Monoclonal Antibodies: Monoclonal antibodies are antibodies that are created by identical immune cells that are all clones of a singular parent cell. Being having monovalent affinity, they bind to the identical epitope (the part of an antigen recognized by the antibody).
Polyclonal Antibodies: Polyclonal antibodies are antibodies that bind to multiple epitopes and are typically created by many totally different B-cells.
Hybridoma Technology: Hybridoma technology is the methodology of the production of large number of identical antibodies known as monoclonal antibodies.
César Milstein and Georges J. F. Köhler invented the production of monoclonal antibodies in 1975 for which they got the Nobel Prize of 1984 for Medicine and Physiology along with Niels Kaj Jerne, who made other contributions to immunology. The term hybridoma was introduced by Leonard Herzenberg in 1976–1977.
Principle: This method starts by injecting a mouse (or different mammal) with immunogen in mice that provokes immunogenic reaction. A kind of white somatic cell, the B cell, produces antibodies that bind to the immunogen. These recently created antibodies are then harvested from the mice. These isolated lymph cells are successively amalgamated with immortal B cells (a myeloma), to provide a hybrid cell line known as a Hybridoma, that has each the antibody-producing ability of the B-cell and also the exaggerated longevity and reproductivity. The hybridomas are fully grown in culture. Every culture beginning with one viable hybridoma cell, manufacturing cultures every of that consists of genetically identical hybridomas that manufacture one antibody per culture (monoclonal) instead of mixtures of various antibodies (polyclonal). In distinction to polyclonal antibodies, that are mixtures of the many totally different protein molecules, the monoclonal antibodies created by every hybridoma cell line are all chemically identical.
Hybridoma Production of Monoclonal Antibody: The production of monoclonal antibody requires several steps involving,
1. Immunization: Immunogen is injected into the laboratory mice. It is mixed with suitable adjuvant and injected intradermally or subcutaneously. This injection procedure should be in repeated manner. Blood sample is taken and assayed for the presence of the desired antibody. If the antibody is present and is in desired amount then the mice is sacrificed and it’s spleen is disintegrated into spleenocytes.
2. Cell Fusion: In this step the acquired spleenocytes are fused with plasmacytoma cells. This fusion takes place in a suitable medium like PEG (Polyethelyn Glycol). The concentration of the medium must be high (about 50%). This mixture will later form the Hybridoma.
3. Selection: In the selection step the fused cells are incubated in HAT medium (Hypoxanthine-Aminopterin-Thymidine medium) for kind of 10 to 14 days. The viable Hybridoma cells are formed in this medium. Afterwards they are transferred to a regular culture medium. The incubated medium is then diluted into multi-properly plates (96-well plastic culture plate) to such an volume that every well contain only one cell. The medium in each well is examined periodically.
4. Screening: The next step is a speedy primary screening system, in which the most effective hybridomas that produce antibodies of suitable specificity are identified. This screening is done by ELISA test. Antigen is applied in the bottom of the well of the culture plates. Samples of produced antibody is incubated in the wells. We know that antigen-antibody bind together. So, if the desired antibody is present in the sample then it’ll bind with the antigen and stay in the plates. While washing the bound materials are retained and the unbound materials are washed away.
5. Cloning: The B cell that produces the desired antibodies can be cloned to produce many same daughter clones. The cloning techniques are Limiting Dilution Method and Soft Agar Method. They can be used individually but most of the cases combined.
In limiting dilution method, 96-well plastic culture plate is used for cloning. The hybridoma cells are diluted and each cell is planted in each well. This process is repeated and it ensured that monoclonality is attained.
In the alternative method hybridoma cells are cloned in a single plate containing soft agar medium where the cells form spherical colonies.
6. Characterization and Storage: The monoclonality of the antibodies is established by biomedical and biophysical characterization of the antibodies using spectrometric, electrophoretic and chromatographic methods. The suitability and stability of the antibodies for therapeutic and diagnostic purpose is determined through this process.
Antibodies are preserved in frozen liquid nitrogen. Some are stored for running the further cloning process.