
There are several steps involved in production of monoclonal antibodies by using hybridoma technology-
1. Immunization of a mouse
2. Isolation of B cell
3. Preparation of myeloma cells
4. Cell fusion
5. Selection of hybrid cells
6. Screening the products
7. Cloning and propagation
8. Characterization and storage
The total process is shown below in a figure –

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Figure: Production of monoclonal antibody

1.Immunization:
Immunization is done by injecting antigen with Freund’s adjuvant or other adjuvant or by homogenizing a gel slice containing antigen.Sometimes whole membrane or intact cells or microorganisms are used as immunogens. Generally within 2-3 weeks mice are immunized and produced desired antibody but it highly varies among investigators. 2

2.Isolation of B cell :
After some weeks of immunization ,for the measurement of serum antibodies blood samples are taken. By following various techniques serum antibody titer is determined , ELISA or enzyme linked immunosorbent assay is one of them. If the titer is low then the mice are boosted by injecting antigen without adjuvant via the tail veins for the desired immunization. But if the antibody concentration is high enough then the mouse is sacrificed and the is taken to isolate the splenocyte by using mechanical method or enzymes.3

3.Preparation of myeloma cell:
The fused antibody producing spleen cell has a limited life span, with an immortal tumor or lymphocytes known as myeloma cell that ultimately results in formation of a hybridoma . These cells are capable of ultimated growth. Myeloma cells are isolated from bone marrow and are immortalized cells . these cells are cultured in 8-azaguanine to ensure the sustainability with HAT(hypoxanthine-aminopterin-thymidine) medium used after cell fusion.Myeloma cells are cultured in 8-azaguanine a week before cell fusion.3
4.Cell Fusion:
The B cells with myeloma cells combination should be done utilizing electrofusion. The B cells and myeloma cells to adjust and combine with the utilization of an electric field thus electrofusion. 2 then it is exposed to PEG(polyethylene glycol) for short period of time, though it is toxic.Through washing PEG can be removed and fresh medium is used to keep the cells . This is a mixture of free myeloma cells,hybridoma cells(fused cells) and free lymphocytes.4
5.Selection of Hybride Cells:
The HAT(hypoxanthine-aminopterin-thymidine) medium only allows the hydribe cells to grow and the rest will disappear slowly. This phenomena can occur in 7-10 days of culture. This is very important to select only a single antibody producing hybride cell and this can possible only by isolating and cultivating them individually. For this it is necessary to dilute the suspension of hybridoma cells to have on an average one cell in each individual aliquots. These cells are then capable of producing desired antibody.4
6. Sceenring and Products:
Hybridoma cells must be screened properly to check the secretion of antibody of desired specificity. There are many methods used to do screening .ELISA or RIA are most commonly used for testing the desired antibody specificity from each hybridoma culture. From the both assays, we will found that the antibody only binds to specific antigen and unbound ones are washed off from the medium. The desired antibody can be identified by screening and the specific antibody secerated cells are called monoclonal antibody.4
7.Cloning and Propagation:
The single hybride cell shoul;d be isolated and cloned .Two techniques are commonly used-
Limited Dilution Method:
In the process hybridoma cells will be diluted in a fashion so that each aliquot can contain only one single cell. This ensures the monoclonal antibody production.4
Soft Agar Method:
Soft agar medium is provided for the growth of hybridoma cells . The monoclonal colonies will be formed showing desired specificity. Practically both techniques are used for the production of monoclonal antibody.4
8.Characterization and Storage:
The monoclonal antibodies must have the property to show desired specificity . It is more important to bring out the Mab for immunoglobulin class.the stability of monoclonal antibodies are also important. The monoclonal cell should be characterized to check their ability to withstand freezing and thrawing. At several stages of cloning and culture the cell lines are frozen with liquid nitrogen. 4

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