Hybridoma technology is applied to create a hybrid cell by fusing beta lymphocytes with myeloma cell. By using this technology these hybrid cells obtain power to generate antibodies because of beta lymphocyte genetic components and also gain capability to multiply indefinitely resulting from the attendance of myeloma cells. This hybrid cells generated by using Hybridoma technology that can be cultured in a laboratory or subcultured in a mouse bodily cavity. Monoclonal Antibody are produced from these hybrid cells and this technology is referred as Hybridoma technology.
The production of MAbs by using Hybridoma technology involves following steps:
2. Cell fusion
3. Selection of Hybridomas
5. Cloning and propagation
6. Characterization and storage
An appropriate adjuvant is composited with microgram or milligram amounts of immunogen and injected at multiple site of the mouse at various times in a repetitious manners. At times the is bled and test for antibodies of aimed specificity is made. Highest concentration of antibodies are established after the mouse is immense with 2-3 individual miniature doses of antigen. When concentration of antibodies are maximum the animal is sacrificed and the spleen is taken. Then it is separated into individual spleenocyte by applying enzyme or instrumental process. Density gradient centrifugation is mostly used to separate the lymphocyte of the spleen from the rest of cells organelles.
2. Cell fusion
In this step HGPRT defective myeloma cells are mixed with thoroughly clean lymphocytes. The mixture of the cells are then exposed to a strong concentration of PEG and fusion is allowed to occur. The PGE ejected by washing and the cells are maintained In a well nurse medium. Three types of cells are found in the medium. They are –
? mixture of Hybridomas
?free myeloma cells
For selection of hybrid cells hypoxanthine aminopterin thymidine medium (HAT) is used. In HAT medium the cellular synthesis of purines and pyrimidines from simple sugar is blocked by aminopterin. But some cells can flourish by taking advantage of hypoxanthine and thymidine existent in the medium by salvage pathway utilizing hypoxanthine guanine phosphoribosyl transferase (HGPRT). But HGPRT is deficient in myeloma cells. So when aminopterin block denovo pathway myeloma cells can not survive in HAY medium. Beta cell can survive in the HAT medium as they are HGPRT+ . Beta cell go through natural cell death following some division.
For the secretion of the antibody of aimed specificity, the Hybridomas must be screened. For the aimed antibody specificity the culture from individual Hybridoma culture is being tested periodically. ELIAS and RIA are mostly used technique for this reason. In these experiments, the antibody is attached to the specific antigen and unwind antibody and remaining components of the medium can be sweeped away. By this way, using screening we can identify the Hybridoma cells which can produces desired antibody. The antibody produce by the hybrid cells are monoclonal antibody.
5. Cloning and propagation
The individual Hybrid cells which produce the intended antibody are isolated and cloned. For cloning hybrid cells two methods are usually used.
Limiting dilution method: In this method, gradual dilution of the suspension of Hybridoma cells is made and aliquots of individual dilution are place down into micro culture wall. The dilution are made to point that every aliquot in a wall contains just one individual hybrid cell. This guarantees the immunglobin which has delivered is monoclonal.
Soft agar method: In this procedure the Hybridoma cells are refined in soft agar. It is conceivable to develop numerous cells at the same time in semisolid medium to form colonies. These colonies will be monoclonal in nature.
In genuine practice both above system are consolidated and utilized for the maximum production of MAbs.
6. Characterization and storage :
The monoclonal antibody must be exposed to biochemical and biophysical portrayal for the intended specificity. It is likewise vital to clarify the MAbs for the immunoglobulin class or sub-class, the epitope for which it is particular and quantity of binding site it has . The strength of the cell lines and the MAbs are essential. The cells must be described for their capacity to withstand solidifying and defrosting. The intended cell lines are solidified in liquid nitrogen at various phase of cloning and culture.