Antibody or immunoglobulin (Ig) is a large, Y shaped glycoprotein which can bind to antigen and found in the B cell membrane and it is secreted by plasma cells. Two types of antibody are available. One is monoclonal antibody and other is polyclonal antibody. Monoclonal antibody is a homogeneous antibody formed from the clone of single B cell and it contains specific antigen binding sites and isotype. It develops certain immunoassay systems. On the other hand, hybridoma technology is a method which is used for producing large numbers of antibody. It is a useful technique for the production of monoclonal antibody. For greater supply of monoclonal antibody, the clone of B cell would be done in artificial medium but B cell cannot stay in this medium and died. To make B cell immortal we use hybridoma technology to produce desired monoclonal antibody.
In this technology immortalization of B lymphocytes cell is occurred which has the capacity to produce antibody but in limited growth. The lymphocytes are attached to cells which come from a non-antibody conducting and growing cell line of tumor (myeloma) and the hybrids secrete antibodies. Monoclonal antibody is one kind of hybrid cell which is made by the fusion of two cells. First one is myeloma cell which has genetic potential to multiply continuously and the second one is gene that make codes for the production of desired antibody. (Anon, 2018)
Figure – Production of monoclonal antibodies
Adopted from – https://med.unr.edu/ddl/technology/monoclonal-antibodies
Production of monoclonal antibody by using hybridoma technology requires some steps.The steps are-
6.Characterization and storage
1. Immunization- Immunogen about microgram to milligram amounts and a compatible adjuvant mixed together. This mixture injected to the mouse either by intradermally or by subcutaneously at different location and at various times and the process is done repetitively.Then the animal is bled and trial it for antibody specificity. When the concentration of antibody is optimum then the animal is dedicated & we take the spleen to make single spleenocytes with the help of enzymes and mechanical systems.
2. Cell fusion- Spleenocytes & plasmacytoma are mixed together in an adequate medium manifested to a 50% concentration of PEG(Polyetylene Glycol) & fusion is occurred in a time period.Then the hybridoma will be formed.
3. Selection & Screening- HAT medium (a medium which has Hypoxanthine, Aminopterine & Thymidine) helped in the selection of hybridoma and the medium is copied by screening the hybridomas for the antibodies specificity. After this method, cells are shifted to medium & incubated. The cells which are durable called hybridomas.By removing them from the medium, we shifted them to constant culture medium. Then a large portion of sample are served in the 96 well plastic culture plates. ELISA is the most ordinary screening experiment. In the base of 96 plates the antigen is adsorbed and we incubated the samples in the well over a period of time. If the sample has antibody in can bind to the antigen & stay in the well as the loose compound is washed off. Then the antibody is found out by an immunoconjugate which hold two covalently attached compounds. First compound is antibody specific & second compound is an enzyme. After second washing, we use a colorless enzyme in the wells & it convert to a colored components. The enzyme action is stopped after incubation & we measure optical density by ELISA reader.
4. Cloning- The aimed antibody is secreted by single cell from cultures & disclosed in cell lines. Cloning method are 2 types- one is limiting dilution method & other is soft agar method. In first method cells present in culture are counted, diluted & divided into new cells as every well has one cell, Then regrow of cells is occurred in a repetitive manner & all cells in the well are monoclonal. In soft agar method the medium is semisolid which has less quantity of agar which can make spherical colonies. Then we applied single cells to the medium so that colony is formed & all the colony is monoclonal. Both methods are used together in the cloning process. The culture of separate colonies is done & shifted it to larger vessels for characterization.
5. Characterization & Storage- In characterization process we set up the monoclenality of antibody. Three methods are used in this process. They are- spectrometric, electrophoretic & chromatographic. Class or subclass of antibody, epitope & number of binding sites etc. are also characterized by this method. The antibody is stored in a frozen state in liquid nitrogen & some antibody is kept for the further procedure.