Monoclonal LO2

Overview of Monoclonal Antibodies Production

  • Focus on the production of monoclonal antibodies, using hybridoma cell lines as the primary method.

Definition of Monoclonal and Polyclonal Antibodies

  • Monoclonal antibodies: Identical antibodies produced from a single clone of B cells.

  • Polyclonal antibodies: Antibodies produced by multiple B cell clones in response to an antigen. They require immunization of an animal and are thus produced in a limited quantity.

Advantages of Monoclonal Antibodies

  • Unlimited Production: Hybridoma cell lines allow for immortal cell lines resulting in continuous production of monoclonal antibodies.

  • Custom Production: Example: Monash University provides a facility for custom monoclonal antibody production based on specific antigens.

Historical Context and Development

  • Historical Background:

    • Kola and Milstein invented the hybridoma technology in 1975.

    • Received a Nobel Prize within ten years for this invention.

    • The technique led to the discovery of various leukocyte surface antigens, some of which were previously unknown.

  • Key Contributor: Alan Williams, an Australian immunologist at Oxford, collaborated with Milstein to use the hybridoma technique to differentiate leukocytes.

    • Immunized mice with rat thymocyte membranes to provoke an immune response.

    • Led to the identification of antibodies such as CD4 in 1977.

Hybridoma Technology Process Overview

  • Basic Steps in producing monoclonal antibodies via hybridoma technology include:

    1. Immunization of a mouse.

    2. Fusion of spleen cells with myeloma cells to create hybridomas.

    3. Selection and cloning of hybridomas producing desired antibodies.

    4. Culture for unlimited antibody production.

Step-by-Step Hybridoma Production Process

  1. Immunization

    • Immunize a mouse with antigen X.

    • The mouse mounts an immune response against the foreign antigen.

  2. Isolation of Spleen Cells

    • Extract the spleen, a crucial lymphoid organ rich in plasma cells (antibody-producing cells).

    • Culture the spleen cells to obtain polyclonal antibodies (temporary as these cells are not immortal).

  3. Fusion with Myeloma Cell Line

    • Characteristics of Myeloma Cells: Cancer cells that are immortal but do not secrete antibodies.

    • Myeloma cells lack an enzyme known as HGPRT (hypoxanthine-guanine phosphoribosyltransferase).

    • Fusion Process: Use a chemical agent to fuse spleen cells (immunized, antibody-secreting) with myeloma cells to create hybridomas.

  4. Selection of Hybridomas

    • Culture cells in HAT media (Hypoxanthine-Aminopterin-Thymidine media).

    • Aminopterin selectively kills myeloma cells (due to lack of HGPRT), allowing only the hybridomas to thrive.

    • Spleen cells will die off after a short time because they are not immortal.

    • Hybridomas survive due to their immortal myeloma origin and may use the salvage pathway for purine synthesis due to the presence of HGPRT.

  5. Cloning and Expansion

    • Isolate hybridoma cells into individual wells for cloning.

    • Allow the cells to grow and secrete antibodies needed for testing.

  6. Antibody Selection

    • Use ELISA (Enzyme-Linked Immunosorbent Assay) to test for binding to antigen X.

    • Identify the optimal hybridoma clone producing the desired antibody (e.g., the first clone in our example).

  7. Expansion of Selected Clone

    • Clone of B cell gives rise to monoclonal antibodies with identical specificity, isotype, and affinity.

    • Different isotypes have varying effector functions, crucial for selecting appropriate antibodies based on intended use.

Production Scales

  • Tissue Culture: Hybridomas can be cultured in research laboratories at small scales.

  • Bioreactors: For larger-scale production, hybridomas can be cultured in bioreactors to yield significant quantities of monoclonal antibodies suitable for clinical or commercial applications.

Conclusion on Hybridoma Technology

  • Hybridoma technology allows for the sustained, unlimited production of monoclonal antibodies, revolutionizing biomedical research and therapeutic developments.