Monoclonal antibodies & Hybridoma technology- CSIR-NET/ICMR/DBT (Life Sciences)

Monoclonal antibodies & Hybridoma technology

Monoclonal antibodies (mAb)

• Antibodies produced by infection or immunization are polyclonal because natural antigens have multiple epitopes, each o which generates clones of lymphocytes.

• This results in antisera containing antibodies from different clones of lymphocytes with specificities against different epitopes of antigens.

• Monoclonal antibodies (mAb) are monospecific antibodies. These antibodies are produced from a clone of single lymphocytes directed against a single antigenic determinant or epitope.

Hybridoma technology: 

• It is a method of forming hybrid cell lines (called hybridomas) by fusing a specific Ab-producing B-cell with myeloma (cancerous B-cell).

• The Ab produced by the hybridoma are all of a single specificity and are therefore monoclonal antibodies (mAb).

• The production of mAb was invented by Cesar Milstein and Georges J.F Kohler in 1975, and they shared the Nobel Prize in 1984 for Medicine & Physiology.

• A hybridoma is somatic cell hybrids produced by fusing antibodies forming spleen cells with myeloma cells.

• Ab- producing B-cells normally die after several weeks in cell culture, in vitro. Therefore, Ab- producing B-cells are fused with B-cell tumors called myelomas. These myelomas are capable of dividing indefinitely and are called immortal cell lines.

• The immortal cell lines that result from the B cell-myeloma fusion are hybrid cell lines are called hybridomas.

• The hybridoma cell lines share the properties of both fusion partners. They grow in vitro and produce antibodies.

• Hybridomas are selected by the use of a selective medium in which the myeloma cells die, but hybridomas survive. The most widely used selective medium involves the inclusion of the antibiotic aminopterin in the growth medium.

Aminopterin (Selective medium):

• It is a synthetic derivative of pterin. 

• This folate analog acts as a competitive inhibitor for the enzyme dihydrofolate reductase which catalyzes the reduction of dihydrofolate into tetrahydrofolate.

• Normal animal cell synthesizes purine nucleotides and thymidylate for DNA synthesis by a de novo pathway requiring tetrahydrofolate. Thus, the addition of aminopterin inhibits the de novo nucleotide synthesis pathway.

• However, normal cells survive in this medium as they are able to use the salvage pathway for nucleotide acid synthesis.

• But, if the cells are unable to produce the enzyme Hypoxanthine-Guanine Phospho Ribosyl Transferase (HGPRT), they are unable to utilize the salvage pathway, therefore, die in the aminopterin-containing medium.

Production of monoclonal antibodies (mAb) step-to-step:

• To produce a monoclonal antibody, a mouse is immunized with the antigen of interest.

• During the next several weeks, antigen-specific B-cells proliferate and begin producing antibodies in the mouse.

• Spleen tissue, rich in B-cells, is then removed from the mouse, and the B-cells are fused with myeloma cells.

• Myeloma cells are engineered to be deficient in the enzyme Hypoxanthine-Guanine Phospho Ribosyl Transferase (HGPRT -ve).

• After fusion of lymphocytes with HGPRT negative myeloma cells, aminopterin-containing medium, supplemented with hypoxanthine and thymidine to ensure an adequate supply of substrate for the salvage pathway (HAT medium) is added, which kills myeloma cells but allows hybridomas to survive as they inherit HGPRT from the lymphocyte present.

• Unfused lymphocytes die after a short period of culture, which results in a pure preparation of hybridomas.