Hybridoma technology is applied to induce a hybrid cell by fusing beta lymphocytes with myeloma cell. By employing this technology these hybrid cells obtain power to generate antibodies because of beta lymphocyte genetic components and also gain ability to multiply indefinitely as a consequence of involvement of myeloma cells. Hybrid cells propagate by employing Hybridoma technology that may be cultured in a laboratory or subcultured in a mouse bodily cavity. These hybrid cells produced monoclonal antibody and this process is point out 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 suitable adjuvant is blended 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 mice is bled and examination for antibodies of purposed specificity is made. Highest concentration of antibodies are established after the mouse is immense with 2-3 individual delicate doses of antigen. At the point when concentration of antibody are most extreme the mouse is yielded and the spleen is taken. Then it is separated into individual spleenocyte by applying enzyme or instrumental process. Lymphocytes of the spleen is separated from rest of the organelles by Density gradient centrifugation .
2. Cell fusion
Thoroughly clean lymphocytes are blend with HGPRT deteriorative myeloma cells in this stage . Next the combination of the cells are disclosed to a strong concentration of PEG and unification is occur. The PGE Chuck out by washing and the cells are maintained In a well nurse medium. Three types of cells are found in the medium. They are –
? combination of Hybridomas
?individual myeloma cells
HAT medium ( hypoxanthine aminopterin thymidine medium) is usually used for the selection of hybrid cells. 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 instinctive cell death following some division.
For the emission of the immune response of pointed specificity, the Hybridomas must be screened. For the pointed 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 sweep away. By along these lines, utilizing screening we can distinguish the Hybridoma cells which can produces wanted antibody.
5. Cloning and propagation
The individual Hybrid cells which generate the planned antibody are isolated and cloned. For cloning hybrid cells two methods are usually used.
Limiting dilution method: In this method, progressive 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 immunoglobulin 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.