Production of Monoclonal Antibodies | 5 Steps and Applications

Monoclonal antibodies are a set of antibodies that 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.

Production of Monoclonal Antibodies

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

The production of monoclonal antibodies is done by either of two methods.

  1. In Vitro Method (Using cell lines in glassware)
  2. 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:

  1. These cells do not produce antibodies themselves.
  2. 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.

Self- fusion

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:

  1. The hybridoma cells are suspended suitably diluted and distributed into two microwells.
  2. The cell in each microwell is allowed to grow. The cells grow and secrete antibodies into the medium.
  3. 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 a suitable antigen that corresponds to the 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 a few weeks until the antibody concentration in the mouse blood reaches the desired level.

3. After several weeks, the blood test is done to see antibody titer 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 grows indefinitely to produce a huge number of monoclonal antibodies. The bunch of fused cells is called a hybridoma.

6. These formed tumor cells are injected into mouse peritoneal cavity or grown in vitro by tissue culture technique.

8. On injection into the mouse peritoneal cavity, the cell grows rapidly and releases them in the form of liquid into the abdomen which appears 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 the in-vitro 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.

2 thoughts on “Production of Monoclonal Antibodies | 5 Steps and Applications”

  1. Hi Larry, as per my understanding, monoclonal antibodies do not replicate in the body once administered. Hence, they need to be given repeatedly with caution and may require plasma level monitoring.

    Vaccination help the body generate its own antibodies. When the body immune system is weak, Mab’s are preferred.

  2. Do monoclonal antibodies replicate themselves within the body of a patient who received them — or continually stimulate other appropriate immune system response(s)? If not, does that then mean that the patient must receive periodic continued injections until the totality of the mAbs have destroyed or disabled the target molecules/cells of the therapy? (therein being a very significant difference between mAb therapy and vaccination)

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