Title: Monoclonal antibody production by hybridoma technology
Introduction: Hybridoma technology plays significant action for the generation of monoclonal and polyclonal antibody. Here, monoclonal antibody production will be discussed only. In this way, a normal B-cell and a cancer cell are used. B-cell has antibody production capability and myeloma cell has immortality and high proliferation rate. Produced antibodies are specific in action. So, Identical antibodies production process is known as hybridoma technology.
The process includes six stages.
1. Immunization :First, mice is immunized. Then antibody is produced against the immunization in the mice’s body. When the amount of antibody is optimum in the mice. It is sacrificed and spleenocytes are taken out from it. Spleenocyte contains antibody producing B-cell.
2. Fusion :Here, the spleenocyte is fused with myeloma cell.50% polyethylene glycol(PEG) is used to fuse the cell. Fused cell is referred as hybridoma cell.
3. Selection: Selection is done in hypoxanthine aminopterine thymidine(HAT) medium. Here, mainly three types of cell are found.
*unfused B-cell: which will die in the medium in short time as it has short life span.
*unfused myeloma cell: which will die in the medium because of synthesis stoppage as it is HGPRT- and Ig-.
*hybridoma cell: it will survive the medium because of B-cell activity.
So, hybridoma cell is selected by this way.
4. Screening :It is done by ELISA method. The selected cells are transferred to 96 plastic well plates. Each well contains one cell. Specific antigens are adsorbed at the bottom of the plates. If the cells produce desired antibody, it will bind to that antigens. This antibody is then detected by immunoconjugate which contains two component. One component of the immunoconjugate is specific for epitope and antibody is immobilized because of this component. Another component is enzyme which brings color to the well. After incubation enzyme activity is stopped and optical density is measured by ELISA reader.
5. Cloning : After screening, cloning of the antibody can be done in interleukin-6 media for further growth and production of the antibodies.
6. Characterization and storage: After characterization the antibodies can be stored in liquid nitrogen media.