Hybridoma Method for Monoclonal Antibody Production

Monoclonal antibodies (mAbs) are identical antibodies produced by a single clone of B lymphocytes. They are highly specific and have revolutionized research and medicine. The hybridoma method, developed by Köhler and Milstein, provides a robust way to produce these antibodies.

Background: Antibody Diversity

The immune system produces a vast array of antibodies to recognize and neutralize pathogens. This diversity arises from gene rearrangement during B cell development. Each B cell expresses a unique antibody with a specific antigen-binding site.

The Hybridoma Process

The hybridoma method involves fusing a B lymphocyte (plasma cell) with a myeloma cell (cancerous B cell) to create a hybrid cell – the hybridoma. This hybridoma inherits the antibody-producing ability of the B cell and the immortality of the myeloma cell.

Step-by-Step Outline

1. Immunization: A mouse is immunized with the target antigen. This stimulates the mouse's immune system to produce antibodies against the antigen.
2. Plasma Cell Isolation: Plasma cells, which are antibody-producing B cells, are harvested from the mouse's spleen or lymph nodes. These cells are rich in antibody genes.
3. Myeloma Cell Preparation: Myeloma cells are selected for their ability to proliferate indefinitely in culture. These cells are typically immortalized cancerous B cells.
4. Cell Fusion: The spleen cells (plasma cells) are fused with the myeloma cells using a fusion agent, typically polyethylene glycol (PEG). This process creates hybridoma cells.

5. Selection: The fusion results in a mixture of fused and unfused cells. A selective medium, such as HAT medium (Hypoxanthine, Aminopterin, Thymidine), is used to eliminate unfused myeloma cells and spleen cells.

   HAT medium contains:

   • Hypoxanthine (Hyp): A purine base.
   • Aminopterin: Blocks the *de novo* pathway of purine synthesis.
   • Thymidine: Provides thymidine for DNA synthesis.

   Myeloma cells, lacking functional enzymes for *de novo* purine synthesis, will die in HAT medium. Spleen cells, having a limited lifespan, will also die. Only the hybridoma cells, which have inherited the immortality of the myeloma cells and the antibody-producing ability of the plasma cells, will survive.

6. Cloning: The surviving hybridoma cells are cloned by limiting dilution to obtain a population of cells that all produce the same antibody. This ensures that the resulting antibody is monoclonal.
7. Antibody Production: The cloned hybridoma cells are cultured in large quantities, and the culture supernatant (the liquid surrounding the cells) is collected. This supernatant contains the monoclonal antibody. The antibody can be purified from the supernatant using techniques like affinity chromatography.

Table Summarizing the Hybridoma Process

Applications of Monoclonal Antibodies

Monoclonal antibodies have numerous applications, including:

• Research: Used as tools for detecting and quantifying specific antigens.
• Therapeutics: Used to treat various diseases, including cancer, autoimmune disorders, and infectious diseases.
• Diagnostics: Used in diagnostic tests to detect the presence of specific antigens.