Production of Monoclonal Antibodies | 5 Steps and Applications
Monoclonal antibodies are set of antibodies which are target specific against a single source of antigen.
A recent development in cell biology has allowed the fusion of different cells to form a “hybrid’ cell, which expresses the properties of both parental cell lines. The fusing agent can be a defective virus (Sendai virus that characteristically causes cell fusion) or various chemicals (such as polythene glycol. And this gives rise to cells called hybridomas.
In a hybridoma, clone produces antibodies of a single specificity. Since the clone is derived from the fusion of a single differentiated (antibody-forming) B-cell with a myeloma cell, i.e., it is a clone of a single of B-cell. Therefore, such antibodies are known as monoclonal antibodies. All molecules of a monoclonal antibody will have the same specificity.
In 1975, the two scientists Georges Kohler and Cesar Milstein, devised a revolutionary method for preparing monoclonal antibodies, which quickly become one of the immunology’s key technologies. They were awarded Nobel Prize for the same in medicine and physiology in the year 1984.
Production of monoclonal antibodies
Production of monoclonal antibodies is done of by either of two methods.
- In Vitro Method (Using cell lines in glass ware)
- In Vivo method (Using living animals like Mice)
In Vitro Method
In this method, the monoclonal antibodies are produced by fusing a normally activated antibody-producing B-cells with myeloma cells. The hybrid cell, which is produced as the result of this fusion is called a hybridoma. And this hybridoma possesses the immortal growth properties of the myeloma cell and antibody-secreting property of the B-cell.
The steps to produce the monoclonal antibodies are as follows:
Isolation of B-lymphocytes
The isolation of B-lymphocytes takes place from the spleen of an animal (e.g., Mouse), which has been immunized with the antigen against the monoclonal antibodies is to be raised. The immunization of animals is achieved by injecting the antigen, with a suitable adjuvant (non-antigenic preparation known to stimulate an immune response ) either simultaneously or in peritoneal cavity followed by booster doses of antigen.
The immunization enhances the population of B-lymphocytes producing antibodies which are specific to the antigen used i.e., called as clonal selection (clonal selection: where the antigen reacts to the cell surface receptor of B-lymphocytes) it proliferates rapidly and to yield a population (clone) of B-cells, all of which produce antibodies of the same specificity. This whole process is called clonal selection.
Isolation of myeloma cells
The myeloma cells are fast-growing large cells of a hemopoietic portion of bone marrow. It is capable of multiplying indefinitely. The myeloma cells are selected for only two features which are as follows:
- These cells do not produce antibodies themselves.
- The cell contains a genetic marker (HGPRT –hypo xanthine phosphor ribosyl transferase), which permits an early solution of the resulting hybrid cells.
The myeloma cells are taken into account for the production of monoclonal antibodies.
The next step is the fusion of HGPRT cells and B-lymphocytes. After the fusion of these two, the mixture is produced which is treated further with PEG (polyethylene glycol). The cell mixture is shaken well for 3min. PEG brings the cells together and induces the fusion. The resulting cell population is the fusion unit of hybrid cells, which is called a hybridoma. i.e., myeloma cells and B-lymphocytes.
Selection of hybridoma cells
The resultant cell population is now cultured or cultivated in the HAT medium (HAT medium is supplemented with hypoxanthine aminopterin thymidine) . The unfused myeloma cells can’t grow in the mixture because they lack HGPRT and thus cannot replicate. The myeloma cells which contain HGPRT only can grow and replicate henceforth. The hybridoma cells are able to grow indefinitely in the media because the spleen cell partner supplies HGPRT and the myeloma partner has traits that make it immortal similar to a cancer cell.
Then, the cultures are screened well for the selection of hybrids producing antibodies that are specific for the immunizing antigen.
The B-cells do not grow for a long period and eventually dies due to a shorter lifespan.
The HGPRT myeloma cells are unable to divide in the HAT medium due to aminopterin because the HGPRT enzyme is responsible for purine synthesis. However, aminopterin inhibits the purine biosynthesis in myeloma cells by alternate pathways.
Screening of hybridoma
The next step is the identification and isolation of hybridoma cells, which are specific to the antigen used for animals. It involves:
- The hybridoma cells are suspended suitably diluted and distributed into two microwells.
- The cell in each microwell is allowed to grow. The cells grow and secrete antibodies into the medium.
- The supernatant from each microwell is sampled for the presence of antibodies specific to the antigen is studied.
Using one of the methods based on either precipitation or agglutination caused by the antibodies specific to the antigen. Most times, ELISA is done in the process of extracting the monoclonal antibodies. This way the monoclonal antibodies are produced.
In Vivo method
This method involves following steps
1. First an animal like a mouse, rabbit etc. is immunized with suitable antigen which corresponds to desired monoclonal antibody we are interested in. This is done by injecting antigens which are previously emulsified with some adjuvants like Freund’s adjuvant.
2. This leads to enhanced production of desired antibodies in the mouse body. The immunization is done for few weeks till the antibody concentration in the mouse blood reaches a desired level.
3. After several weeks, the blood test is done to see antibody titre using suitable techniques like ELISA or flow cytometry etc.and are obtained from mouse spleen.
4. Then the obtained monoclonal antibodies are fused with a cancer cell (i.e. myeloma not just tumor) as they are immortal and can divide indefinitely.
5. So the fused cell grow indefinitely to produce huge number of monoclonal antibodies..The bunch of fused cells is called hybridoma.
6. These formed tumor cells are injected into mouse peritoneal cavity or grown in vitro by tissue culture technique.
8. On injection into mouse peritoneal cavity, the cell grow rapidly and release them in the form of liquid into the abdomen which appear as bulged fluid bags (ascites).
9. These monoclonal antibodies from ascites are extracted to harvest the formed antibodies.
Another method is growing these hybridoma cells invitro but is not easy and also is expensive than the mouse method. But sometimes the mouse may experience pain, irritation due to ascites. Then they go for invitro cell culture technique.
Application of monoclonal antibodies
Monoclonal antibodies find their application in diagnosis, imaging, and therapeutic agents in clinical medicine. Such hybridoma – derived monoclonal antibodies are becoming increasingly important in diagnostic areas. For example: in cancer therapy, monoclonal antibodies can be used directly to attack and destroy tumor cells. They can be labeled with radioactive isotopes to locate tumors and to deliver specifically lethal doses of radiation to inaccessible tumors. They can be used to deliver anticancer drugs to tumor cells in a similar manner.