HYBRIDOMA TECHNOLOGY INTRODUCTION

HYBRIDOMA TECHNOLOGY

INTRODUCTION:
The antigen has multiple epitopes. For producing multiple antibody series of B-cell is stimulated. B cell recognizes and reacts with the antigens and activated cells. These activated cells are matured in plasma cell. Then they synthesizes antibody of specific epitope. This is called polyclonal activation. As polyclonal do not have single specificity, it is not preferred. Monoclonal antibody is preferred for single specificity. To produce monoclonal antibody an artificial medium is needed where B-cell will be destroyed. In this media myeloma cell is fused with B-cells to immortalize the B-cell. This technique which is used to produce monoclonal antibody is called “Hybridoma Technology”. Kulkarni, G. (2002)

Adopted from: https://www.researchgate.net/figure/Illustration-showing-the-production-route-of-hybridoma-technology-Monoclonal-antibodies_fig3_315849444

METHOD:
The production of monoclonal antibody in hybridoma technology involves following steps-
1. Immunization
2. Cell fusion
3. Selecting
4. Screening
5. Cloning
6. Characterization and storage

Immunization:Immunogen of microgram to milligram quantities are mixed with adjuvant and injected to mouse. It is given subcutaneously or intradermally at multiple sites. It is given at different times in a repetitive manner. Then the animal is bled and assayed. When concentration of antibody is optimum, mouse is sacrificed and spleen is taken. Then single spleenocyte is dissociated.
Cell fusion: Spleenocytes are mixed with plasmacytoma cells in an medium which contains high PEG ( Polyethyleneglycol) concentration and fusion is done to take place over a period of time. In this step hybridoma is formed.
Selection:A HAT medium (Hpoxanthene Aminopterine Thymidine) is selected for antibodies which will provide desired specificity. After fusion, cells are transferred to this medium. Then incubated the cells. After that they are removed from the medium and transferred into regular culture medium.
Screening:In screening, the cells are distributed among wells of 96-well plastic culture plates. Most common screening assay is ELISA. Antigens will be absorbed to the bottom of 96 well plates. If sample have the desired antibody , it will bind with the antigen and will remain in the well. The unbound material will be washed off. After that antibody is detected by an immunoconjugate which contains two components – one is specific for an epitope another is an enzyme.
Cloning:In cloning two methods are involved-
1) Limiting dilution method
2) Soft agar method

1) Limiting dilution method: In this method first cell are diluted and aliquoted into wells. Each well contains only one cell. Then cells are regrowed& method is applied repeated to ensure that cells are monoclonal.
2) Soft agar method: A semisolid medial which contains less amount of agar is taken. This
forms spherical colonies. In this method, Single cells are dispersed in culture so that they can form colony with sufficient space. Every colonies will produce monoclonal.Kulkarni, G. (2002)

Characterization& storage: Monoclonality of the antibody is established by characterization process. It requires biochemical & biophysical characterization. Chromatographic, Spectrometric electrophoretic methods are used in this case. Suitability of antibody is tested.
Antibodies are stored in liquid nitrogen to avoid degradation of clones. Stocks should contain maximum number of clones so that it can be used for further production.Kulkarni, G. (2002)
Application:.
1) Monoclonal antibody is used in cardiovascular diseases.
2) Used in cancer treatment.
3) Used in organ transplantation.
4) Used in autoimmune diseases.