I shall explain the hybridoma technique used in production of monoclonal antibodies in a simplified way.

An antigen (unless it is a small peptide) is a complex molecule with several antigenic determinants (or epitopes). When the immune system encounters such an antigen, it is usually processed to result in several fragments. Humoral (antibody-mediated) response may occur against some of these fragments. There are multiple clones of B cell, each against a specific epitope; resulting in production of antibodies against several epitopes. Such a response is said to be polyclonal. This is what that happens when our body encounters a microbial antigen following infection or immunization.

There are situations when it becomes necessary to have antibodies against a single antigenic determinant produced by a single clone of B cell. Such a response is said to be monoclonal. In order to produce monoclonal antibody, it is necessary to possess a purified antigen.

Hybridoma technique was developed by Georges Kolher and Cesar Milstein in 1975, for which they were awarded the Nobel Prize. The chief participants of this technique are the B cells and the myeloma cells. The B cells are obtained from the mouse which has been immunized with the antigen of choice. Myeloma cells are malignant B cells that are immortal and multiply continuously. Myeloma cells that have lost the ability to produce antibodies are chosen for this technique. In addition, these cells lack the ability to produce hypoxanthine-guanine phosphoribosyl transferase (HGPRT-) and thymidine kinase (TK-) enzymes through an induced mutation. Normal B cells have functional enzymes (HGPRT+ and TK+) and are able to produce antibodies. However, their life span is not beyond two weeks. Hybridoma technique involves physical fusion of both these cells so that the resulting hybrid (called hybridoma) has the features of both these cell types.

The first step towards the production of monoclonal antibodies is the immunization of the animal by antigen of choice. Mouse is the commonly used animal, but rat or hamster too can be used. Following repeated immunization (booster doses) the blood of the animal is tested for antibodies against the immunized antigen. Once it is determined that the animal has produced sufficient antibodies, it is killed and its spleen removed. The spleen is rich in B cells and would contain B cells specific to the immunized antigen among B cells of other specificities. The B-cells are separated from other cells and cultured. They are then mixed with cultured myeloma cells and allowed to fuse. Fusions of the cells are aided by polyethylene glycol (PEG). Not all cells fuse; present in the reaction mixture are unfused B cells, unfused myeloma cells and fused hybridoma cells. The next step involves separation of hybridoma cells from the unfused cells using a special selective medium.

In order to understand the functioning of selective medium, one must be aware of the following facts. Multiplying cells need to produce their DNA. Most cells produce their purines nucleotides and thymidylate (both precursors of DNA) utilizing tetrahydrofolates by a De-Novo pathway. This can be blocked using anti-folate drugs such as Aminopterin. The cells can then adopt Salvage pathway to synthesize DNA if hypoxanthine and thymidine are exogenously supplied. Purine nucleotides are produced from hypoxanthine using hypoxanthine-guanine phosphoribosyl transferase enzyme and thymidylate is produced from thymidine using thymidine kinase. The selective medium (HAT medium) used to select the hybridoma contains aminopterin, hypoxanthine and thymidine. Amiopterin inhibits the De-Novo pathway and presence of hypoxanthine and thymidine facilitates salvage pathway.

Normal unfused B cells can’t produce DNA by De-Novo pathway because of aminopterin but are able to undertake salvage pathway. This is because they contain functional enzymes (HGPRT+ and TK+). However, since they are mortal, they die after few multiplications. Unfused myeloma cells too can’t use De-Novo pathway because of aminopterin. They are unable to utilize the salvage pathway either because of deficient enzymes (HGPRT- and TK-). These cells die despite being immortal. However, fused hybridoma cells receive (HGPRT+ and TK+) trait from normal B cells and immortality from myeloma cells. These cells can utilize salvage pathway for DNA synthesis and yet be immortal. After two weeks, only the hybridoma cells survive in the selective medium.

The surviving hybridoma cells would have formed against different epitopes. The next step is to select the hybridoma produced against the desired antigen. The cultures are diluted to such an extent that only a single cell gets transferred to the wells of microtitre plate. The cells are allowed to multiply. These cells produce antibodies that can be readily detected in their supernatant fluids. Supernatant fluids from all the wells are tested for antibodies against the antigen of choice and the well that contains desired antibodies is selected and the rest may be discarded. Finally, a hybridoma cell producing antibodies against the epitope of choice is available.

These hybridoma cells may be lyophilized, cultured in vitro or injected intra-peritoneally into a mouse and monoclonal antibodies raised whenever required.